IMPERIAL COLLEGE LONDON

Faculty of Engineering

Department of Civil and Environmental Engineering

Protozoa in Anaerobic Waste Treatment

Systems
Brenda Mary Owomugisha
September 2015

Submitted in fulfilment of the requirements for the MSc and the Diploma of Imperial College
London

DECLARATION OF OWN WORK

Declaration:
This submission is my own work. Any quotation from, or description of, the work
of others is acknowledged herein by reference to the sources, whether published
or unpublished.

Signature: ___________________________________

Abstract
Protozoa play an important role in the environment as a link between primary producers and
decomposers, and higher organisms in the ecosystem. They are also common in anaerobic
environments because they have hydrogenosomes that enable them carry out anaerobic
fermentation. It therefore important to understand their physiological behaviour and the roles
they play in different environments including aerobic wastewater treatment and anaerobic
environments like the rumen and sediments and harnessing this to increase CH 4 production
and substrate utilization in anaerobic digestion. Furthermore, it is essential to understand the
role they play in enteric pathogen inactivation in soil.
Protozoa have several behavioural traits that enable them take part both directly and
indirectly in the anaerobic degradation of waste and generation of CH 4. They enhance
bacterial activity by grazing on them and supplying essential growth nutrients as byproducts
of their metabolism thereby increasing substrate utilization. Furthermore, protozoa have the
ability to degrade recalcitrant cellulose mainly intracellularly through phagocytosis as
observed in the rumen. In addition to this, they are directly involved anaerobic fermentation
by ingesting particulate and soluble organic matter. They contribute to the generation of CH 4
by enhancing methanogenic bacterial activity and through their relationship with
endosymbiotic methanogens that utilize the byproducts of their metabolism to generate CH4.
Cellulose is common in sewage sludge and municipal solid waste but is not readily degraded
in anaerobic digesters therefore the use of protozoa as a biological pretreatment of waste for
anaerobic digestion has been proposed because of their efficient degradation of cellulose. In
the soil, protozoa preferentially graze on gram-negative bacteria, which are the major human
gastro intestinal pathogens and therefore play a biotic role in their inactivation. However,
they sometimes internalise pathogens therefore contributing to their continued persistence
and spread. Further research should be carried out to determine combined effect of this
behaviour to establish a scientific approach to land restrictions on the use of biosolids.

Acknowledgments
There are several people I would like to thank for helping me successfully complete my
dissertation.
I am sincerely grateful for the guidance given by Prof. Stephen Smith, my supervisor,
throughout my research. His insightful mentorship was paramount in enabling me conduct
well rounded research. I am also grateful to Dr. Hannah Rigby, my co-supervisor, for her
comments that greatly improved my dissertation.
In addition, I would like to thank my family and friends for their moral support and
encouragement throughout this period.
Last but not least, I thank the Lord Almighty who preserved me in good health throughout.

Table of Contents
1 Introduction...........................................................................................................1
1.1

Background................................................................................................................... 1

1.2

Thesis Structure............................................................................................................2

1.3

Aims and objectives.......................................................................................................3

1.3.1 Aim.......................................................................................................................... 3
1.3.2 Objectives................................................................................................................3

2 Aerobic Protozoa..................................................................................................4
2.1

Role in aerobic wastewater treatment............................................................................4

2.2

Protozoa as indicators of activated sludge plant performance.......................................5

3 Anaerobic Protozoa..............................................................................................7
3.1

Oxygen effects............................................................................................................... 7

3.1.1 Adaptation to oxygen...............................................................................................7

3.2

Anaerobic Metabolism...................................................................................................8

3.2.1 Anaerobic metabolic pathways in protozoa.............................................................9

3.3

Symbiosis.................................................................................................................... 12

3.3.1 Phototrophic endosymbionts.................................................................................12

3.3.2 Methanogens.........................................................................................................13
3.3.3 Ectosymbiotic sulphate reducers...........................................................................14
3.4

Anaerobic Digestion.....................................................................................................14

3.4.1 Anaerobic Reactors...............................................................................................16

3.4.2 Factors affecting anaerobic digestion....................................................................17
3.5

Ammonia toxicity..........................................................................................................18

3.5.1 Perturbation of electrochemical gradients and pH.................................................19

3.5.2 Interaction with enzymes.......................................................................................20
3.6

Ammonia influence on methanogenesis in anaerobic digesters..................................21

3.7

Role of protozoa in anaerobic wastewater treatment...................................................21

4 Fundamental Characteristics of Protozoa........................................................24

4.1

Classification............................................................................................................... 24

4.1.1 Subphylum Sarcomastigophora.............................................................................24

4.1.2 Subphylum Sporozoa............................................................................................25

4.1.3 Subphylum Cnidospora.........................................................................................26

4.1.4 Subphylum Ciliophora...........................................................................................26
4.2

Nutrition....................................................................................................................... 26

4.2.1 Feeding mechanisms............................................................................................28

4.3

Movement.................................................................................................................... 28

4.3.1 Pseudopodial movement.......................................................................................29

4.3.2 Ciliary movement...................................................................................................29
4.3.3 Flagellar movement...............................................................................................30
4.4

Reproduction............................................................................................................... 30

4.5

Protozoan in aquatic food webs...................................................................................30

4.5.1 The food web dynamics.........................................................................................31

5 Protozoa activity in ruminant animals..............................................................34

5.1

Movement of digesta through the reticulorumen..........................................................35

5.2

Role of protozoa in the rumen......................................................................................38

5.2.1 Entodiniomorphida protozoa..................................................................................38

5.2.2 Holotrich protozoa.................................................................................................38
5.2.3 Cellulose, hemicellulose and pectin degradation...................................................39
5.2.4 Storage and soluble carbohydrates.......................................................................42
5.2.5 Protein................................................................................................................... 43
5.3

Methane production.....................................................................................................45

5.4

Ammonia in the rumen.................................................................................................45

6 Protozoa in water and terrestrial environments..............................................46

6.1

Marine and Freshwater Sediments..............................................................................46

6.1.1 Distribution of protozoa..........................................................................................48

6.1.2 Environmental factors affecting protozoa in sediments..........................................48
6.1.3 Metabolism and nutrient cycling of protozoa in sediments.....................................49
6.1.4 Grazing of protozoa...............................................................................................49
6.2

Methane production.....................................................................................................50

6.3

Ammonia in sediments................................................................................................50

6.4

Soil Protozoa............................................................................................................... 51

6.4.1 Role of protozoa in the soil....................................................................................51

6.4.2 Factors affecting protozoa activities in soil............................................................52
6.5

Selective feeding on bacteria by protozoa...................................................................53

6.6

Internalisation of bacteria by soil protozoa...................................................................55

6.6.1 Effects of internalization on bacteria......................................................................55

6.6.2 Survival of bacteria within protozoa.......................................................................56

7 Protozoa as a pretreatment................................................................................57
7.1

Hydrolytic pretreatment reactor design........................................................................57

7.1.1 Reactor temperature..............................................................................................58

7.1.2 Reactor pH............................................................................................................ 59
7.1.3 Mechanical pretreatment unit................................................................................59
7.1.4 A recirculation stream............................................................................................60
7.1.5 Controlled micro-aerobic conditions......................................................................61
7.1.6 Inoculation............................................................................................................. 62
7.2

The effects of hydrolysis on the proposed digester......................................................62

8 Discussion...........................................................................................................64
9 Conclusions.........................................................................................................67
9.1

The role and behaviour of protozoa in anaerobic digestion..........................................67

9.2

Methane production.....................................................................................................68

9.3

Protozoa and pathogens..............................................................................................68

9.4

Future research and recommendations.......................................................................69

References................................................................................................................70
Appendix A................................................................................................................78

List of Figures
Figure 3.1: Anaerobic respiration with lactic acid fermentation (Fenchel & Finlay, 1995)........9
Figure 3.2: Metabolism in the hydrogenosome of Tritrichomonas foetus (Priya, 2009).........11
Figure 3.3: Metabolism in a hydrgenosome of Daystricha ruminantium (Fenchel & Finlay,
1995)............................................................................................................................ 11
Figure 3.4: Metabolism in cytosol of Giardia lamblia (Fenchel & Finlay, 1995).....................12
Figure 3.5: Endosymbiotic production of CH4 (Fenchel & Finlay, 1995)................................13
Figure 3.6: A schematic of the steps in anaerobic digestion (van Haandel & Van Der Lubbe,
2007)............................................................................................................................ 15
Figure 3.7: Perturbation of electrochemical gradients and pH as NH3 diffuses out of the
mitochondria (Schneider et al., 1996)...........................................................................19
Figure 3.8: Perturbation of electrochemical gradients and pH as NH3 diffuses into the
mitochondria (Schneider et al., 1996)...........................................................................20
Figure 4.1: Degradation of a food particle by phagocytosis (Funke et al., 2004)..................27
Figure 4.2: Amoeboid movement (Zug, 2015)......................................................................29
Figure 4.3: Protozoa in the planktonic food web (Pauli et al., 2001; Porter et al., 1985).......32
Figure 5.1: The ruminant stomach (Schoenian, 2014)..........................................................34
Figure 5.2: A schematic of the reticulorumen compartments (Worfolk, 2015).......................36
Figure 5.3: Schematic structure of ciliate (Jouany & Ushida, 1999)......................................40
Figure 6.1: Schematic representation of the three major layers in sediments (yellow, grey
and black), the redox discontinuity layer, sediment surface, as well as some compounds
and ions in sediments; modified from (Fenchel, 1967)..................................................47
Figure 7.1: A pretreatment hydrolytic plug flow reactor with a mechanical treatment unit with
a solids recycle stream.................................................................................................58
Figure 7.2: A comparison between the degradable substrates concentration in sludge before
and after hydrolysis......................................................................................................63

List of Tables
Table 2:1: The effect of ciliate protozoa on the effluent quality in activated sludge plants
(Pike & Curds, 1971)......................................................................................................5
Table 3:1: A summary of the concentrations at which ammonia is beneficial, inhibitory or
toxic to the AD process (Rajagopal et al., 2013)...........................................................21
Table 4:1: Taxonomic classification of the single phylum Protozoa according to the Report
published by Honigberg et al (1964) (Corliss, 2001).....................................................25
Table 4:2: Classification of the aquatic microbial community by size (Porter et al., 1985).. . .31
Table 5:1: A summary of some of the approximate physical, chemical, and microbiological
conditions in the rumen (Moran, 2005).........................................................................37
Table 5:2: A summary of the protozoa that act on the different plant polysaccharides in the
rumen (Wang & McAllister, 2002).................................................................................42
Table 7:1: Composition of mixed sewage sludge (Haghighatafshar et al., 2014)..................63

List of Appendix Tables

Table A.1: A table showing the substrate composition for mixed sewage sludge from
Haghighatafshar et al (2014)........................................................................................78
Table A.2: A table showing the degradable portion of the sewage sludge, the hydrolysis rate
constants, the hydrolysis rate per day and the % hydrolysed after 14 days..................78
Table A.3: A table showing the hydrolysis rate constants, the hydrolysis rate per day and the
% hydrolysed after 14 days..........................................................................................78
Table A.4: A table showing the concentrations of the hydrolysis end products and the
amounts hydrolysed.....................................................................................................79

List of abbreviations and symbols

AD
ADP
AS
ASL
ATP
BOD
C:N
CoA
COD
CSTR
DOM
Fdox
Fred
GAC
GDH
HPH
HRT
HS
IBR
KH
LC50
LS
MLSS
MMAD
MS
NAD
NADP
OFMSW
OLR
PFOR
POM
RPD
SLP
SOD
SRT
TAN
TPAD
TS
UASB
VFA
VS
WWTP
Xs
hyd

Anaerobic Digestion
Adenosine diphosphate
Activated Sludge
Amino acids, sugars and Long chain fatty acids.
Adenosine triphosphate
Biological Oxygen Demand
Carbon to Nitrogen ratio
Coenzyme A
Chemical Oxygen Demand
Continuous Stirred Tank Reactor
Dissolved Organic Matter
Oxidized Ferredoxin
Reduced Ferredoxin
Granular Activated Carbon
Glutamate dehydrogenase
High Pressure Homogenizer
Hydraulic Retention Time
High Solids
Induced Blanket Reactor
Hydrolysis constant
50% lethal concentration
Low Solids
Mixed liquor Suspended Solids
Membrane Micro Aerated Digester
Medium Solids
Nicotinamide adenine dinucleotide
Nicotinamide adenine dinucleotide phosphate
Organic Fraction of Municipal Solid Waste
Organic Loading Rate
Pyruvate: ferredoxin oxidoreductase
Particulate Organic Matter
Redox Potential discontinuity
Substrate Level Phosphorylation
Superoxide dismutase
Solid Retention Time
Total Ammonia Nitrogen
Temperature Phased Anaerobic Digestion
Total Solids
Up flow Anaerobic Sludge Blanket
Volatile Fatty Acid
Volatile Solids
Waste Water Treatment Plant
Degradable particulate organic matter
Hydrolysis rate

1 Introduction
1.1

Background

Human impacts on the climate through fossil fuel consumption for energy generation is the
main cause of the current global warming problem according to most climate scientists
(NASA, 2015; Appels et al., 2011); human beings will need to rely more on renewable
energy sources for the future to offset this problem. Anaerobic digestion (AD) is able to
generate renewable energy in form of biogas from the treatment of waste therefore it is
considered as one of the most important potential renewable energy sources (Appels et al.,
2011). In addition to generation of biogas, AD is advantageous because of its low energy
and space requirements as compared to aerobic digestion. This makes it possible to apply it
on both a large and small scale.
There are a variety of potential feedstock sources for anaerobic digesters such as sewage
sludge, municipal solid waste and agricultural waste. Sludge has the highest biogas
production capacity worldwide at approximately 0.590 m3 kg-1 organic dry solids but its
disposal accounts for up to 50% of the current operational costs of a wastewater treatment
plants (Appels et al., 2011). Optimizing the AD process would greatly cut down on the
operation costs of waste treatment plants by utilizing the biogas produced as a source of
energy for the plant operations and selling off the excess.
AD is a biological process of treating waste and bacteria are the most important group of
microorganisms that have been recognized in the process although other microorganisms
like protozoa potentially have a critical supporting role. Protozoa are a diverse group of
unicellular microorganisms that have the ability to thrive and survive in diverse
environmental conditions. They are unique because of their ability to degrade cellulose that
is common in municipal solid waste and sewage sludge but is not readily degradable in
anaerobic digesters (Noike et al., 1985). They have also been observed in soils where they
preferentially graze on gram-negative bacteria (Perez-Viana, 2010). It is therefore useful to
know the precise role protozoa play in AD waste treatment systems and soil by studying their
behaviour and harnessing this to increase CH 4 production, substrate utilization and enteric
pathogen inactivation.

1.2

Thesis Structure

Chapter 2 examines aerobic protozoa and the role they play in aerobic wastewater treatment
as well as their ability to be used as indicator organisms for activated sludge system
performance.
Chapter 3 focuses on anaerobic protozoa i.e. their metabolism, the effects of oxygen on their
behaviour, their symbiotic relationship with bacteria and their role in anaerobic wastewater
treatment. Furthermore, the dynamics of AD, the different types of digesters and factors that
affect their performance are discussed.
Chapter 4 is a review of the fundamentals of protozoa including classification, nutrition,
reproduction, movement and their role in the aquatic food web.
Chapter 5 discusses the role of protozoa in the rumen highlighting their contribution to
digestion and CH4 production.
Chapter 6 examines marine and terrestrial habitats including sediments and the soil to
consider the role of protozoa within them. Additionally, the effect of protozoa on bacteria and
their role in pathogen inactivation and persistence in soil is discussed.
Chapter 7 discusses how protozoa can be used as a biological pretreatment of waste for AD
by mimicking their behaviour in the rumen.
Chapter 8 is analyses the role protozoa play in the various environments studied and the
potential effect on AD of waste and the inactivation of pathogens.
Finally, Chapter 9 is a summary of the key findings of the thesis and future research and
recommendations are made.

1.3

Aims and objectives

1.3.1 Aim

To investigate the role of protozoa in anaerobic waste treatment systems, including their
influence on CH4 production, substrate utilization and pathogen inactivation.

1.3.2 Objectives

i.

Provide fundamental and process level understanding of the role and behaviour of

ii.
iii.
iv.

protozoa in municipal wastewater treatment.

Complete a critical review of literature of protozoa behaviour and metabolism.
Review the role of protozoa in ruminants and sediments.
Examine the different types of nutrition represented and the potential impact on
enteric bacteria inactivation and on CH4 production.

