Accepted Manuscript

Co-composting of organic fraction of municipal solid waste mixed with different

bulking waste: Characterization of physicochemical parameters and microbial
enzymatic dynamic

Mukesh Kumar Awasthi, Akhilesh Kumar Pandey, Pushpendra Singh Bundela,

Jamaluddin Khan

PII: S0960-8524(15)00124-8
DOI: http://dx.doi.org/10.1016/j.biortech.2015.01.104
Reference: BITE 14536

To appear in: Bioresource Technology

Received Date: 21 November 2014

Revised Date: 23 January 2015
Accepted Date: 24 January 2015

Please cite this article as: Awasthi, M.K., Pandey, A.K., Bundela, P.S., Khan, J., Co-composting of organic fraction
of municipal solid waste mixed with different bulking waste: Characterization of physicochemical parameters and
microbial enzymatic dynamic, Bioresource Technology (2015), doi: http://dx.doi.org/10.1016/j.biortech.
2015.01.104

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
 Co-composting of organic fraction of municipal solid waste mixed with different bulking

waste: Characterization of physicochemical parameters and microbial enzymatic

dynamic

Mukesh Kumar Awasthia-c,*, Akhilesh Kumar Pandeyd, Pushpendra Singh Bundelab, Jamaluddin

Khanc

*Corresponding author. Dr. Mukesh Kumar Awasthi

Department of Biotechnology

Amicable Knowledge Solution University

Satna, India

Tel.: +917672-4037776, 09407141306,

Fax: +917672-4037776,

E-mail address: mukesh_awasthi45@yahoo.com

 Co-composting of organic fraction of municipal solid waste mixed with different bulking

waste: Characterization of physicochemical parameters and microbial enzymatic

dynamic

Mukesh Kumar Awasthia-c,*, Akhilesh Kumar Pandeyd, Pushpendra Singh Bundelab, Jamaluddin

Khanc
a
Department of Biotechnology, Amicable Knowledge Solution University, Satna, India
b
Regional Office, Madhya Pradesh Pollution Control Board, Jabalpur, India
c
Mycological Research Laboratory, Department of Biological Sciences, Rani Durgavati

University, Jabalpur, India

d
Madhya Pradesh Private Universities Regulatory Commission, Bhopal, India

Abstract

The effect of various bulking waste such as wood shaving, agricultural and yard trimming waste

combine with Organic Fraction of Municipal Solid Waste (OFMSW) composting was

investigated through assessing their influence on microbial enzymatic activities and quality of

finished compost. All three piles of OFMSW with different bulking waste were inoculated with

microbial consortium. The results revealed that OFMSW combine with wood shaving and

microbial consortium (Phanerochaete chrysosporium, Trichoderma viride and Pseudomonas

aeruginosa) were helpful tool to facilitate the enzymatic activity and shortened composting

period within four weeks. Major fluctuations of enzymatic activity was observed in pile 1 and 3

during the first 3 weeks, while in pile 2 relatively very low through composting. But phosphatase

activity was relatively higher in all piles until the end of the process. Maturity parameters of

compost quality also favored the pile 1 as the best formulation for OFMSW composting.

Keywords: Compost, wood shaving, yard trimming, agricultural waste, microbial consortium.
1. Introduction

Most of human activities worldwide produce over million tonnes of municipal solid waste

(MSW) each year now which create serious health hazards problems from both environmental

and economic points of view (Castaldi et al., 2008; Adam et al., 2009). From long time ago

landfill is one of the way for MSW management process in all over the world, which takes up

broad land area and generate huge quantity of numerous auxiliary pollutants including leachate,

greenhouse gases and odorous compounds (Zeng et al., 2010). Owing to rapid increasing

population, rising MSW landfill costs and expanded environmental attention, the Indian

Environmental Ministry has begun to consider alternative solid waste disposal methods

(Gabhane et al., 2012; Awasthi et al., 2014). Many alternatives encouraged as successful strategy

for the proper disposal of OFMSW have been proposed so far. But the use of aerobic windrow

composting is one of the most attractive method due to its insignificant environmental impact,

easy operational procedures and low cost technology (Liu et al., 2011). Because MSW contains

more than 50% moisture and 60% of heterogeneous organic matter content; therefore,

composting mixed with bulking agent is a possible alternative ecofriendly method, after

elimination of metal, plastic and glass (Castaldi et al., 2008; Echeverria et al., 2012).

But the compactness and high moisture content of raw materials means interrupt the efficacy

of composting process, due to OFMSW cannot be properly composted alone and they need to be

mixed with dry bulking agents must be added to provide the structural support to absorbing the

moisture, improving the aeration and the end product quality (Eftoda and Mc-Cartney, 2004;

Vargas-Gracia et al., 2010