2 Aerobic Protozoa

2.1

Role in aerobic wastewater treatment

An aerobic wastewater treatment system consists of a combination of physical, chemical,

and biological processes to remove solids, organic matter and sometimes nutrients from
wastewater. The biological stage involves bacterial utilization of oxygen to degrade organic
matter and other pollutants. It can be regarded as a man-made ecosystem subjected to
extreme conditions due to a strong flow of organic load into the system, accelerated
decomposition processes, short biomass turnover time, and the prevalence of heterotrophic
organisms (Madoni, 2011; Antonietti et al., 1981). Biological wastewater treatment aims at
enhancing the self-purification ability of microbial communities and protozoa are part of
these microorganisms. The process can either be a suspended growth (activated sludge) or
attached growth system whereby the former have freely suspend microorganisms in water
while the latter use a medium to retain and grow microorganisms. Rotating biological
contactors and trickling filters are examples of commonly used attached growth systems for
treating wastewater.
Protozoa have been recognised in wastewater treatment for their role in producing good
quality effluent (Priya, 2009), which is achieved by:
i.

Grazing on bacteria thus preventing them from reaching self-limiting numbers by

keeping them in the log growth phase (Porter et al., 1985), therefore preventing
ageing cells from accumulating by renewing the bacterial biomass. The activity of
bacteria and assimilation rate of organic materials is therefore greatly increased
(Schmidt et al., 1992).

ii.

Enhancing flocculation by attaching to flocs and in certain species the secretion of a

mucus-like substance from the peristome region (the channel leading to the
cytosome or mouth in certain types of protozoa) has been observed to bring about
flocculation or using a polysaccharide (Curds, 1982; Pike & Curds, 1971).

iii.

Ingesting suspended solids and dissolved organic matter and indirectly by the
excretion of mineral nutrients as products of their metabolism; N and P are released

in the form of

NH 4

and orthophosphate resulting in an accelerated usage of

organic matter by bacteria and by excretion of growth-stimulating compounds such

as vitamins, amino acids, and nucleotides, which enhance bacterial activity (Madoni,
2011; Pogue & Gilbride, 2007).
iv.

Additionally, it has been observed that protozoa increase the rate of nitrification
because of their ability to influence bacterial growth (Madoni, 2011).

These effects are shown by the studies of Curds et al (1968) on activated sludge (AS) with
and without ciliates, which found that protozoa in the AS process lead to a reduction in
Biological Oxygen Demand (BOD), suspended solids, Chemical Oxygen Demand (COD),
bacterial numbers and organic nitrogen. This is shown in Table 2.1 where the effects of
ciliates on activated sludge are represented.
Table 2:1: The effect of ciliate protozoa on the effluent quality in activated sludge plants
(Pike & Curds, 1971)
Parameter
BOD (mg/l)
COD (mg/l)
Organic nitrogen (mg/l)
Suspended solids (mg/l)
Optical Density at 620nm
Viable bacterial count

Without ciliates
5370
198250
1421
86118
0.95 1.42
106160

With ciliates
724
124142
7 - 10
2634
0.23 0.34
19

(cfu/ml 106)

2.2

Protozoa as indicators of activated sludge plant

performance

The colonization dynamics of ciliates show determinate temporal successions of groups and
species, from plant starting phases to the stabilization of the AS system. This succession
limits the number of species present in each phase of the biological waste treatment
process; therefore ciliates are considered as potential indicators of plant performance (Mara
& Horan, 2003). Three major phases have been recognized during the succession, as
shown in Figure 2.1. The first phase is characterised by a high number of free-swimming
bacteriophagous species; in the transition phase the free-swimming forms are substituted by
attached and crawling forms (the decomposers) and the maturity phase is characterized by a
stable ciliate community whose composition reflects the stable condition of the biological

plant (Mara & Horan, 2003). Flagellates are not linked to the presence of sludge therefore
they are not considered when looking at indicators or performance although they are present
in the system.

Figure 2.1: Microorganism succession during the colonization of activated sludge (Mara &
Horan, 2003)

Due to the fact that each phase has a specific ciliate community, a fully functioning plant
should not have not host species characteristic of one of the colonization phases. However,
this can occur if dysfunctions in the degree of aeration, the amount of sludge, sewage
retention time or organic loading cause regression in the environmental conditions (Mara &
Horan, 2003).

3 Anaerobic Protozoa

3.1

Oxygen effects

Anaerobic protozoa fall into several categories depending on their response to oxygen
including obligate, facultative and microaerophilic anaerobes. The point at which facultative
anaerobes convert from aerobic respiration to a fermentative metabolism is called the
Pasteur point. It is usually set at 1% atm.sat although it is not a constant as it varies between
species. For example, many anaerobes are inhibited at oxygen tensions < 0.1% atm.sat
while some tolerate normal atmospheric oxygen tension. A partial pressure of oxygen < 0.1%
atm.sat is considered an anaerobic condition and 0.1 5% atm.sat microaerobic (Fenchel &
Finlay, 1995). Organisms are considered anaerobic if they have an energy metabolism
independent of oxygen and can complete their entire life cycle in the absence of oxygen;
facultative if they are capable of oxidative phosphorylation but oxygen is not a requirement
for growth; strict anaerobes if they are inhibited below the detection limit of oxygen and
microaerophilic if they are sensitive to oxygen above a certain threshold value (Fenchel &
Finlay, 1995).

3.1.1 Adaptation to oxygen

Anaerobes are periodically exposed to free oxygen in their habitats for example in the steep
but fluctuating redox gradients in sediments. Free oxygen inhibits some of the enzymes that
take part in the metabolism of anaerobic organisms for example pyruvate: ferredoxin
oxidoreductase (PFOR) and hydrogenase. Due to this, the anaerobes develop chemical and
behavioral adaptations to the oxygen. The behavioural adaptations include the phobic and
kinetic response whereby in the latter the anaerobes increase their swimming velocity with
increasing oxygen tensions taking a minimum value in anoxia while in the former there is
rapid reversal as they swim away from a zone with detectable oxygen to anoxia (Fenchel &
Finlay, 1995).
Anaerobes consume oxygen, which at low tensions of approximately 1% atm. sat is used by
their cells as a sink for reducing power during fermentation. However, reduction of O 2 to H2O
is inefficient, therefore toxic oxygen radicals and other compounds that are potentially

harmfully for example H2O2 or singlet oxygen are produced. The enzymes superoxide
dismutase (SOD) and catalase are used to protect the anaerobes from the toxins. SOD
catalyses the dismutation of

O2 :

2O -2 + 2 H+ O 2 + H2 O2 ( SOD )

Equation 3.1

2 H2 O2 2 H2 O + O 2 (Catalase)

Equation 3.2

On the other hand, oxygen consumption can be beneficial by balancing inwards diffusion so
the cells retain a constant anaerobic interior as observed in Metopus contortus (Fenchel &
Finlay, 1995).

3.2

Anaerobic Metabolism

Anaerobic metabolism occurs in the absence of oxygen, therefore organic or inorganic

molecules are used as the final electron acceptors. It is comprised of two major phases:
glycolysis and fermentation. Glycolysis is the backbone of all eukaryotic metabolism in which
one molecule of glucose is broken down to produce produce 4 ATP, 2 NADH, and 2 pyruvate
molecules with the help of nicotinamide adenine dinucleotide (NAD+). Fermentation is a
series of reductions and oxidations that occur on a substrate to yield energy for anaerobic
organisms (Fenchel & Finlay, 1995).
There are two major types of fermentation: alcoholic and lactic acid however only the latter
occurs in animals including protozoa. In simplest lactic acid pathway glucose is broken down
into 2 molecules of pyruvate and lactate is produced as the end product, shown in Figure
3.1. During this process, an oxidation reaction occurs where H 2 is removed and transferred
to NAD followed by the conservation of released energy in form of ATP (adenosine
triphosphate). The conversion of ATP to ADP (adenosine diphosphate) serves as the primary
source of free energy in all living cells. ATP is therefore important as it provides the energy
that drives most biological processes. The process following the production of pyruvate
exists to ensure the re-oxidation of NAD so lactate acts as a sink for reducing power and is
then excreted (Fenchel & Finlay, 1995).

Glucose
2 NAD+

2 ADP

2 NADH

2 ATP

+ 2 H+
2 Pyruvate

2 NAD+
2 Lactate

Figure 3.1: Anaerobic respiration with lactic acid fermentation (Fenchel & Finlay, 1995).

After pyruvate, the pathway is modified in different fermenting organisms. The lactic acid
fermentation process breaks down glucose to yield 2 mol ATP, but fermentation pathways
are more efficient and yield 4 ATP/glucose, therefore either incorporation of another
substrate level phosphorylation (SLP) occurs or electron transport to an organic electron
acceptor. The additional SLP depends on the production of Acetyl-Coenzyme A (acetyl-CoA)
and a greater variety of end products can be formed for example ethanol, CO 2, H2, butyrate,
propionate etc. (Fenchel & Finlay, 1995). Certain prokaryotes that are found in soil,
sediments and in the digestive tracts of ruminants, such as cows and sheep use other
electron acceptors for example rumen protozoa harbour methanogenic endosymbiotic
bacteria that utilize H2 to reduce CO2 to CH4 (Fenchel & Finlay, 1991). Similarly, sulphatereducing bacteria reduce

SO2-4

to H2S in sediments (Fenchel & Riedl, 1970). Protozoa

have several metabolic pathways that are discussed in Chapter 3.2.1 below.

3.2.1 Anaerobic metabolic pathways in protozoa

Although protozoa are eukaryotes, which usually dictates obligate aerobic metabolism, they
demonstrate more diverse and ancient forms of anaerobic metabolism similar to
prokaryotes, enabling them to flourish in oxygen deficient environments. They carry out
anaerobic fermentation of reduced organic compounds mostly with the help of
hydrogenosomes, which are membrane-bound organelles found in the cytoplasm of
eukaryotic cells that evolved from mitochondria, to produce ATP and H2 (Rogers, 2010;
Embley et al., 2003). In the absence of hydrogenosomes, metabolism occurs in the cytosol.
The main function of hydrogenosomes is to ferment pyruvate into acetate and H 2, coupled
with the production of ATP (Fenchel & Finlay, 1995).

In Trichomonads, which are anaerobic flagellates shown in Figure 3.2, the enzyme pyruvate:
ferredoxin oxidoreductase (PFOR) catalyzes the oxidative decarboxylation of pyruvate to
acetyl-CoA and CO2; see Equation 3.3. PFOR assumes a reduced state by the transfer of
electrons from pyruvate to the ferredoxin portion of the enzyme (Embley et al., 2003).

pyruvate + CoA + oxidized ferredoxin acetylCoA + CO2 + reduced ferredoxin + 2 H+

Equation 3.3
The reduced ferredoxin is reoxidized by the enzyme hydrogenase in a reaction that reduces
protons, H+, to H2; see Equation 3.4 (Embley et al., 2003).

2 H + 2e H2

Equation 3.4

Energy resulting from the activity of PFOR is conserved in one molecule of ATP, which is
formed by the enzymes acetate:succinate CoA transferase and succinate thiokinase
(succinyl-CoA synthetase) that catalyze the subsequent metabolism of acetyl CoA into
acetate and ATP (Rogers, 2010), refer to Figure 3.2. Malate, which is produced in the
ctytosol, is oxidatively decarboxylated to pyruvate when it enters the hydrogenosome
(Fenchel & Finlay, 1995).
In Daystricha ruminantium, which is a common rumen ciliate, metabolism occurs in the
hydrogenosome, see Figure 3.3; therefore, its metabolic pathways are similar to
trichomonads. However, unlike the trichomonad it is able to produce short chain fatty acids

10

especially butyrate from the condensation of 2 molecules of acetyl-CoA. Acetyl-CoA is

Fdox
Fdred

CoA
ATP
ADP+ Pi

2H+
Pyruvate

ADP
ATP

CoA

produced from pyruvate in the hydrogenosome. Some of the enzymes e.g. malate
dehydrogenase are located in the cytosol. Additionally, lactate fermentation can occur in both
the cytosol and inside mitochondria (Fenchel & Finlay, 1995).

Figure 3.2: Metabolism in the hydrogenosome of Tritrichomonas foetus (Priya, 2009). Fdox is
oxidized ferredoxin and Fdred is reduced ferredoxin.

11

AcetylCO2

2NADH

Acetyl-CoA

NAD

Aceto-acetyl-CoA
Butryl-CoA

Figure 3.3: Metabolism in a hydrgenosome of Daystricha ruminantium (Fenchel & Finlay,

1995). Fdox is oxidized ferredoxin and Fdred is reduced ferredoxin.

12

NAD(P)H
NAD(P)

NADPH

NADP

Giardia lamblia is an example of an anaerobe that commonly occurs in vertebrate hosts and
whose enzymes of metabolism occur in the cytosol, as represented in Figure 3.4. The basic
fermentation pathway occurs to produce acetate. However, in this case, PFOR is not
coupled to a hydrogenase but rather transfers its reducing power on organic compounds to
produce ethanol and alanine. PFOR is associated with either the plasma or the endoplasmic
reticulum membrane.

Figure 3.4: Metabolism in cytosol of Giardia lamblia (Fenchel & Finlay, 1995). Fdox is
oxidized ferredoxin and Fdred is reduced ferredoxin.

3.3

Symbiosis

Most anaerobic protozoa have other microorganisms either living inside of them or attached
to their external surfaces, although some species like Metopus contortus have both. These
microorganisms are either endosymbiotic purple non-sulphur photosynthetic bacteria and
methanogens; or ectosymbiotic sulphate-reducing bacteria. The endosymbionts are
protected from competition with sulphate reducers in such an environment and from oxygen
inactivation (Fenchel & Finlay, 1995).

3.3.1 Phototrophic endosymbionts

13

Alanin

The oligotrich ciliates, which are predominantly found in benthic environments, contain
endosymbionts. Strombidium purpureum is an oligotrich found in the illuminated zone of
anoxic marine sediments and has a purple cytoplasm due to the presence of non-sulphur
bacteria that contain photosynthetic membranes. The bacteria consume H 2 and other
fermentation products as reductants for photosynthesis and transfer a fraction of their
photosynthate to the ciliate. Purple non-sulphur bacteria are sensitive to light and thus able
to carry out oxidative phosphorylation at low oxygen tensions in the dark. This behaviour
relieves the threat of oxygen toxicity (Fenchel & Finlay, 1995).

3.3.2 Methanogens
Anaerobic protozoa sometimes contain methanogens, which release CH4 by using the end
products of metabolism for growth and energy production (Priya, 2009). The endosymbionts
are vertically transmitted: at mitosis, where they are distributed to the daughter cells and
are also retained at encystation (Hackstein, 2010). This accounts for their continued
presence in protozoa. Symbiotic bacteria benefit from protozoa by obtaining growth
substrates (H2, CO2 and acetate) and protection from predators. The most common species
of protozoa that harbour CH4-producing symbionts are the ciliates. The ciliates contain
methanogenic archea for example Metopus contortus, Plagiopyla frontata, Trimyema sp. and
Cyclidium porcatum that utilize H2 to produce energy by the reduction of CO 2 and the
excreted dissolved organics are utilized by the protozoa (Fenchel & Finlay, 1991); see
Equation 3.3 and Figure 3.5 representing the relationship between an endosymbiont and
hydrogenosome in protozoa.

CO2 + 4H 2 CH 4 + 2H 2 O

14

Equation 3.5

Hydrogenosome

Pyruvate
CO2

Protozoa
ATP

H2

Endosymbiotic
Bacteria

CH4

Figure 3.5: Endosymbiotic production of CH4 (Fenchel & Finlay, 1995)

CH4 is sometimes liberated from acetate by acetoclastic methanogens of which only two
genera; the Methanosarcina and Methanosaeta utilize acetate as a substrate. They have
been identified in the ciliate Metopus es although such methanogens are rare in symbiotic
relationships because they compete with their hosts for acetate (Hackstein, 2010).
In some ciliate species, there is an increased growth rate in the presence of methanogenic
symbionts. Fenchel & Finlay (1991) carried out a study to compare the anaerobic ciliates M.
contortus and P. frontata with and without endosymbionts. The endosymbionts were
removed from protozoan cells using bromeothanesulfonic acid. The results showed a 25%
reduction in growth rates and yields in the aposymbiotic cells when compared with the
normal cells with active methanogens. Symbiotic methanogens are responsible for about 5%
of the CH4 produced in freshwater sediments, 15-90% in anaerobic marine sediments and 925% in the rumen. In fresh water sediments anaerobic protozoa play a relatively smaller role
in CH4 production due to their low number and limited vertical distribution (Priya, 2009;
Fenchel & Finlay, 1995).

3.3.3 Ectosymbiotic sulphate reducers

The

2-

SO 4

reducing symbionts are predominant in anaerobic ciliates of marine sediments

although they have been observed in other habitats like the rumen (Fenchel & Finlay, 1995).

15

They attach to the host by various mechanisms for example: insertion into pits in the cell
membrane observed in the ciliate Parablepharisma pellitum, living in sheaths that attach to
the host cell membrane in Metopus contortus and in Sonderia the ectosymbionts lie
embedded in a thin mucous layer which covers the cell surface. The

SO2-4

reducers utilize

the fermentation products from the ciliates for their metabolism (Fenchel & Finlay, 1995).

3.4

Anaerobic Digestion

Anaerobic digestion is a biological process in which microorganisms break down

biodegradable material in the absence of oxygen leading to the production of biogas (Zheng
et al., 2013). The AD process is divided into four major steps, shown in Figure 3.6. During
hydrolysis, complex organic matter is broken down into soluble fatty acids and other organic
monomers like amino acids and sugars by hydrolytic enzymes for example proteases to
degrade protein, lipases for fats, cellulases for cellulose, amylases for starch etc. The
hydrolysis products (amino acids, fatty acids and sugars) are then converted into volatile
fatty acids, alcohols, CO2 and H2 during acidogenesis. In acetogenesis the products from
acidogenesis are converted into acetic acid, CO2 and H2 (Griffin, 2012). Finally, CH4 is
produced during methanogenesis from acetic acid and H2.

Proteins

Carbohydrates

Fats

Hydrolysis
Amino acids, sugar

Fatty acids

Acidogenesis
Intermediates
Propionic acid, butyric acid

Acetogenesis

Acetic acid

Hydrogen

Methanogenesis

16

Methane

Figure 3.6: A schematic of the steps in anaerobic digestion (van Haandel & Van Der Lubbe,
2007).

Acetoclastic methanogens and H2 utilizing methanogens are the two main types of
microorganisms responsible for the formation of CH 4. The acetoclastic methanogens split
acetate into CO2 and CH4; see Equation 3.6 (Griffin, 2012).

2CH3 COOH CH 4 + CO 2

Equation 3.6

The H2 utilizing methanogens generate CH4 by using H2 to reduce CO2; see Equation 3.7
(Mara et al., 2003).

CO2 + 4 H2 CH4 + 2 H2 O

Equation 3.7

The dominant CH4 forming mechanism in AD reactors is the acetoclastic pathway, which
accounts for approximately 70% of the total CH 4 formed, because H2 is limited in AD (Griffin,
2012).

3.4.1 Anaerobic Reactors

Waste undergoes a pretreatment step prior to digestion to improve digestate quality and
increase biogas yield. Pretreatment is useful to remove non-biodegradable materials,
provide a uniform small particle size feedstock, protect the downstream plant from damage
and to remove materials that may decrease the digestate quality. Digestion then proceeds
and is classified depending on the reactor configuration (batch or continuous), solids content
(low, medium or high solids), and the number of stages (single or multiple) (Griffin, 2012;
Monnet, 2003).
i.

Reactor configuration

The key difference between batch systems and continuous systems is that in batch systems
the reactor is loaded with feedstock after which it is run to completion, emptied and reloaded
while continuous systems have the reactors being continuously fed with feedstock; this
allows for both a steady- state to be achieved in the reactor as well as for a constant gas

17

yield. Batch reactors have low operation costs and are simple to operate although they have
a large footprint and suffer from instabilities in the microbial population. In contrast,
continuous systems have higher operating costs but are able to maintain microorganisms
within the system (Griffin, 2012).
ii.

Total Solid Content

The total solid content (TS) influences the size of the digester where by the higher the solids
content, the small the reactor will be because of lower water requirements. Low solids (LS) is
10% of TS, Medium solids (MS) is 15 20% and high solids (HS) is from 22-40% (Monnet,
2003).

iii.

Number of Stages

The number of stages can either be single or multiple. In a single stage reactor all the
digestion processes take place simultaneously and this approach generally has the lowest
operating and capital costs. The most common multiple stage reactor is a two-stage reactor
whereby the first vessel is used for hydrolysis and acidogenesis while the second is for
acetogenesis and methanogenesis. The first reactor is limited by the rate of hydrolysis of
cellulose while the second by the rate of microbial growth (Monnet, 2003).

3.4.2 Factors affecting anaerobic digestion

Several variables including temperature, pH, retention time, mixing, organic loading rate, the
carbon:nitrogen ratio and NH3 concentration affect the performance of an anaerobic digester.
i.

Temperature - There are two main temperature ranges: mesophilic (20-45 C) and
thermophilic (50-65C) (Monnet, 2003). Higher temperatures increase the reaction
rates in a digester resulting in more efficient operation (increased loading rate and
gas production) and lower retention time requirements (Cloete & Muyima, 1997).

ii.

pH - It is important to keep the pH of an anaerobic digester at or near a neutral pH of

7 for stable operation. Methanogenesis thrives at pH within the range of 6.5 to 7.5 but

18

if acid accumulates in the system the pH drops to between 4.5 and 5 causing
process inhibition (van Haandel & Van Der Lubbe, 2007).
iii.

Retention time - this is the time needed to achieve complete degradation of organic
matter and it varies with parameters such as temperature or the waste composition.
In a mesophilic digester the retention time is typically 15-30 days while in a
thermophilic it is 12-14 days (Monnet, 2003).

iv.

Mixing - This improves the contact between microorganisms and the substrate,
improves homogeneity of the feedstock and prevents the formation of scum and
development of temperature gradients within the digester. Slow mixing is preferred as
excessively high speeds can disrupt the microorganisms (Monnet, 2003).

v.

Organic Loading Rate (OLR) This is a measure of the biological conversion

capacity of the AD system. If the system is fed above its OLR, the biogas yield is
lower due to accumulation of fatty acids (Monnet, 2003).

vi.

Carbon to Nitrogen ratio (C:N) This is the relationship between carbon and nitrogen
present in organic matter whereby carbon serves as an energy source for
microorganisms while nitrogen enhances microbial growth. Therefore, in situations of
limited nitrogen availability (a high C:N), the microbial populations will be smaller and
take a longer time to decompose the available carbon resulting in a low gas yield.
However, a low C:N causes high NH3 levels because the excess nitrogen is excreted
as NH3 gas. The optimum ratio is 30:1 in an AD digester and can be achieved by
mixing material of low and high C:N ratios (Igoni et al., 2008; Monnet, 2003).

vii.

Ammonia It is inhibitory when present in high concentrations in the form of NH 3 at

concentrations between 1500 and 3000 mg/L of total ammonia nitrogen (TAN) and at
a pH greater than 7.4. However, at concentrations above 3000 mg/L
becomes toxic regardless of the pH (Cloete & Muyima, 1997).

3.5

Ammonia toxicity

19

NH +4

Ammonia is the end product of digestion of proteins, urea and nucleic acids in the absence
of oxygen. It is present in wastewater in either the ionized

( NH +4 )

or non-ionized form

(NH 3 ) and is the most significant inhibitor of the AD process (Rajagopal et al., 2013). The
toxicity of total TAN is primarily due to NH 3 because of its lipophilic properties which enable it
+

NH 4

to diffuse through the cell membrane while

contributes to a smaller proportion. The

chemical equation below shows the relationship between

NH 3

and

NH 4

with the

direction mainly dependent on pH.

NH 3 + H2 O NH4 + OH

Equation 3.8

When the pH is low, the reaction is driven to the right, and when the pH is high, the reaction
is driven to the left (Sawyer, 2015). In addition to pH, temperature is another major factor
influencing of NH3 concentrations. At a constant pH, there will be higher concentrations of
NH3 at higher temperatures. A study by Kayhanian (1999) showed that the concentration of
NH3 at thermophilic (55 C) temperatures is expected to be six times higher than under
mesophilic conditions at the same pH (Rajagopal et al., 2013).
NH3 toxicity can lead to detrimental effects on the growth and reproductive activities of
animals, sometimes leading to death (Xu et al., 2004). It is thought to be brought about in
cells is by perturbation of electrochemical gradients and pH and interaction with enzymes
(Schneider et al., 1996).

3.5.1 Perturbation of electrochemical gradients and pH

20

+
NH 4 diffuses across the cell membrane very slowly, but is actively transported across the

cell by specific transport proteins and by facilitated diffusion (Schneider et al., 1996).

NH +4

ions compete with the transport of K ions into the cytoplasm resulting in an

increased demand in maintenance energy and reduced cell growth (Hauser & Wagner,
2015). On the other hand, diffusion of NH 3 across the cell membrane follows its chemical
potential gradient, which can be approximated by the gradient of its partial pressure. It is
produced in the mitochondria by the action of enzymes and then diffuses out through the
membrane into the cytoplasm pool and environment leading to a drop in the mitochondria
pH as shown in Figure 3.7. This leads to acidic conditions in the mitochondria and alkaline
conditions in the environment and cytoplasm.

Figure 3.7: Perturbation of electrochemical gradients and pH as NH3 diffuses out of the
mitochondria (Schneider et al., 1996).
It will then be actively transported back to the cell as

NH +4 and converted into NH3 in the

cytoplasm. This leads acidic conditions in the cytoplasm leading to an increase in pH in the
mitochondria and other organelles when NH3 diffuses into them; see Figure 3.8.

mitochondrion
pH

cytoplasm

Glutamine catabolism
mitochondrion
diffusion
pH
diffusion
diffusion

cytoplasm

active transport

diffusion
active
transport
ammonium
addition
Figure 3.8: Perturbation of electrochemical gradients and pH as NH3 diffuses into the
mitochondria (Schneider et al., 1996).

21

The constant NH3 diffusion and

NH4

transport across membranes leads to futile energy

consuming cycles and continuous changes in pH. An increase in pH of the cell is harmful to
some organelles for example lysosomes with a naturally low internal pH. Additionally, a low
pH results in the inactivation of some key enzymes in glycolysis like of PK-1
(Phosphofructokinase-1) (Hauser & Wagner, 2015).

3.5.2 Interaction with enzymes

NH3 and

NH+4

can participate in enzyme reactions and displace equilibria or interact with

regulatory sites of enzymes. The main source of the NH3 in cell cultures is glutamine and it
has been suggested by Schneider, Marison & von Stockar (1996) that NH3 might drive a
futile cycle of glutaminase and glutamine synthetase thus affecting ATP production.
Glutaminase catalyzes the deamidation of glutamine to yield

NH +4

and glutamate, while

glutamine synthetase catalyzes the ATP-dependent reverse reaction (Schneider et al.,

1996). Furthermore,

NH+4

may reduce or reverse the conversion of glutamate to -

ketoglutarate thus reducing ATP generation (Hauser & Wagner, 2015).

3.6

Ammonia influence on methanogenesis in anaerobic

digesters

High NH3 concentrations are one of the main causes of digester failure because of its
inhibitory effects on microbial activity. Inhibition is demonstrated by a decrease in the steady
state rate of CH4 production and toxicity is manifested by a total cessation of methanogenic
activity (Rajagopal et al., 2013). The decrease in CH4 production may happen due to TAN
levels between 15007000 mgL-1. The wide range of inhibiting concentrations exists due to
the differences in nature of substrates, microorganisms, environmental conditions
(temperature, pH) and acclimation periods (Rajagopal et al., 2013). A summary of the
beneficial, inhibitory and toxic levels of NH3 in AD is given in Table 3.1.
Table 3:2: A summary of the concentrations at which ammonia is beneficial, inhibitory or
toxic to the AD process (Rajagopal et al., 2013).

Effect on AD process

Ammonia (mg NH4N/L)

22

References

Beneficial
No antagonistic effect
Inhibition (especially at
higher pH values)
Complete inhibition or toxic

50200
2001000

McCarty (1964)
Hobson and Shaw (1976)

15003000

Angelidaki and Ahring (1993)

3000

Sung and Liu (2003),

at any pH

3.7

Prochzka et al (2012)

Role of protozoa in anaerobic wastewater treatment

Protozoa in anaerobic wastewater treatment lead to a reduction Chemical Oxygen Demand

(COD), Mixed Liquor Suspended Solids (MLSS), and an increase in CH4 production. This is
mainly attributed to the direct consumption of particulate and soluble organic matter by
ciliates and their symbiotic relationships with bacteria (Priya et al., 2008).
Priya et al (2008; 2007) carried out experiments to determine the role of protozoa in AD
systems using both batch and continuously stirred tank reactors (CSTRs). The reactors were
fed with either a suspension (colloidal sodium oleate) or solution (sodium acetate) as the
carbon source.
The CSTRS were used to study the changes in protozoa populations in response to feed
composition (Priya et al., 2007). The number and diversity of anaerobic protozoa were
observed to vary significantly as the feed composition and physicochemical conditions
changed (Priya et al., 2008). At all COD loading rates and retention times, the start up phase
had the least number of ciliates and there was poor COD removal while the steady state had
a high diversity and number of ciliates and achieved greater than 75% COD removal (Priya
et al., 2007).
The change in population is attributed to pH and generation times or the species. Flagellates
and amoeba are usually present during the plant start-up phase because they are less
affected by the acidic conditions created by the predominance of volatile fatty acids (VFAs)
while ciliates thrive best at neutral pH and are sensitive to changes in pH. Furthermore,
flagellates have a shorter generation time than ciliates and benefit from an energy
advantage by directly utilizing dissolved substrate like VFAs (Priya et al., 2008). These
colonization dynamics suggest that protozoa can be considered as good indicators of
anaerobic system performance (Priya et al., 2008).

23

An experimental set up using CSTRs was made to determine the effect of protozoa on
methanogenesis and COD removal whereby protozoa were inhibited using cycloheximide
(Priya et al., 2008). Ciliate density and COD removal had a strong positive correlation, with a
correlation coefficient of R2 = 0.974 for particulate oleate and R2 = 0.966 for soluble acetate.
The strong relationship could imply either that ciliates enable COD removal or that COD
removal enables ciliate growth (Priya et al., 2008). Although the difference between oleate
and acetate COD removal was not significant, it was more consistent and stable with the
oleate suspension. Additionally, the reactor fed with particulate COD had a higher number
and diversity of ciliates indicating a possible direct uptake of particulate oleate by ciliates
(Priya et al., 2007).
Changes in the MLSS concentrations in CSTRs in the presence and absence of protozoa
were studied. In the presence of protozoa, the MLSS concentration was observed to decline
as ciliate numbers increased. The correlation coefficient was R2 = 0.87 and MLSS
concentration was 1634% lower in the CSTR with protozoa. This can be interpreted as the
grazing activity of ciliates reducing the sludge biomass while enhancing COD removal
therefore protozoa may play a direct role in anaerobic degradation and production of CH 4
(Priya et al., 2008; 2007).
Batch tests were used to determine the contribution of protozoa in AD by inhibiting the
growth of protozoa and measuring CH4 production and COD removal, which were then
compared with results from a control. The reactor with ciliates had greater than 95% COD
removal while the one without had less than 66% COD removal and CH4 produced was 29 33% lower in the absence of protozoa (Priya et al., 2007).
Bacteria suppression was achieved by the addition of 1ml of antibiotic solution to the
reactors. Direct utilization of soluble and colloidal COD by flagellates and ciliates was
observed. In the reactor with particulate sodium oleate, ciliates thrived while flagellate
species were dominant in the culture with soluble acetate (Priya et al., 2007). The results
indicate the direct consumption of colloidal substrates by ciliates. Furthermore, filtered COD
removal was 69% in the case of oleate and 65% in the case of acetate. These results
confirm the direct participation of protozoa in anaerobic degradation (Priya et al., 2007).
Biagini et al (1998) studied the behaviour of bacteria feeding on plant material in anaerobic
microcosms by monitoring the CH4 and sulphide amounts produced. The ciliate Metopus
palaeformis was introduced into the environment leading to a reduction in bacteria numbers.
However, bacteria activity increased resulting in an increase in bacteria CH 4 and the rate of

24

activity was positively correlated with ciliate numbers. The contribution of the endosymbiontic
methanogens from M. palaeformis was not significant at 3-8% of the total CH 4 generated.
The increase in CH4 production was mainly attributed to the excretions of organic acids by
protozoa like acetate and propionate that stimulate bacteria activity. It was also due to
protozoa grazing that resulted in nutrient cycling.

25

4 Fundamental Characteristics of Protozoa

Protozoa are single celled eukaryotic organisms that exhibit numerous mechanisms of
motility (Mara & Horan, 2003). There are over 35,000 species that are very diverse in
structure and their sizes range in diameter from 1m to as large as 2000m (Diaz, 2014;
Birkett & Lester, 2002). The majority of them employ chemoheterotrophic nutrition, but
certain species possess chloroplasts therefore they can practice photoautotrophy (Mara &
Horan, 2003).

4.1

Classification

There are several classifications of protozoa however the classification system of Honigberg
et al (1964) is widely accepted and divides the phylum Protozoa into four subphyla:
Sarcomastigophora, Sporozoa, Cnidospora and Ciliophora as shown in Table 4.1.

4.1.1 Subphylum Sarcomastigophora

The protozoa are characterized by the presence of pseudopodia or flagella for locomotion
and includes three super classes: Mastigophora, Opalinata and Sarcodina.
i.

Superclass Mastigophora These are protozoa in which the organs of motion and
food capture in the adult are the flagella for example euglena (Minchin, 2003). The
body is covered by a pellicle and they undergo asexual reproduction by binary
fission. It has two classes: Phytomastigophorea and Zoomastigophorea.

ii.

Superclass Opalinata These protozoans are found in the intestinal tracts of

amphibians and some other animals, they have oblique rows of cilia covering their
bodies and their nuclei vary in number from two to several. Asexual reproduction is
by longitudinal binary fission and plasmotomy while sexual reproduction is by
syngamy.

iii.

Superclass Sarcodina - These protozoa move and feed by the use of pseudopodia.
The protoplasmic layer is non-corticate hence the body tends to be spherical in

26

floating forms or continually changing in the creeping species (Minchin, 2003). It has
three major classes: Class Actinopodea, Piroplasmea and Rhizopodea.

Table 4:3: Taxonomic classification of the single phylum Protozoa according to the Report
published by Honigberg et al (1964) (Corliss, 2001).

Subphylum I. Sarcomastigophora
Superclass 1. Mastigophora

Subphylum III. Cnidospora

Class 1.

Class 1. Myxosporidea

Phytomastigophorea
Class 2. Microsporidea
Class 2. Zoomastigophorea
Superclass 2. Opalinata

Subphylum IV. Ciliophora

Superclass 3. Sarcodina

Class 1. Ciliatea

Class 1. Rhizopodea

Subclass (1) Holotrichia

Class 2. Piroplasmea

(2) Peritrichia

Class 3. Actinopodea

(3) Suctoria

Subphylum II. Sporozoa

(4) Spirotrichia

Class 1. Teleosporea
Class 2. Toxoplasmea
Class 3. Haplosporea

4.1.2 Subphylum Sporozoa

These protozoa always occur as parasites without definite organs for locomotion or ingestion
of food. In the adult and the body is covered with a thick pellicle (Minchin, 2003). The
asexual phase involves multiple nuclear fissions to produce daughter cells. The sexual

27

phase is by generation of spores and is followed by the production of sexually reproductive

cells. All Sporozoa have a cellular structure known as apical complex, which enables them to
invade the host cells (Lerner & Lerner, 2003). It has three classes: Telosporea, Toxoplasmea
and Haplosporea.

4.1.3 Subphylum Cnidospora

These are parasitic protozoa that form complex spores containing polar filaments. The
filaments can be extruded and used for attachment to the host. It includes two classes:
Myxosporidea and Microsporidea.

4.1.4 Subphylum Ciliophora

This subphylum consists of ciliates whereby the organs for locomotion and food capture are
cilia. Ciliates are the most important protozoan grazers in any environment (Priya et al.,
2007). There are four distinct subclasses under Class Ciliatea based on locomotion and
arrangement of cilia (Mara & Horan, 2003).

i.

Holotrichia free-swimming protozoa, which have cilia uniformly arranged over their
whole bodies (Mara & Horan, 2003).

ii.

Spirotrichia these have a flattened body with cilia found mainly on the lower surface
for locomotion (Mara & Horan, 2003).

iii.

Petitrichia these have inverted, funnel or bell-shaped bodies which are mounted on
a stalk. The wide end of the bell acts as an oral aperture and they have cilia arranged
around this to aid feeding (Mara & Horan, 2003).

iv.

Suctoria these have cilia when young that enable them disperse from their parents,
after which they lose their cilia and develop a stalk and feeding tentacles (Mara &
Horan, 2003).

4.2

Nutrition

28

Protozoa exhibit diverse forms of nutrition, four of which are represented in wastewater
treatment systems (Mara & Horan, 2003). The ability to utilize organic carbon for growth by
these organisms is important in the treatment of waste because they are responsible for
biochemical oxygen demand (BOD) removal. Chemoheterotrophs demonstrate three major
forms of substrate intake:
i.

Phagocytosis This is the predominant mechanism where particulate organic matter

is ingested and enclosed in a membrane to form a food vacuole where digestion
occurs (Priya et al., 2007), as shown in Figure 4.1. Certain species demonstrate
pinocytosis, which is a method of ingesting nutrient materials whereby fluid is drawn
through small temporary openings in the body wall membrane to form a food vacuole
(Priya, 2009).

Figure 4.9: Degradation of a food particle by phagocytosis. 1) Chemotaxis and adherence of

microbe to phagocyte. 2) Ingestion of microbe by phagocyte. 3) Formation of a phagosome.
4) Fusion of the phagosome with a lysosome to form a phagolysosome. 5) Digestion of
ingested microbe by enzymes. 6) Formation of residual body containing indigestible material.
7) Discharge of waste materials (Funke et al., 2004).
ii.

Saprozoic intake In this method nourishment is obtained by absorption of dissolved

organic matter through diffusion or active transport across the cell.

29

iii.

Predation This is mainly exhibited by ciliates through feeding on algae, flagellates,

bacteria and other ciliates.

4.2.1 Feeding mechanisms

Protozoa exploit different mechanisms to capture suspended particles and they are grouped
into filter feeding, diffusion feeding and direct interception/raptorial feeding (Priya, 2009).
i.

Filter feeding This involves the transport of water through a filter created by cilia or
pseudopodial tentacles and microvilli (Boenigk & Arndt, 2002). A feeding current is
created which is then passed to a membranelle that sieves out solid particles based
the size of its spaces (Mara & Horan, 2003). Filter feeding is responsible for the clear
effluent produced when ciliates are present in activated sludge systems because the
sieving process typically retains particle sizes between 0.3 and 1.5 m.

ii.

Diffusion feeding This relies on the motility of the prey and not the predator and is
practiced by the sarcodines (Rnn et al., 2012; Mara & Horan, 2003). In the
suctorians, which are common in AS and mainly feed on ciliates, the swimming prey
collides with their sticky tentacles through which the cell contents are withdrawn
(Mara & Horan, 2003).

iii.

Direct interception This is the interception of particles carried along the flow line
due to motility of the predator (Boenigk & Arndt, 2002). Each particle is captured
separately so protozoa are able to discriminate what is ingested based on prey size
or type. It mainly occurs in small flagellates and amoeba (Mara & Horan, 2003).
Protozoa are able to preferentially choose between different bacterial species for
example gram-negative bacteria that are preferentially selected in soil (van-Elsas et
al., 2006).

4.3

Movement

There are three major organs of locomotion utilized by protozoa whereby the Sarcodina
utilizes pseudopodia, ciliates utilize cilia and the flagellated protozoa use flagella (Mara &

30

Horan, 2003).

4.3.1 Pseudopodial movement

This is achieved by pseudopodia and involves the flow of cytoplasm as extensions of the
organism. This movement is characteristic of all Sarcodina, certain Mastigophora and
Sporozoa (Khanna, 2004). The Sol-gel theory is widely accepted as the cause of this motion
(Khanna, 2004). In the amoeba for example, the cytoplasm is divided into two parts namely:
the ectoplasm that is made of gel and encloses an inner mass, and the endoplasm that is
made of sol (a fluid containing suspended particles) see Figure 4.2. Part of the ectoplasmic
gel is converted to sol as the pseudopodium forms. Once this process has been completed,

endoplasmic sol begins flowing towards the area has changed from gel to sol causing the
cell wall to expand and the pseudopodium to extend forward. Upon reaching the tip, the
endoplasmic sol extends laterally and is transformed into a gel (Zug, 2015).
Figure 4.10: Amoeboid movement (Zug, 2015)

4.3.2 Ciliary movement

This movement is characterized a metachronal rhythm that propels the protozoa through the
water by overcoming the viscous forces that act on the cilia (Diaz, 2014). During the
metachronal rhythm coordination is achieved by a wave of simultaneously beating groups of
cilia that move from the anterior to the posterior end of the organism. This wave produces
continuous forward locomotion because there are always groups of cilia beating backward
and in this way it is possible to avoid interference between adjacent cilia (Zug, 2015).

31

4.3.3 Flagellar movement

Several theories have been proposed for the movement of protozoa using the flagellum;
sidewise lash during rapid movement that propels the body forward, screw propeller
whereby forces act on the flagellum causing the protozoa to drive forward while rotating
along its axis, circular beat in which the flagellum beats in a circle tracing a cone producing
suction to drive the microorganism forward and undulating where the flagellum undergo
spiral undulations from head to tip propelling the animal forward like a screw (Khanna,
2004).

4.4

Reproduction

The growth curve of the free-living protozoa involves an increase in cell size followed by
some form of asexual reproduction (Mara & Horan, 2003). Asexual reproduction occurs
under favourable conditions, the most common form of which is binary fission where the
organism divides into two equal sized daughter cells. Multiple fission, plasmogamy and
budding (demonstrated by the Suctoria) also occur (Priya, 2009). Sexual reproduction,
mainly through syngamy and conjugation occurs during periods of environmental stress
(Priya, 2009; Mara & Horan, 2003).

4.5

Protozoan in aquatic food webs

Protozoa act as a link between the classical food chain and the microbial food web and
phagocytosis underpins their role in the latter (Priya et al., 2007). They have two major roles
in the planktonic food web:
i.

They are a link between the smaller phytoplankton and bacteria and the larger
zooplankton that cannot effectively feed on small zooplankton (Porter et al., 1985).
Protozoa provide particulate organic matter (POM) to higher trophic levels by
repacking the organic material into edible portions and thus making it available to
crustaceans, rotatoria, and other metazoans (Pauli et al., 2001).

32

ii.

They are involved in nutrient cycling of carbon compounds and mineral elements (N
and P) by the direct and indirect effects of their metabolism (Porter et al., 1985).

Plankton is composed of the phytoplankton (the plants of the sea) and zooplankton, which
are typically the tiny animals found near the surface in aquatic environments. The
zooplanktonic community is categorized by size of organisms, whereby the size classes
relate to the pore size of the filters used in feeding to collect and separate the components.
The classification is shown in Table 4.2.
Table 4:4: Classification of the aquatic microbial community by size (Porter et al., 1985).

Size Class
Picoplankton

Heterotrophs
Bacteria

Autotrophs
Cyanobacteria

0.2 2.0m

Microflagellates

Chemolithotrophic bacteria

Nanoplankton

Microflagellates

Eukaryotic algae
Phytoflagellates

2 20m

Naked ciliates

Non-flagellate algae

Microplankton

Naked Ciliates

Smaller diatoms
Larger diatoms

20 200m

Tintinnids

Larger dinoflagellates

Larger dinoflagellates
Amoeboid protozoa
Copepod nauplii
Rotifers
Other metazoa

4.5.1 The food web dynamics

Algae, which are photoautotrophs and form part of the phytoplankton, are the producers in
the food web and utilize sunlight to photosynthesise their own food. Picoplankton form the
base of the microbial food web deriving their energy from dissolved organic matter (DOM),
from sunlight that is used by cyanobacteria and small eukaryotic algae to fix CO 2 and from
NH3 and CH4 in the chemolithic bacteria (Porter et al., 1985). Heterotrophic picoplankton
obtain DOM from both algae and the autotrophic picoplankton (Pauli et al., 2001); see Figure
4.3. Most of the nanoplankton cells in the ecosystem are pigmented flagellates and diatoms
although nonpigmented flagellates exist as well and are known as the heterotrophic
microflagellates (Porter et al., 1985).

33

Higher Trophic Levels

Autotrophic Algae

Macrozooplankton

DOM/POM

Microzooplankton

Myxotrophic Algae

34
Bacteria

Heterotrophic Microflagellates and

Small Ciliates

(autotrophic pikoplankton-cyanobacteria)

Figure 4.11: Protozoa in the planktonic food web (Pauli et al., 2001; Porter et al., 1985)

The colourless microflagellate protozoa have been proposed as the predominant grazers of
bacteria and autotrophic pikoplankton (Porter et al., 1985). Small ciliates, which form part of
the nano plankton, feed on bacteria and pico and nano autotrophs. Myxotrophic algae graze
on picoplankton and utilize DOM and POM for energy in addition to being photosynthetic
The nanoplankton are fed on by the microzooplankton, for instance large ciliates graze on
nanoplanktonic algae and are in turn fed on by the macrozooplankton, that are 20-200 mm
in size. Flagellates graze on bacteria and inhabit a central position in the transfer of organic
carbon to higher trophic levels due to their high turnover rates (Pauli et al., 2001). This food
web is graphically represented in Figure 4.3.
Protozoa nurture the growth of bacteria by grazing on them thus keeping them in the log
growth phase and preventing ageing cells from accumulating therefore minimizing the
possibility of substrate limitation and by providing dissolved organic carbon and minerals
through their activities (Porter et al., 1985). In addition to this, the P excreted by protozoa is
beneficial to phytoplankton (Pauli et al., 2001). Protozoa are decomposers associated with
the decay of organic matter and enhance the decomposition process because they increase
the turnover of the bacterial population (Priya et al., 2007).
The contribution of different groups of organisms is seasonally variable and the presence or
absence of a single species can modify the resource transfer pathways of the food web
(Pauli et al., 2001), therefore the links in Figure 4.3 are simply a representation of the
various possible pathways.

35

5 Protozoa activity in ruminant animals

Protozoa are ubiquitous and are able to survive in a variety of anaerobic environments
including marine and fresh water sediments, wet landfills, aerobic and anaerobic sewage
plants and the rumen (Priya, 2009). The rumen is not a completely anaerobic environment
because O2 makes up approximately 1% of the gas content. Oxygen enters the rumen in a
variety of ways such as through food, saliva, by eructation and by diffusion from the blood
(Bayan & Guiot, 2011). Ruminants consume forage rich in cellulose but they do not have
the ability to degrade it because they lack the enzymes to do this in their digestive tract.
They therefore utilize microorganisms in the stomach, which has a series of cavities, to
achieve this. The microorganisms convert forage into energy and protein that are both
necessary for metabolism, growth and multiplication (Moran, 2005).
The stomach has four major compartments adapted for a forage-based diet. The first three
compartments are the reticulum, rumen and omasum that are known as the fore stomach
and the fourth is the abomasum as shown in Figure 5.1.

Figure 5.12: The ruminant stomach (Schoenian, 2014)

i.

The Rumen this is the largest compartment, and it harbours the microorganisms
responsible for fermentation. A stable pH is maintained by the absorption of the acidic

36

end products of fermentation via the rumen wall and bicarbonate from saliva. A
ruminant produces approximately 150L of saliva per day, which contains Na and K
that buffer against acidity (Moran, 2005). The walls of the rumen continuously move,
churning and mixing the ingested food with rumen fluids and microbes. The finer food
passes to the omasum, while the rest is regurgitated to the mouth for further chewing
to break it down into smaller particles, which increases the surface area available for
enzymes and microorganisms, hence increased fermentation. The cuticle layer on
plants and legumes, which is resistant to microbial and enzyme action, is destroyed
through repetitive masticating (Wang & McAllister, 2002).
ii.

Reticulum this allows the animal to regurgitate and reprocess its food (Moran,
2005).

iii.

Omasum this lies in between the reticulum and abomasum. The material entering
the omasum is 90 to 95% water, so its primary function is to absorb some of the
water and further break down feed that is then directed to the abomasum (Moran,
2005). It is responsible for absorption of fatty acids and bicarbonate. The removal of
fermentation products such as VFAs is important for an efficient activity of cellulolytic
microorganisms (Bayan & Guiot, 2011).

iv.

Abomasum this chamber is the true stomach with an acidic pH of about 2, therefore
microbes are destroyed here. Additionally, pepsin is released that carries out the
initial digestion of protein (Moran, 2005).

5.1

Movement of digesta through the reticulorumen

The reticulorumen, represented in Figure 5.2, is composed of the reticulum and the rumen.
Rumen fermentation combines both homogenization and stratification of the digesta, due to
its compartmentalization. Flow of digesta through a ruminant can be classified into three
major stages:
i.

Mastication

Digestion in ruminants begins with mechanical grinding and moistening of the ingested
material in the mouth after which it enters the rumen (Bayan & Guiot, 2011).

37

ii.

Mixing

Digesta is inoculated with microorganisms in the reticulorumen (Bayan & Guiot, 2011).
Upon entering the rumen, fiber floats on the surface of the liquid of the rumen content. Flow
starts with contraction of the reticulum, which directs liquid flow to the cranial sac and fiber
flow to the dorsal rumen. Contraction of the cranial sac and cranial pillar directs flow into the
dorsal sac. The dorsal sac then contracts to direct flow into the ventral sac. Finally,
contraction of the ventral sac moves the rumen content forward over a relaxed cranial pillar
toward the reticulum. The contraction cycles are initiated approximately every minute
(Gookin et al., 2011). Feed particle sizes are reduced and their densities increased during
the digestion process. The smaller, more dense particles produced during this process are
able to settle and reach the ventral sac after which they are returned to the cranial sac where
they are aspirated through the rericulo-omasal orifice into the omasum (Bayan & Guiot,
2011). Refer to Figure 5.2 for a schematic of the reticulorumen compartments.

Figure 5.13: A schematic of the reticulorumen compartments (Worfolk, 2015)

iii.

Rumination this enables fiber in the reticulorumen to be moved into the

oesophagus, delivered to the mouth and remasticated (Gookin et al., 2011).

38

The speed of digestion in the rumen is influenced by type and number of microorganisms,
feed composition, pH and growth limiting nutrients. The diverse microbial community
consists of anaerobic protozoa, bacteria and fungi and a summary of some of the
approximate physical, chemical, and microbiological conditions is provided in Table 5.1.
(Moran, 2005).
Table 5:5: A summary of some of the approximate physical, chemical, and microbiological
conditions in the rumen (Moran, 2005)

Characteristic
Physical
pH
Temperature
Dry matter

Property
5.5 6.9 (mean 6.4)
38 - 41C
10 18%

Chemical
Gas phase (%)
Volatile Fatty Acids (mmol L-1)

CO2 - 65, CH4 - 27, N2 - 7, O2 - 0.6, H2 - 0.2

Acetate 60 - 90
Propionate 15 - 30
Butyrate 10 - 25
Lactate < 10
< 1 mmol L-1 present 2 3 h post feeding
2 12 mmol L-1
< 1 mmol L-1 present 2 3 h post feeding

Non-volatile acids (mmol L-1)

Amino acids and oligopeptides
Ammonia
Soluble carbohydrates
Insoluble polysaccharides
Dietary (cellulose, hemicellulose, pectin)
Endogenous (mucopolysaccharides)
Lignin
Minerals
Trace elements/vitamins

Always present
Always present
Always present
High Na; generally good supply
Always present; good supply of B vitamins

Microbiological
Bacteria
Ciliate protozoa
Anaerobic fungi

1010 1011 g-1 ( 200 species)

104 106 g-1 (25 genera)
103 105 g-1 (5 genera)

39

5.2

Role of protozoa in the rumen

Protozoa are often found in large numbers in the rumen of ruminants resulting in more stable
fermentation, higher levels of NH3, reduced number of bacteria, higher ruminal and tract
digestion of organic matter and cellulose (Veira, 1986). Protozoa are 104 106 per g in the
rumen as shown in Table 5.1, and represent approximately 50% by mass of the microbial
biomass (Jouany & Ushida, 1999). There are more than 100 species that have been
identified although no more than 15 to 20 species are found in an individual ruminant
(Hobson & Stewart, 1997). The predominant group of protozoa in the rumen are the ciliates
although

some

flagellates

have

been

identified

including

Trichomonas

spp.,

Monoceromonas sp. and Chilomastix sp. from the Class Zoomastigophorea. The two
principal ciliate groups in the rumen are Entodiniomorphida and Holotrichs under subclass
Trichostomatia are although their roles differ significantly because of their different metabolic
behaviour (Veira, 1986).

5.2.1 Entodiniomorphida protozoa

The Entodiniomorphida have a hard pellicle and an extrudable U-shaped peristome with a
band of cilia. There are 11 major subclasses including the genus Entodinium, Eodinium,
Diploplastron,

Metadinium,

Epidinium,

Enoploplastron,

Ophryoscolex,

Epiplastron,

Elytroplastron, Caloscolex, Opisthotricum and Parentodinium. Entodiniomorphida are strict

anaerobes that principally feed on particulate matter by engulfing it although they also have
the ability to take up soluble compounds (Hobson & Stewart, 1997). Feeding can be inhibited
when the food source is moving rapidly therefore they do not grow effectively in continuous
culture apparatus (Hobson & Stewart, 1997). The reproduction mechanism is binary fission
and in the endotina the duration of this process is 15 minutes. In some cases especially with
cultures that grow greater than 27 years, sexual reproduction by conjugation is observed
(Hobson & Stewart, 1997).

5.2.2 Holotrich protozoa

Several Holotrich families are present in the rumen including: Microcetus, Buetschliidae,
Blepharoconus, Paraisotrichidae, Isotrichidae etc. although the most common genera are
the Isotricha and Dasytricha from the Isotrichidae family and they commonly occur together

40

(Williams, 1986). The three principal species are Isotricha intestinalis, Isotricha protosoma
and Dasytricha ruminantium. There is a diurnal variation in the number of Isotricha,
Dasytricha, and Buetschlia spp. whereby the numbers increase before feeding and decline
after feeding, which is attributed to post feed increase in rumen outflow rates, protozoal
settlement, disintegration and sequestration on food particles or the reticulum wall where
there are higher oxygen levels.

Holotrich protozoa are aerotolerant anaerobes and commonly survive longer than other
ciliates when oxygen is present. Oxygen gradients along the gastrointestinal tract may
stimulate protozoan activity and in turn improves their hydrolytic capacity (Bayan & Guiot,
2011). The major organelle of carbon metabolism is the hydrogenosome, which produces H 2
under anaerobic conditions but in presence of oxygen acts as a respiratory organelle
(Williams, 1986). Oxygen affects the proportion of metabolites formed and the effects are
dependent on its concentration. Atmospheric levels are toxic while low oxygen tensions have
pronounced energetic benefits. The metabolic processes in the hydrogenosomes of
holotrichs are influenced by physiological levels of oxygen which inhibit H 2 production,
increase acetate production, reduce butyrate synthesis, increase glucose uptake and in the
absence of glucose, increase extracellular lactate consumption (Hobson & Stewart, 1997).

5.2.3 Cellulose, hemicellulose and pectin degradation

Carbohydrates, which comprise 75% of the total nutrient intake of ruminant animals, are of
three types: the soluble, storage and structural (composed of lignocellulose and pectin).
Lignocellulose is cellulose and hemicellulose linked with lignin (Bayan & Guiot, 2011). It is
difficult to degrade because of the bonds between different cellulose chains and the
presence of the polymer lignin that slows down the breakdown process (Montgomery &
Gnther, 2014). Soluble carbohydrates are digested 100 times faster than storage
carbohydrates by rumen microorganisms, while storage carbohydrates are digested about
five times as fast as the structural carbohydrates (Moran, 2005).
Ruminal microorganisms degrade plant cell wall materials using three mechanisms: (1)
production of the enzymes required to break the numerous bonds within cell walls, (2)
attachment and colonization of feed particles and (3) synergetic interactions among ruminal
species (Wang & McAllister, 2002).

41

i.

Enzymes and feed colonization

Ciliate protozoa are responsible for 30-40% of fiber degradation in the rumen (Bayan &
Guiot, 2011). There are two forms of protozoa are found in the rumen: the free-swimming
species associated with ruminal fluid, which preferentially digest soluble feed components,
and those that are attached either loosely or firmly to the substrate (Wang & McAllister,
2002). Protozoa have generation times of 5 to 14 h so it is important for them to attach to the
substrate to be retained in the rumen for them to successfully degrade plant material (Wang
& McAllister, 2002). The Entodiniomorphida are responsible for the larger fraction of plant
cell breakdown (Jouany & Ushida, 1999). Ingestion of plant particles is achieved by various
mechanisms, which are species dependent. Small particles are wafted into the oesophagus
by ciliary action while large flexible particles are surrounded by the vestibule, which opens
up around the plant material. Additionally, large rigid particles are grasped by the vestibular
lips that adhere strongly to the ingested material, detaching some of the plant debris from
large plant material (Jouany & Ushida, 1999). Refer to Figure 5.1 for a schematic description
of the ciliate structure.

Figure 5.14: Schematic structure of ciliate (Jouany & Ushida, 1999).

42

Protozoa are efficient at hydrolysing cellulose because they carry out phagocytosis and can
localise substrates within internal food vacuoles, thus minimizing wastage of enzymes. In
addition, some ciliates excrete cellulases into the surrounding medium for partial degradation
of plant fragments to make them easier to digest e.g. Epidinium caudatum (Fenchel &
Finlay, 1995). In Epidinium caudatum, the partially degraded cellulosic material is enclosed
in a primary vacuole immediately after ingestion, which then migrates from the pharyngeal
cytoplasm to the general cell endoplasm. Bacteria are also ingested with plant particles
contained in the primary vacuole. Pseudopodial tongues emerging from the pharyngeal
cytoplasm separate the bacteria from the plant material leading to the formation of
secondary vacuoles containing the separated the bacteria and food particles. Protozoa
enzymes are believed to be concentrated in the cytoplasm and are released into the vesicles
to degrade plant fragments (Jouany & Ushida, 1999). The enzymes involved in cellulose
digestion are cellulases while the hemicellulase enzymes break down hemicellulose and
pectinases degrade the pectin (Mackie et al., 2001).
Products from substrate degradation are found in vesicles that move from the endoplasm to
the ectoplasm and undigested cellulose and bacteria are released and discharged from the
cell through the cytopract. In some species like Polyplastron and Eudiplodinium, which have
a similar method of digestion, the digestion products are passed from the vacuoles to the
adjacent cytoplasm (Jouany & Ushida, 1999). In vitro studies by Castillo-Gonzlez et al
(2014) show that Polyplastron and Eudiplodinium are the major degraders of cellulose.
All rumen entodiniomorphid protozoa except the small Entodinium spp. contain the enzyme
cellulase, with the highest activities occurring in Eudiplodinium maggii, Epidinium ecaudatum
caudatum and Ostrcodinium obtusum bilobum (Hobson & Stewart, 1997). Holotrichs do not
contain cellulases but they have hemicellulolytic and pectolytic activities that enable them to
weaken plant cell walls and release the more readily fermentable carbohydrates (Hobson &
Stewart, 1997). A summary of the protozoa that act on the different plant polysaccharides is
given in Table 5.2.
ii.

Synergies

Protozoa stabilize the rumens pH by engulfing starch, making it unavailable to rapidly

fermenting bacteria therefore slowing the production of VFAs. This results in a reduction in
the redox potential of rumen digesta thus stabilizing ruminal fermentation and stimulating
bacterial cellulolytic activity because bacteria are sensitive to these two factors (Jouany &
Ushida, 1999; Veira, 1986). A high redox potential inhibits the growth of some obligate

43

anaerobes for example methanogens that grow at a redox potential lower than -0.3 V (Kim &
Gadd, 2008). Other synergies include the release of valuable nutrients and bacterial gazing,
which further increases bacterial activity.
Table 5:6: A summary of the protozoa that act on the different plant polysaccharides in the
rumen (Wang & McAllister, 2002).

Protozoa

Cellulolytic

Eudiplodinium maggii
Ostracodinium dilobum
Epidinium caudatum
Metadinium affine
Eudiplodinium bovis
Orphryoscolex caudatus
Polyplastron multivesiculatum
Diplodinium pentacanthum
Endoploplastron triloricatum
Orphyroscolex tricoronatus
Ostracodinium gracile
Entodinium caudatum
Isotricha intestinalis
Isotricha prostoma

+
+
+
+
+
+
+
+
+
+
+
+
+
+

Hemicellulolyti

Pectinolyti

c
+
+
+
+
+
+
+

+
+

+
+
+

+
+
+

+
+
+
+

5.2.4 Storage and soluble carbohydrates

Rumen microbial fermentation degrades carbohydrates into simple sugars that are used by
the microbes for energy. These are converted to volatile fatty acids (VFA) and gases for
example H2 and CH4. The proportion of VFAs produced is dependent on the composition of
the feed for instance, large amounts of acetic acid will be produced from structural
carbohydrates i.e. fibre, while more propionic acid will be produced from storage
carbohydrates i.e. starch and sugar (Mackie et al., 2001).
All entodiniomorphid protozoa engulf starch while the holotrichs have considerable capacity
for taking up soluble compounds, mainly sugars, from the rumen medium (Jouany & Ushida,
1999; Jouany, 1996). The entodiniomorphid protozoa render starch unavailable to bacteria
by engulfing it, and slowly fermenting it to mainly H 2, CO2, acetic acid, butyric acid and
glycerol, with the relative proportions dependent on the concentrations of O2 and CO2 in the
gas phase (Hobson & Stewart, 1997). They have high amylase activity (Jouany & Ushida,
1999), and the rate of starch breakdown is proportional to the initial amount of starch or

44

amylopectin inside the protozoa. Ingested starch is degraded in the endoplasm where about
50% is released in the medium in form of maltose and glucose. The other part is stored in
granules or in the skeletal plates inside the cells as amylopectin-like polysaccharide (Jouany
& Ushida, 1999).
Holotrichs are attracted by chemotaxis to sources of soluble sugar, which they assimilate
non-selectively. They convert the sugars to starch and ferment them to acetic, butyric and
lactic acid. The rate of sugar uptake is dependent on the nature and concentration of the
sugars, temperature and pH. Glucose uptake is inhibited by lactic acid but is stimulated by
ambient concentrations of oxygen (Hobson & Stewart, 1997). The range of saccharides
fermented is genus dependent whereby Isotricha spp. utilizes starch of a suitable grain size
and D. ruminantium readily ferments sucrose, fructose, D-glucose, raffinose and inulin.
The rate of carbohydrate utilization and nature of products formed is dependent on the
substrate (Williams, 1986). Protozoa contain a wide variety of carbohydrase enzymes and
polysaccharide degrading enzymes. Isotricha and Daystricha spp. have amylolytic enzymes,
which are inhibited by short chain oligomeric degradation products. They reduce the rate of
fermentation by uptake of readily fermentable sugars and starch thereby removing them
from immediate fermentation by bacteria, coupled with the reduced numbers of bacteria in
the rumen of faunated animals. This is important because it stabilizes ruminal fermentation
and prevents excessive amounts of lactate by increasing the number of lactate utilizing
bacteria. This enables the rumen system to successfully adapt to a change in feed
composition (Veira, 1986).

5.2.5 Protein
Protozoa do not have the ability to synthesize amino acids de novo from NH3 (Jouany &
Ushida, 1999), therefore they rely on bacteria and plant proteins. Digestion of engulfed
protein and bacteria occurs intracellularly. Protozoa release exo and endo-peptidases into
the medium, and produce peptides that become available to the bacteria and themselves
(Jouany & Ushida, 1999).
i.

Bacterial Protein

All ciliates ingest bacteria as their principal source of protein amino acids. Some protozoa
species feed on all types of bacteria present for example Entodinium caudatum while others

45

selectively ingest bacteria. The rate of uptake of bacteria is pH sensitive with an optimum pH
of 6 because uptake ceases at pH 5, and is reduced to 75% at pH 7 and 30% at pH 8
(Hobson & Stewart, 1997). Small entodina have been shown to be the major contributor to
bacterial protein turnover in the rumen (Jouany, 1996). Protozoa actively graze on bacteria
and a single protozoan can ingest 10 2 104 bacteria per hour, therefore, by applying a value
of 109 bacteria/mL to the rumen, predation could almost renew the entire bacteria biomass
every 12 hours in a rumen harbouring a high concentration of protozoa (10 5-106/mL)
(Jouany, 1996).
Bacteria are degraded within protozoa into small peptides, then to free amino acids that are
incorporated into protozoal protein without further interconversion. Parts of the peptides are
excreted from the protozoa as amino acids into the ruminal juice. Release of small molecular
weight nitrogenous products may account for as much as 50% of the bacterial protein
ingested by protozoa (Jouany, 1996). Protozoa deaminate amino acids intracellularly and
released NH3 is excreted into the rumen medium (Jouany & Ushida, 1999).
Ciliate protozoa ingest rumen bacteria resulting in increased recycling of microbial N in the
rumen (Jouany, 1996). Constituents of bacterial nucleic acid are incorporated into the nucleic
acid of the protozoa (Hobson & Stewart, 1997). Holotrichs obtain the nucleotide precursors
for nucleic acid synthesis by assimilation and digestion of ingested bacterial nucleic acid
material (Williams & Coleman, 1992).
ii.

Plant protein

Protozoa play a major role in the ingestion both particulate and soluble protein (Mackie et al.,
2001). All entodiniomorphid protozoa contain proteolytic enzymes and protease activity is
present in several holotrichs including the D. ruminantium, Isotricha spp. etc. (Hobson &
Stewart, 1997). Holotrichs degrade soluble proteins easily while the Entodiniomorphida do
not metabolize soluble proteins and do not grow unless insoluble proteins are present
(Jouany & Ushida, 1999). According to the studies by Jouany et al (1992, 1993), all ciliates
have leucine aminopeptidase (a proteolytic enzyme that hydrolyzes the peptide bond
adjacent to a free amino group) and the enzyme exopeptidase (it releases a single amino
acid or dipeptide), which were found in mixed ciliate protozoa especially in entodina. The
active proteolytic enzymes from the ciliates and their ability to engulf feed particles are
factors that contribute to increased dietary protein breakdown (Veira, 1986).

46

5.3

Methane production

Methanogenesis is the dominant terminal mineralization step in the rumen. The

entodiniomorphid and holotrich protozoa have bacteria in vesicles in their cytoplasm most of
which are methanogenic and survive digestion by the production of a polysaccharide
capsule (Hobson & Stewart, 1997; Fenchel & Finlay, 1995). As discussed in Chapter 3,
protozoa produce H2 as a metabolite, which the endosymbiotic methanogens utilize as the
main electron donor to reduce CO2 thus producing CH4. Formate is another important
electron donor used by many rumen methanogens and may account for up to 18% of the
CH4 produced in the rumen (Morgavi et al., 2010). Finlay et al (1994) showed that each
sheep rumen ciliate contained several hundred methanogens and in total contributed to 37%
of the CH4 produced by the ruminant per day. Protozoa from the genera Entodinium,
Polyplastron, Epidinium, and Ophryoscolex are the most common protozoa with
endosymbiotic methanogens in the rumen and the methanogens most often associated with
them are from the orders Methanobacteriales and Methanomicrobiales (Hook et al., 2010).

5.4

Ammonia in the rumen

Ruminal NH3 is produced due to microbial degradation of nitrogenous compounds such as

protein, peptides, amino acids and nucleic acids and by microbial hydrolysis of urea (Abdoun
et al., 2006). There is an increase in NH3 production in the presence of protozoa because of
greater recycling of microbial N, higher production of free amino acids fewer bacteria to
utilize the NH3 and an increased dietary protein breakdown (Hobson & Stewart, 1997).
Numerous studies have been carried out that show effect of protozoa in TAN levels in the
rumen. Following defaunation, Abou Akkada & el-Shazly (1964) showed a 47% decrease in
TAN levels, Christiansen et al (1965) 52%, Klopfenstein et al (1966) 44%, 50%, and 39%
and 49 and 50% by Chalmers et al (1968) as compared to control animals that had
protozoa. This decrease is attributed to a more efficient utilization of NH3 by the bacteria, as
they are significantly higher in number in the absence or protozoa (Males & Purser, 1970).

47

6.1

Protozoa in water and terrestrial environments

Marine and Freshwater Sediments

Marine sediments are reducing environments covered only by a thin oxic surface layer
(Kristensen, 2000). POM provides a food source for bacteria and it accumulates in the
sediments by the effect of gravity. Protozoa are attracted to these bacteria and graze upon
them (Fenchel, 1974a). Sediments are anoxic at a certain depth beneath the surface
because the high consumption rate of oxygen (as it is the most favourable electron acceptor
for microbial respiration) combined with its low solubility in water usually prevents deep
penetration of oxygen into sediments (Kristensen, 2000). Additionally, when the food content
(organic material) present is higher than the oxygen input needed for its oxidation, anaerobic
conditions will form; therefore the anoxic depth depends on the food to oxygen flow into
interstices (Fenchel & Riedl, 1970). The penetration depth of oxygen is controlled by the
balance between downward transport of oxygen from above and by consumption processes
of all benthic organisms and their metabolic products within the sediment (Kristensen, 2000).
This is dependent on external parameters including the influx of organic matter, the
mechanical properties of the sediment, water turbulence, light, temperature and bioturbation
(Kristensen, 2000; Fenchel, 1974a).
Marine sediments are composed of three distinct recognizable layers as shown in Figure
6.1. The layer closest to the surface is yellow usually due to the presence of Fe 3+ and
contains free O2, which declines with increasing depth. The grey layer has O 2 and reduced
compounds like H2S in small amounts while the black zone is totally devoid of O 2 and has
H2S in large amounts (Fenchel & Riedl, 1970). A sulphide system is established under the
cover of oxidized marine sediments and the boundary is definable by a redox-potentialdiscontinuity (RPD) (Fenchel & Riedl, 1970); represented in Figure 6.1 i.e. that depth below
the sediment-water interface marking the transition from chemically oxidative to reducing
processes. In the black layer found below the RPD, the initial degradation of organic matter
is due to fermenting bacteria, which generate H 2 and low molecular weight organic
compounds (Fenchel, 1974a). Further decomposition may take place by the activity of
certain bacteria that utilize inorganic compounds other than O2 as H2 acceptors. Sulphate
reduction dominates in marine sediments although

48

NO-3 , CO2, and H2O may be used as

H2 acceptors and the end products of the processes are reduced compounds such as H 2S,
NH3, and CH4 (Fenchel & Riedl, 1970).

Sediment Surface
Aerobic Decomposition

Yellow

Oxidized Compounds:
Grey
Redox Discontinuity Layer
Reduced Compounds:
Simple Inorganic compounds

Black

Anaerobic Decomposition
Figure 6.15: Schematic representation of the three major layers in sediments (yellow, grey
and black), the redox discontinuity layer, sediment surface, as well as some compounds and
ions in sediments; modified from (Fenchel, 1967).

H2S is present in different forms depending on the pH of the water but predominantly as HS (Fenchel, 1974a). The inorganic end products may be oxidised as they diffuse through the
sediments by free O2, chemoautotrophic bacteria or anaerobically by photoautotrophic
bacteria thus utilizing H2S as a H2 donor for the reduction of CO2 to organics (Fenchel &
Riedl, 1970). Sulphureta have the highest concentrations of animals i.e. more than 10 7
ciliates per m2. Sulphur bacteria may constitute 25-50% of the food for ciliates for example
immediately beneath the chemocline in sediments, where photosynthetic purple and green
sulfur bacteria that are predominantly consumed by protozoa are active (Fenchel, 1974a).
Diurnal vertical migration of these chemical zones and the associated microorganisms is
observed due to external parameters (Fenchel, 1974a), i.e. influx of organic matter, the
mechanical properties of the sediment, water turbulence, light, temperature and bioturbation.
Reduced permeability brings the RPD closer to the surface, higher temperature causes
higher position of RPD, mixing by winter storms lowers RPD level, whereas, input of organic
matters causes it to rise again (Fenchel & Riedl, 1970). All these processes, as well as the
spatial and temporal patterns which arise from them, allow for the great diversity of protozoa
associated with marine sediments (Fenchel, 1974a).

49

6.1.1 Distribution of protozoa

The mechanical properties of the sediments are important in influencing the species
composition, quantity and distribution of protozoa as shown in the studies by Fenchel
(1974a) and Hamels et al (2004) on experiments in benthic sediments. Fenchel (1974a)
states that ciliates are the dominant protozoa of sandy sediments, although phagotrophic
flagellates (in particular dinoflagellates, euglenids, and amoebae occur as well while Hamels
et al (2004) showed that nanoheterotrophs were the most dominant protozoa at the
sampling stations especially in the silty sediments, both in terms of abundance and biomass,
which shows the effect of sediment type on the species of protozoa present.
Benthic protozoa live in the interstitial of sand grains, and most of them have adapted to
living in sediments through shape and locomotion for example by being more slender or
flatter, or more contractive to facilitate swimming among sand grains (Lei et al., 2014). Wellsorted sands with small amounts of silt and clay harbour protozoa and other organisms in
the space between the individual mineral grains. The largest numbers (> 107 per m2) are
found in fine sand with a median grain size of 125-250m, in localities with strongly reducing
sediments and a rich growth of sulphur bacteria (Fenchel, 1967). In sediments with a smaller
than average grain size, only smaller protozoa are capable of moving in the interstitial space,
while in very fine-grained or poorly sorted sediments, protozoa (except for some
foraminifera) are confined to the sediment surface because the interstitial spaces are too
small to allow ciliate movement and they are incapable of burrowing (Fenchel, 1974a).

6.1.2 Environmental factors affecting protozoa in sediments

The marine and freshwater protozoa species are different whereby the freshwater protozoan
appear less distinct and diverse than that of marine ones (Fenchel, 1974a). The typical
marine ciliate observed by Hamels et al (2004) were species such as Tracheloraphis
longicollis and Chlamydodon triquetrus while freshwater taxa such as Pseudochilodonopsis
fluviatilis and Trithigmostoma cucullulus were seen, with the difference mainly attributed to
compliance with the general pattern of species turnover along estuarine salinity gradients.
However, the species difference is not limited only to salinity because other factors like
degree of organic pollution and/or oxygen concentrations influence the protozoan
microbenthos (Hamels et al., 2004).

50

Ciliate biomass is usually driven by chlorophyll a in typical marine systems however,

multivariate analyses by Lei et al (2014) showed that other factors like temperature, salinity,
silt/clay, carbon and nitrogen are important to varying degrees in determining the ciliate
community composition, most especially the combination of salinity, temperature, and
silt/clay. In addition cysts are usually regarded as the most important factor for species
dispersal because they are important for survival particularly in harsh environments and
ensure the persistence of the population, therefore many protozoa may pass through a
resting stage during their life cycle in form of resting cysts (Lei et al., 2014).

6.1.3 Metabolism and nutrient cycling of protozoa in sediments

Protozoa excrete mineral nutrients such P as

PO 3-4

and N2 as NH3 or

NH +4

as a by-

product of their metabolic activities (Pogue & Gilbride, 2007). Furthermore, the contribution
of protozoa to benthic energetics is disproportional to their biomass because of a higher
turnover of smaller cells (Fenchel, 1974b); making them very important in nutrient cycling in
sediments. This is shown in the studies by Hamels et al. (2004) who studied protozoa
behaviour in 2 sandy and 2 silty sediment stations to determine their relative contribution to
metabolism. Protozoan biomass (ciliates and nanoheteretrophs) exceeded metazoan
biomass at one of the sandy stations. However, at the rest of the stations the protozoan
biomass was less than 5% of the combined biomass of meio- and macro- benthos. The
biomass-metabolic rate equation R = a W -0.249 was applied to calculate the relative metabolic
rates for ciliates, nanoheterotrophs, and metazoan; where the metabolic rate per unit weight
(R) increases with decreasing body weight (W) (Fenchel, 1974b). The results showed that
protozoa contributed more to sediment respiration than the metazoa especially at the sandy
stations, with the nanoheteretrophs contributing the largest proportion. This shows that their
metabolic contribution is disproptional to biomass but related to individual microorganism
weight. Protozoa represented 29 to 96% of the combined metabolic rate of benthic
consumers in the sandy stations observed in late spring/early autumn (Hamels et al., 2004).

6.1.4 Grazing of protozoa

The two most important foods for the ciliates are bacteria and protophytes whereby among
the latter, diatoms are most important followed by flagellates and other unicellular algae.
Carnivores, which feed on other ciliates, contribute about 10% (Fenchel, 1969). Sulphur
bacteria may contribute 25 50% of the food for ciliates in certain regions indicating a large

51

trophic role of reduced low molecular end products of anaerobic decomposition (Fenchel,
1969).

Methane production

6.2

2-

SO 4

In marine anoxic environments,

reduction is the dominating terminal mineralization

step accounting for 90 99% of activity. However, some CH 4 is produced because of the
existence of non-competitive substrates for methanogens like methanol, the existence of
2-

SO 4

free micro niches inside detrital particles and protozoa with endosymbionts.

Anaerobic protozoa with endosymbiotic methanogens account for 15 -90% of the CH 4

produced (Priya, 2009; Fenchel & Finlay, 1995). The endosymbionts are protected from
2-

SO 4

competition with

reducers in the sediments and their substrates are mainly H 2 and

acetate. In marine sediments the contribution of anaerobic ciliates to the CH4 production (1590%) is greater than the freshwater sediments (maximum of 5%) where it is generally
marginal throughout the year (Priya, 2009; Van Hoek et al., 2006; Fenchel & Finlay, 1995).
In a microbial succession based on the accumulation of organic matter, there is a sequential
depletion of O2,

NO-3 and SO2-4 and eventually methanogenesis prevails as the terminal

mineralization process. The anoxic layers in marine sediments can eventually become
depleted of

2-

SO 4

allowing free-living methanogens to increase in abundance, and the

endosymbiotic methanogenesis decreases to a figure typical of any methanogenic system.

However, CH4 can be oxidised anaerobically by

2-

SO 4

reducers in the sediments (Fenchel

& Finlay, 1995).

6.3

Ammonia in sediments

Microbial reduction of

NO-3

in anoxic marine sediments can be divided into two major

pathways: denitrification and nitrate ammonification. In the former

(NO-3 )

is reduced to

(NO-2 ) , which is then further reduced to N 2O or N2 while in the latter, the dissimilatory
pathway,

(NO-3 ) is reduced to

(NO-2 ) that serves as an electron sink and then to

(NH +4 ) in a fermentative reaction (Blackburn, 1985). The dissimilatory pathway requires a

52

supply of organic substrate and dominates nitrate and nitrite reduction in anaerobic
environments like marine sediments (Bothe et al., 2007). Denitrifying and nitrate
ammonification bacteria are the key microorganisms responsible for nitrate reduction
(Vymazal & Krpfelov, 2008). Protozoa in the sediments graze on bacteria thus stimulating
their activity and release

NH 4

as a by-product of their metabolism (Pogue & Gilbride,

2007).

6.4

Soil Protozoa

Protozoa are 103 104 cell g-1 of soil (Hoorman & Islam, 2010), and live in the soil water films
and water-filled pores of soil aggregates. Most of the activity occurs in the rhizosphere next
to roots, which typically contains 1,000 to 2,000 times more microorganisms than the typical
soil without roots (Hoorman, 2011b). The lifecycle of soil protozoa consists of an active
phase that involves feeding and multiplication and a cyst stage where the cell produces a
thick coating during times of environmental distress. The cyst stage enables the cell to
survive for many years during harsh environmental conditions after which it reverts to the
active phase when environmental conditions improve (Hoorman, 2011b). The cysts have a
high dispersal potential, and are easily spread by aerosolization; therefore protozoa can
colonize new substrates rapidly (Rnn et al., 2012). The majority of the protozoa in soil are
bacteriovorus while only one amoebae group, the vampyrellids, feed on fungi. Fungaldominated soils tend to have more testate amoebae and ciliates compared to other protozoa
types while the bacterial-dominated soils predominantly have flagellates and naked
amoebae (Hoorman, 2011b).

6.4.1 Role of protozoa in the soil

Protozoa are a key link in the terrestrial food chain as a predator of bacteria and prey for
higher organisms like nematodes. Through their predatory activities, the main roles they play
in the soil ecosystem are nutrient cycling and regulating the microbial community activity
through grazing (Lal, 2006). Bacterial activity is stimulated by predation and in turn
decomposition rates and soil aggregation increase; the predatory behavior or protozoa is
also useful in enteric bacteria inactivation (Perez-Viana, 2010).

53

The C:N ratio for protozoa is 10:1 or more, while that of the bacteria on which they graze lies
within the range of 3:1 up to 10:1. This implies that the protozoa take in more much more N
than is necessary for their carbon needs when they graze on bacteria (Hoorman, 2011b).
The excess N is released in form of

NH +4 , which will be available for plant uptake and

other microorganism needs in the soil. Their contribution to total net nitrogen mineralization
is 12 - 30% (Hoorman, 2011b).

6.4.2 Factors affecting protozoa activities in soil

There are several factors that affect the growth and distribution of soil protozoa including
substrate supply, pH, temperature, moisture content and the soil structure and texture:

i.

Soil texture and structure

The soil texture determines the habitable soil pore space and the water retention capacity.
Coarse soils like sand have larger pore spaces than finer soils like clay therefore the latter
contain a higher number of smaller protozoa (flagellates and naked amoebae), while coarser
textured soils contain more large flagellates, amoebae, and ciliates (Hoorman, 2011b).
Pore size limits the grazing activities of soil protozoa whereby small flagellates and naked
amoebae can only access bacteria in water-filled pores with openings of a minimum size 2-3
m (Rnn et al., 2012). This protects bacteria from predation (Perez-Viana, 2010). In a study
by Rutherford & Juma (1992), bacteria numbers in soils with protozoa were reduced by 68%,
50% and 75% in the silty clay, clay loam and sandy loam respectively compared. Coarse
soils drain water faster than the fine soils and as soil drains, the size of the water filled pores
decreases (Rnn et al., 2012). At the wilting point water filled pores measure approximately
0.3 m therefore excluding protozoan microbial activity (Perez-Viana, 2010).
ii.

Moisture content

Protozoa live in the thin films of water in the soil pores and are not active when confined to
the colloidal film (Hoorman, 2011b). At typical temperate ambient temperatures, a water
content of 30-50% supports the activity of soil protozoa. However, protozoa are inhibited at
lower soil moisture contents and water contents of 20% only support bacterial growth
(Fenchel, 1994).

54

iii.

Substrate supply

Protozoa are found in greatest abundance near the surface of the soil, particularly in the
upper 15 cm (Hoorman, 2011b), and decrease with soil depth from approximately 10 6 g-1 at
0.1m to 10 g-1 below 1m. This is due to decreasing levels of organic matter in the soil profile
(Lal, 2006).
iv.

pH

Protozoa have the ability to survive in a wide pH range but their prey do not. Acidic soil pH
reduces bacterial populations and the substrate supply for growth thus indirectly affecting
protozoa (Perez-Viana, 2010).
v.

Temperature

High temperatures favour metabolism and reduce doubling times, leading to an increase in
protozoan activity. However, the upper temperature limit that they can safely withstand is
30C with 35 40C causing death. They are able to survive extremely high and low
temperatures by encysting (Perez-Viana, 2010).

6.5

Selective feeding on bacteria by protozoa

Protozoa ingest bacteria by phagocytosis and influence the species composition in soil
because of their high consumption rates and feeding preferences. They select bacteria
based on the following factors:
i.

Cell surface characteristics

Gram-negative bacteria are preferred to the gram-positive as protozoan food (van-Elsas et

al., 2006). The latter have a thick cell wall (due to the presence of a peptidoglycan layer) and
have a negative charge on the outer surface, while the former have a thinner cell wall
(Hoorman, 2011a). Gram-positive bacteria are less edible because their cell wall is less
digestible, which may enable survival during passage through the protozoan cell (Ronn et
al., 2002). In a study by Ronn et al (2002) to determine the impact of protozoa grazing on
bacteria in soil, the flagellates Ochromonas and Spumella rapidly egested the indigestible

55

bacteria. Furthermore, some gram-positive bacteria like Mycobacterium chlorophenolicum

were completely unaffected by grazing of the indigenous soil protozoa during the study.
Gram-negative bacteria are the main types of human enteric pathogens such as Salmonella,
Escherichia coli, Shigella etc. Protozoa and nematodes are the main groups of soil
organisms that prey on enteric pathogens (Opperman et al., 1989). It is therefore important
to understand the mechanisms that contribute to the fate of enteric pathogens when
introduced in soil to develop management practices that will mitigate the risk they pose to
health based on scientific evidence (Lang et al., 2007).
Habte & Alexander (1977) showed the predatory relationship of protozoa on bacteria.
Rhizobium was added to soil and decreased from 109 cell g-1 to 109 cell g-1 while protozoa
numbers increased up to 104 cell g-1. A protozoa inhibitor (actidione) was then added to the
soil, which resulted in reduced protozoan numbers but the bacteria population remained
constant. After 4 days, protozoa resistant to the inhibitor emerged and the bacteria numbers
subsequently declined. The ability of bacteria to maintain their populations in the absence of
protozoa signifies that protozoa are the major regulators of bacteria populations (PerezViana, 2010).
ii.

Cell size

Selection by size is typical for filter feeding ciliates and interception feeding flagellates, which
feed most efficiently on intermediate sized bacteria (Rnn et al., 2012). Small and large
filamentous cells may escape predation and the study carried out by (Ronn et al., 2002),
found that the protozoa preferentially fed on cells larger than 0.5 m.
iii.

Bacterial growth yields

Bacteria vary in their nutritional value and protozoan growth rates strongly depend on the
type of bacteria they feed on; therefore they will preferentially graze on bacteria with higher
nutritional value (Rnn et al., 2012). Many gram-negative bacteria are good food sources for
protozoa as they produce high growth yields. This is shown in the studies by Ronn et al
(2002) to determine the impact of protozoa grazing on bacteria in soil whereby protozoa
showed preference for bacteria that had an affiliation with species of Enterobacteriaceae,
which produce high protozoan growth yields.

56

iv.

Bacterial growth rates

Protozoa prefer to graze on bacteria that have high growth rates because they are able to
replace the cells lost to predation (Ronn et al., 2002).
v.

Motility

High bacterial motility reduces protozoan feeding efficiency by reducing the chance of being
consumed by protozoa (Rnn et al., 2012).
vi.

Biochemical processes

Certain bacteria release toxic chemicals that kill protozoan cells therefore it is beneficial for
protozoa to avoid grazing on them. Furthermore, some bacteria form dense micro-colonies
through the excretion of polysaccharides thereby reducing the susceptibility to grazing by
protozoa (Rnn et al., 2012).

6.6

Internalisation of bacteria by soil protozoa

Free-living soil amoebae like Acanthamoeba, Naegleria and Hartmannella can act as
reservoirs and dispersal agents for pathogenic bacteria like Legionella, Chlamydia,
Salmonella etc. (van-Elsas et al., 2006). This is because when soil protozoa graze on
bacteria, some of the bacteria are not digested but rather internalised within the cell. The
fate of the internalised bacteria falls into three categories: bacteria that multiply and cause
lysis of the protozoan cell, bacteria that multiply and do not cause lysis of the protozoan cell
and bacteria that survive without multiplication (Barker & Brown, 1994).
Although protozoa preferentially feed on gram-negative bacteria, some survive protozoa
grazing either because the protozoa are unable to take them up and digest them or because
they have defensive mechanisms like outer membrane structures, production of toxic
chemicals etc. Certain bacteria are able to form an endosymbiotic relationship with the
protozoa as a survival mechanism in harsh environments (Barker & Brown, 1994). Protozoa
egest vesicles as part of their digestion process through which pathogenic bacteria can be
dispersed (van-Elsas et al., 2006).

57

6.6.1 Effects of internalization on bacteria

Protozoa are able to provide protection to the bacteria from environmental antagonists within
their cells. Furthermore, in times of environmental distress protozoa from cysts which in
effect provide protection for the internalised bacteria. These cysts are easily spread by wind
thereby dispersing the bacteria and enabling them to colonize new habitats (Barker & Brown,
1994). Additionally, some protozoa exchange genetic material with the bacteria possibly
leading to the development of new phenotypes (Vaerewijck et al., 2014). A change in
phenotype can lead to changes in physiology, virulence and survival of bacteria. The
physiology of bacteria that grows within protozoa is changed and they are observed to be
more resistant to biocides. For example, a 1000-fold kill was observed for legionellae grown
in vitro when a biocide was applied but only a 10-fold kill was observed for the cells grown
inside amoeba (Barker & Brown, 1994). Furthermore in terms of survival, the bacteria
Legionella pneumophila that were stored at 4C for 6 months after extraction from
Acanthamoeba polyphaga were observed to have small reductions in viable cell count
(Barker & Brown, 1994).

6.6.2 Survival of bacteria within protozoa

Temperature is a major factor that influences the survival of intraprotozoan bacteria. Lower
temperatures are unfavourable for the growth and multiplication of internalised bacteria. For
example, the bacteria Legionella pneumophila in acanthamoebae multiplies at 35 C but is
digested and in some cases has limited intra-amoebal growth at 20C (Vaerewijck et al.,
2014). Additionally, temperature affects the virulence of Leg. pneumophila, whereby a
decrease in temperature from 37C to 24C caused an attenuation in virulence (Barker &
Brown, 1994).
In some bacterial species, the bacteria avoid digestion by receptor-mediated endocytosis.
This method of ingestion directs the organisms into vesicles that do not merge with the
lysosome, therefore avoiding the digestive enzymes. This behaviour is observed in the
virulent Leg. pneumophila inside the amoeba Hartmannella vermiformis where the bacteria
are found in endosomes that fuse with the endoplasmic reticulum of the protozoa; this
becomes the site of bacterial multiplication (Barker & Brown, 1994).

58

7 Protozoa as a pretreatment
Pretreatment systems can be used to enhance the efficiency of the AD process resulting in
improved digestate quality, increased biogas yield and faster digestion (Montgomery &
Gnther, 2014; WRAP, 2012). Biological processes using specific microbial species offer
advantages such as low capital cost, low energy, no chemical requirement, mild
environmental conditions, and no inhibitory compounds formation (Cesaro & Belgiorno,
2014).
Protozoa are able to degrade cellulose intracellularly, excrete nutrients as by-products of
their metabolism and enhance beacterial activity by grazing on them. Additionally, they
maintain a stable rumen environment by engulfing starch, making it unavailable to rapidly
fermenting bacteria therefore slowing the production of VFAs as discussed in the previous
Chapters. These behavioural traits make them useful as a pretreatment for wastes with a
high cellulolytic content like sewage sludge and municipal solid waste.
The physiological and environmental conditions of the rumen are mimicked in the design of
the reactor because the rumen harbours a large population of protozoa. Protozoa
populations are 104 106 per g in the rumen and they represent approximately 50% by mass
of the microbial biomass (Jouany & Ushida, 1999). Furthermore, ruminants consume forage
as part of their diet that is rich in cellulose. Protozoa are shown to contribute to 43% of the
ruminal cellulase activity in a buffalo (Kuhad & Singh, 2007).

7.1

Hydrolytic pretreatment reactor design

A hydrolytic pretreatment treatment reactor is proposed to be able to use protozoa as a

biological pretreatment for wastes that are rich in cellulose. The pretreatment vessel can be
added onto an already existing single or multi stage AD plant to enhance digestibility of
waste. The reactor is a dry digestion horizontal plug flow vessel with a solids recirculation
stream. It will be micro-aerated and have a mechanical pretreatment stage. The returned
solids will go though the mechanical unit to undergo further size reduction and mix with fresh
feed. This is represented in Figure 7.1.

59

Paddle shaft

Dewatering
unit

Mechanical
pretreatment unit

Effluent
to digester

Feed

Plug flow reactor

Solids
to digester
Recirculated solids
Figure 7.16: A pretreatment hydrolytic plug flow reactor with a mechanical treatment unit with
a solids recycle stream

Dry AD is commonly used to treat a wide range of inputs but mainly commercial and
municipal solid wastes. A plug flow system allows for more process control because the
material moves predictably and can be tracked. Horizontal pug flow systems have a
minimum retention time of 14-20 days and operate within a 25-40% DS range. The largest
horizontal modules are able to process 20,000-25,000 tonnes per annum, therefore a largescale project will require more vessels arranged in parallel (ADBA, 2013).
The material is pre-blended with recycled digestate being fed into the reactor resulting in
rapid inoculation at the right temperature (ADBA, 2013). An internal paddle mixer system is
used to homogenize the waste and to ensure maximum contact between the waste and
microorganisms; it also ensures a uniform digester temperature and that the less dense
floating layers are blended back into the mix while the denser particles do not settle as the
waste is moved along the vessel (Evans, 2001).

7.1.1 Reactor temperature

The optimum temperature for ruminal ciliate activity is 39C while temperatures below 25C
and above 42C will inhibit their growth (Williams & Coleman, 1992). The vessel will

60

therefore be operated at mesophilic temperatures with an optimum at 39C. A constant

temperature can be achieved by using insulated hot water heating loops within the digester
body made of either steel or concrete (ADBA, 2013).

7.1.2 Reactor pH
The pH of the system will be kept near neutral (6.5 7), to enhance hydrolysis and prevent
process inhibition due to acidogenesis. In the rumen, cellulolytic activity occurs between pH
6 and 6.9 whereby a pH below 6 leads to a drastic decrease in fibre digestion (Mehra et al.,
2012; Rode, 2000). The entodiniomorphid protozoa and holotrichs are the main rumen
ciliates and the former are observed to degrade cellulose at a pH between 6.5 and 7 while
the latter have maximum productivity at a pH from 6.8 to 7 (Williams & Coleman, 1992). An
anaerobic digester has the ability to buffer its environment therefore providing resistance to
change in pH. Monitoring the ratio of bicarbonate alkalinity to VFAs is used as a control for
the pH whereby the result is used to regulate the feed rate of the digester (ADBA, 2013).

7.1.3 Mechanical pretreatment unit

A mechanical pretreatment unit is used before the hydrolytic vessel and a portion of the
hydrolyzed material will be returned to the unit for further size reduction. It is carried out to
achieve a reduction in particle size and crystallinity of lignocellulosic materials. This leads to
an increase in the specific surface area and a reduction in the degree of polymerization
consequently increasing the rate of enzymatic degradation (Cesaro & Belgiorno, 2014).
Additionally, it reduces viscosity of the waste thus making mixing easier. This is similar to
rumination, which involves the back and forth cycling of feed. The mastication occurring with
rumination helps to disrupt the protective layer of cutin and exposes more digestible portions
of plant particles to hydration and fermentation (Bayan & Guiot, 2011).
Size reduction of lignocellulosic substrates results in a 525% increased hydrolysis yield,
depending on the mechanical methods used and a particle size of 1 to 2 mm has been
recommended for effective hydrolysis of lignocellulose (Montgomery & Gnther, 2014;
Ariunbaatar et al., 2014). The most commonly used mechanical methods for organic solid
waste like lignocellulosic materials, manure and wastewater treatment plant (WWTP) sludge
are maceration, sonication and high-pressure homogenizer (HPH) (Ariunbaatar et al., 2014).
A HPH draws compressed waste through a valve at a high pressure, where the cells are

61

subjected to impact and shear stress and a pressure drop across the valve leading to cell
disruption; sonication liquefies the biomass by destroying part of the cells using ultrasonic
frequencies; while maceration technologies grind food waste into a pulp that can be
pumped.

7.1.4 A recirculation stream

In the rumen, more than 75% of protozoa are tightly attached to the feed particles and
mucus while some microorganisms are retained by the omasum laminae. This ensures the
retention of microorganisms whose doubling time is shorter than rumen turnover time, like
protozoa (5-14h), thus guaranteeing the complete digestion of ingested feed. It also ensures
different turnover rates for solids and liquids (Sun et al., 2013; Bayan & Guiot, 2011).
Furthermore, the rumen stratifies its digesta according to density whereby the less dense
degradable digesta is in dorsal sac and as the particles ferment and become smaller and
denser, they settle to the more liquid levels of the ventral sac (Gookin et al., 2011; Bayan &
Guiot, 2011). Stratification provides different turnover rates for solids and liquids thus
improving fermentation (Teather & Sauer, 1988). The solid phase of a fermenter needs to
have a lower turnover rate than the liquid phase for the protozoa attached to solid particles
are retained longer (Muetzel et al., 2009).
Separating solids from the effluent and recirculating the concentrated sludge back to the
fermenter achieves a cell residence time greater than the hydraulic retention time (HRT).
Additionally, it allows further contact between biomass and raw waste (Mara & Horan, 2003).
The separated biomass system will retain both active microorganisms, like protozoa and the
undigested influent suspended solids (Malina, 1992). Separation can be achieved on a large
scale by a centrifuge where a rapidly rotating bowl throws solids to the outer edge from
which they are scraped off or a filter plate press where sludge is pumped between multiple
vertical plates and dewatered under pressure. The solids to be returned to the mechanical
unit can be wetted with the separated liquids to achieve the desired solids content. Plug flow
systems recirculate 15-30% of digestate or liquids (ADBA, 2013). Mixing the digestate with
fresh waste will prevent excessive acid buildup near the front end of the digester (Rapport et
al., 2008).

62

7.1.5 Controlled micro-aerobic conditions

Low O2 levels exist in the rumen as discussed in Chapter 5. Facultative anaerobes have a
higher yield in the presence of O2, leading to higher populations and more enzymes that
carry out hydrolysis (Botheju & Bakke, 2011). Furthermore, anaerobes are able to adapt to
low O2 concentrations as discussed in Chapter 3.1.1. The introduction of small amounts of
O2 into an anaerobic biochemical process enables both anaerobic and aerobic biological
activities to occur within a single bioreactor (Botheju & Bakke, 2011). Preaeration can be
achieved either by difussed aeration system or through mechanical aeration. Dry AD vessels
have waste that is non-flowable therefore turbulent agitation and mixing is not possible
(ADBA, 2013); for example with mechanical aeration that involves turbulent agitation
therefore a diffused aeration system is used. Diffused aeration systems involve the injection
of pressurized air or O2 below the liquid surface and are classified into three groups: porous,
nonporous and other aerators (ASCE, 1988).
i.

Porous diffusers: These diffusers come in various shapes and sizes, such as discs,
tubes, domes, and plates and have a relatively high oxygen transfer efficiency (EPA,
1999; ASCE, 1988). They are mounted or screwed onto the diffuser pipe.

ii.

Nonporous diffusers: The common types of nonporous diffusers are fixed orifices
which are pipes with holes, valved orifices that contain a check valve to prevent the
backflow of waste into the orifice when aeration stops and static tubes which contain
interference devices that create bubbles by breaking up the air flow. The bubble size
of these diffusers is larger than the porous diffusers therefore they do not clog easily
(EPA, 1999; ASCE, 1988).

iii.

Other diffusion devices: These include jet aerators that discharge a mixture of air and
liquid through a nozzle near the bottom of the tank; aspirators mounted at the basin
surface to supply a mixture of air and water; and U tubes that discharge compressed
air into the down leg of a deep vertical shaft (EPA, 1999).

The effects of limited aeration on AD were tested by (Juanga et al., 2005). A batch test was
carried out with a pre-stage (hydrolytic and acidogenic) and a CH 4 producing stage that
occurred consecutively in each digester. One of the three reactors used had a partially
aerated pre-stage and it showed a considerably improved methanogenic activity. It produced
a higher biogas volume and reached the active CH 4 phase (50% CH4 in gas) quicker than
the rest of the reactors. Increased CH 4 production was attributed to better acidification in first

63

phase. This is an indication of the positive effect that partially aerated pretreatment can pose
on anaerobic digester performance (Botheju & Bakke, 2011; Juanga et al., 2005).

7.1.6 Inoculation
Anaerobic microorganisms exhibit growth rates, which are much slower than those of
aerobes therefore it is important to adequately seed the reactor with beneficial
microorganisms at start up (Stronach et al., 2012). Facultative and obligate anaerobes are
needed for the hydrolysis of particulate and colloidal compounds (Gerardi, 2003). In
ruminants, newborn animals are inoculated by licking their mothers and other members of
the herd (Husvth, 2011). Digesters are usually seeded with microorganisms from actively
digesting sludge, a municipal digester, a well-rotted manure pit or using cow dung slurry. In a
continuous or semi-continuous operation, the seed material should be at least twice the
volume of the fresh feed during the start up phase, with gradual reductions in seed over a 3
weeks period (NRC, 1977).

7.2

The effects of hydrolysis on the proposed digester

The hydrolysis rate (hyd) is determined using the first order model with respect to substrate;
it a product of the hydrolysis rate constant (k H) and the fraction of degradable POM
expressed as COD (Xs) (Siegrist et al., 2002); see Equation 7.1

hyd =

k H Xs

Equation 7.9

Several kH values have been proposed depending on temperature, waste composition, HRT
etc. Siegrist et al (2002) states that the k H for sewage sludge is 0.25 d-1 under mesophilic
conditions; Stamatelatou et al (2005) established kH as 0.35, 0.2 and 0.063 day1 for
carbohydrates, proteins and lipids respectively under mesophilic and thermophilic conditions
(Fezzani & Cheikh, 2008); Noike et al (1985) determined that cellulose has a k H of 0.1 d-1 at
35C (Vavilin et al., 1996) and Haghighatafshar et al (2014) proposed a value of 0.32 d-1 at
35C for a 15 days HRT. The values used for the analysis of the plug flow digester
performance are based on those from Stamatelatou et al (2005) and (Vavilin et al., 1996).
Mixed sewage sludge values from Haghighatafshar et al (2014) are used for the Xs values.
Carbohydrates, fats and lipids represented as 37%, 32% and 30% of the COD fraction refer
to Table 7.1.

64

Table 7:7: Composition of mixed sewage sludge (Haghighatafshar et al., 2014)

Mixed Sludge
Total composition
COD fraction

Fat
(g/L) (%)
5.9
17.1
30

Protein
(g/L) (%)
13
35.4
32

Carbohydrates
(g/L)
(%)
18.2
47.5
37

Inert
(g/L) (%)
13.5 26.5
-

TS
(g/L)
50.5

Hunter & Henkelekian (1965) state that 60 to 80% of the total carbohydrate in sewage
sludge is composed of cellulose (Knapp & Howell, 1978); therefore based on that an
assumption is made that the cellulose content in this sludge is 60 % of the carbohydrates
while starch is the remaining 40%. Applying the model by Siegrist et al (2002) to this sludge
and considering a plug flow retention time of 14 days, cellulose will be 31% hydrolyzed,
protein 89.6%, fats 26.5%, and starch 72.5% to produce amino acids, sugars and long chain
fatty acids; see Figure7.2. The inert fraction of is non-biodegradable and therefore leaves the
system unchanged. Refer to Appendix A for the calculation.
A comparison between the degradable substrates concentration before and after hydrolysis in g COD/L

32.41

Amino acids, sugars

and Long chain fatty acids
13.10

Cellulose

Starch

Protein

Fat

8.74

19.50
9.03
2.40
2.03
17.11

12.58

Before hydrolysis

After Hydrolysis

Figure 7.17: A comparison between the degradable substrates concentration in sludge

before and after hydrolysis.

65

8 Discussion
Protozoa demonstrate numerous behavioural traits that are beneficial for the anaerobic
treatment of waste and wastewater treatment as observed by studying the roles they play in
different environments. They play a key role in stimulating bacterial activity by excreting
beneficial nutrients and grazing on them in both the aerobic wastewater treatment and
anaerobic environments (rumen, sediments and soil) studied. Furthermore, in the food chain
protozoa graze on bacteria and provide POM for higher organisms (Pauli et al., 2001),
showing that feeding on bacteria (smaller cell size) is characteristic of their behaviour in
different habitats. Grazing on bacteria is beneficial because they are kept in their log growth
phase and ageing cells are prevented from accumulating by renewing the bacterial
populations (Porter et al., 1985), hence increasing bacteria activity. The mineral nutrients
such P as

PO 3-4

and N as NH3 or

NH +4

are excreted as products of protozoan

metabolic activities as well as organic acids like acetate (Biagini et al., 1998; Porter et al.,
1985). Bacteria utilize these nutrients resulting in an accelerated degradation of organic
matter. Protozoa therefore play an important supporting role in AD to increase the turnover
rate of waste.
In addition to stimulating bacterial activity, protozoa directly consume particulates and take
up dissolved solids therefore directly taking part in fermentation. This behaviour is noticed in
both the aerobic wastewater treatment and anaerobic environments for example in the
rumen where the entodiniomorphid protozoa predominantly engulf particulate matter and the
holotrichs take up mainly soluble sugars (Jouany & Ushida, 1999; Jouany, 1996).
Furthermore, in a study on the role of protozoa in anaerobic wastewater by Priya et al (2007)
there was a decrease in MLSS and COD in the presence of ciliates. This is an indication of
the direct participation of protozoa in substrate utilization in anaerobic digesters. The direct
uptake of POM makes it unavailable to rapidly fermenting bacteria leading to a more stable
fermentation in the rumen by reducing the rate of VFAs formation showing that protozoa play
a role in regulating the environment in AD. This shows that protozoa contribute directly to the
AD process.
However, increased NH3 concentrations are observed in the presence of protozoa in the
rumen as well as in sediments where they contribute to NH 3 generation by stimulating
bacteria activity (Pogue & Gilbride, 2007; Males & Purser, 1970). This is disadvantageous

66

because NH3 is inhibitory to methanogens in high concentrations (Rajagopal et al., 2013),

leading to low CH4 yield. Several methods can be used to control NH 3 inhibition like reducing
process temperatures, pH control (optimum at 7), controlling the C:N ratio (optimum is 10:1)
and immobilizing microorganisms (Rajagopal et al., 2013).
Appels et al (2011) states that sludge has the highest biogas production capacity worldwide.
However cellulose that is hard to degrade is usually present in the sludge and it limits the
hydrolysis step in AD (Monnet, 2003; Knapp & Howell, 1978), therefore it is important to
degrade it efficiently to maximize the biogas yield potential from AD systems. In the rumen,
protozoa are able to efficiently degrade cellulose intracellularly in vacuoles thus minimizing
enzyme wastage and certain species release cellulases into the surrounding medium
(Fenchel & Finlay, 1995). Enhancing protozoan cellulolytic activity by providing optimum
growth conditions and biomimicking the rumen can enable their use as a biological
pretreatment for waste rich in cellulose. The rumen is imitated because ruminants feed on
forage that is rich in cellulose and they retain a large biomass of protozoa that contribute to
its breakdown (Kuhad & Singh, 2007). However, protozoa have short generation times
(Wang & McAllister, 2002) therefore they have to be retained in the system for as long as
possible to efficiently degrade matter.
Protozoa contribute to CH4 production in anaerobic environments due to their relationship
with endosymbiotic methanogens observed in the rumen and sediments where the
endosymbionts use the H2 from protozoa metabolism to reduce CO 2 in effect generating CH4
(Fenchel & Finlay, 1991). Furthermore, by stimulating bacteria activity there is an increase in
CH4 from the free-living bacteria (Biagini et al., 1998). These behavioural traits are an
indication of a direct and indirect role they play in CH 4 generation in the anaerobic treatment
of wastes. It is affirmed in the experiments carried out by Priya et al (2007), who found that
CH4 production was 29-33% higher and increased linearly in the presence of ciliates in
anaerobic wastewater treatment digester.
A multiple barrier approach is employed when biosolids are applied to soil to prevent the
spread of pathogens to humans, which incorporates minimum acceptable treatment levels
and time restrictions between the time of sludge application to soil and harvest of the cop to
ensure pathogen elimination (ADAS, 2011). Soil protozoa play a biotic role in the inactivation
of pathogens because they preferentially graze on gram-negative bacteria, which are the
major human enteric pathogens (van-Elsas et al., 2006). However, they also contribute to
the survival and spread of pathogens in soil, including gram-negative bacteria by
internalizing them (Barker & Brown, 1994). It is therefore essential to understand the effect

67

of this behaviour so as to establish a scientific approach to the land application controls on

the use of biosolids.
Sulphate is the terminal electron acceptor in sediments leading to large H 2S amounts
(Fenchel & Finlay, 1995). However, protozoa with methanogenic endosymbionts are able to
accept some of the electrons that would otherwise have gone to

2-

SO 4

reduction to

produce CH4 instead therefore reducing the impact of competitive microbial reduction
processes (Fenchel & Finlay, 1995). H2S is a major problem in wastewater treatment as it is
malodorous, toxic and corrosive. The major problem associated with its toxicity is that it
although it is initially pungent; it quickly deadens the sense of smell so detection by sense of
smell is difficult. Furthermore, it weakens the structural integrity of the collection system by
corrosion because it forms sulfuric acid when it interacts with moisture (Carus, 2015).
Protozoa containing endosymbionts in anaerobic digesters can lead to a reduction in H 2S
produced based on these findings but further research needs to be done to determine their
role and the effects of enhancing their activity.
Protozoa can be used as indicator organisms of plant performance because different
species are present during different phases of plant operation (Mara & Horan, 2003). This
colonisation pattern is observed in both the aerobic wastewater treatment process and
anaerobic environments. In a study by Priya et al (2007) on the role of protozoa in anaerobic
wastewater treatment systems, specific protozoan species were observed during the startup
and stabilization phases of the reactors. This behaviour can be used as an empiric tool for
expert system development for anaerobic plants or as an indication of system failure.

68

9 Conclusions
Anaerobic waste treatment systems have the potential to generate biogas as a biorenewable energy source. This dissertation highlights the important role protozoa play in the
AD of waste by increasing substrate utilization and cellulose degradation to enhance CH 4
production, as well as their role in the inactivation of soil pathogen population.

The role and behaviour of protozoa in anaerobic

9.1

digestion
Protozoa contribute both directly and indirectly to the degradation of organic matter with
ciliates being the predominant protozoan group responsible for their activity in the rumen,
sediments and anaerobic wastewater treatment.

Protozoa enhance bacterial activity by grazing on them thus keeping the populations

young and increasing their activity (Porter et al., 1985).

Protozoa are able to efficiently degrade cellulose intracellularly in vacuoles therefore
minimizing wastage of enzymes. This is observed in the entodiniomorphid protozoa
in the rumen. Furthermore, some ciliates secrete cellulases into the surrounding
medium like Epidinium caudatum for partial digestion thus making them easier to

ingest (Fenchel & Finlay, 1995).

They take up both particulate and soluble organic matter thus directly contributing to

fermentation observed in the experiments by Priya et al (2007).

They stabilise the fermentation process by taking up substrate therefore making it
unavailable for the rapidly fermenting bacteria therefore slowing down the rate of

VFAs formation (Jouany & Ushida, 1999; Veira, 1986).

Protozoa excrete beneficial nutrients as part of their metabolism like N and P and
organic acids, which enhances bacterial activity (Pogue & Gilbride, 2007; Biagini et

al., 1998).
The concentration of NH3 is increased in the presence of protozoa in the rumen
(Males & Purser, 1970). Protozoa also stimulate bacterial activity in sediments

leading to increased NH3 production (Pogue & Gilbride, 2007).

There are specific protozoan species in each of the colonization phases in AD where by
in the early stage a few ciliates occur while a large diversity and number of ciliates occur
in the stabilisation phase (Priya et al., 2007).

69

Methane production

9.2

Increased CH4 production is observed in the presence of protozoa in the rumen and they
contribute significantly to CH4 amounts in anaerobic marine sediments. Furthermore, CH4
amounts are observed to increase with increasing ciliate populations in AD (Priya et al.,
2007). Anaerobic protozoa possess hydrogenosomes that are used for fermentation to
produce ATP and H2 as an end product (Fenchel & Finlay, 1995). They contribute to
increased CH4 generation through the following mechanisms:

Protozoa harbour endosymbiotic methanogens that utilize the H 2 produced to reduce

CO2 into CH4. This behaviour is observed in the rumen and sediments where they
contribute 9-25% of the CH4 generated in the former and 15-90% of the latter (Priya,
2009; Fenchel & Finlay, 1995). Protozoa benefit from this symbiotic relationship

through increased growth and yield (Fenchel & Finlay, 1995).

Protozoa increase the activity of free-living bacteria by grazing on them and by the
provision of essential nutrients leading to increased CH 4 generation (Biagini et al.,
1998).

Protozoa and pathogens

9.3

In the soil, protozoa play a biotic role in the inactivation of enteric pathogens but also
contribute to their spread therefore influencing the pathogen dynamics in the following ways:

Protozoa preferentially graze on gram-negative bacteria, the major human

gastrointestinal pathogens, because of their ease of digestibility (van-Elsas et al.,
2006; Ronn et al., 2002), hence contributing to the inactivation of enteric pathogens

in soil.
In some cases, the pathogens are not digested but are internalised within the
protozoan cell which leads to their survival and spread in soil (van-Elsas et al., 2006).

9.4

Future research and recommendations

70

Basing on the discoveries from this research, future research areas and recommendations in
relation to increasing anaerobic digestion performance and the inactivation of pathogens
have been made.

A study into the contribution of protozoa with endosymbionts to H 2S and CH4

production in AD and the effect of increasing their numbers.

A study into the combined effects of internalisation and preferential grazing of gramnegative bacteria by protozoa as well as the factors that influence them. This is
essential to harness the inactivation mechanism during biowaste treatment and at the

point of use in the field.

Protozoa can be used as a pretreatment for waste rich in cellulose. Providing suitable
environmental conditions for optimum yield and biomimicking the rumen in reactor
designs can achieve this because ruminants feed on food rich in cellulose and they
able to retain a large biomass of protozoa.

71

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Appendix A
Hydrolysis Calculations for the plug flow digester
Table A.1: A table showing the substrate composition for mixed sewage sludge from
Haghighatafshar et al (2014).

Mixed Sludge
Total composition
COD fraction

Fat
(g/L) (%)
5.9
17.1
30

Protein
(g/L) (%)
13
35.4
32

Carbohydrates
(g/L)
(%)
18.2
47.5
37

Inert
(g/L) (%)
13.5 26.5
-

TS
(g/L)
50.5

The COD fraction is obtained based on the average chemical composition of each of the
substrates with fat 2.9g COD/g of fat, protein 1.4g COD/g of protein and carbohydrates 1.2g
COD/ g of carbohydrate. 1 % of the VSS as COD is assumed to be inert (Haghighatafshar et
al., 2014). The proceeding calculations are based on the values in TableA1.1.
Table A.2: A table showing the degradable portion of the sewage sludge, the hydrolysis rate
constants, the hydrolysis rate per day and the % hydrolysed after 14 days.
(g/L)
5.9
13
7.28
10.92
13.5

Fats
Protein
Starch
Cellulose
Inert

g COD/g
2.9
1.5
1.2
1.2
-

g COD/L
17.1
19.5
8.7
13.1
-

Cellulose is assumed to be 60% of the carbohydrate content and starch 40%. The inert
fraction of waste is non-biodegradable and therefore not hydrolysed.
Table A.3: A table showing the hydrolysis rate constants, the hydrolysis rate per day and the
% hydrolysed after 14 days.

Fats
Protein
Starch
Cellulose

Xs (%)
30
32
14.8
22.2

KH(1/d)
0.063
0.2
0.35
0.1

hyd (%/d)
1.89
6.4
5.18
2.22

80

Retention time
14
14
14
14

% Hydrolysed
26.46
89.6
72.52
31.08

The kH values used are from literature by Stamatelatou et al (2005) for carbohydrates,
protein and lipids and Vavilin et al (1996) for cellulose. hyd is the hydrolysis rate, Xs is the
biodegradable fraction of substrate in terms of COD and KH is the hydrolysis rate constant.

hyd = k H Xs
The % Hydrolysed is obtained by multiplying hyd by the 14 days plug flow retention time.
Table A.4: A table showing the concentrations of the hydrolysis end products and the
amounts hydrolysed.

% Hydrolysed
Initial (g COD/L)
Amount hydrolysed (g COD/L)
After hydrolysis (g COD/L)

Fat
26.46
17.1
4.53
12.58

Protein
89.6
19.5
17.47
2.03

Starch
72.52
8.7
6.34
2.40

Cellulose
31.08
13.1
4.07
9.03

ASL
32.41

ASL = Amino acids, sugars and Long chain fatty acids.

The produced amino acids, sugars and long chain fatty acids are 32.41 g COD/L and are
obtained from adding up the amount of fat, protein, starch and cellulose hydrolysed.

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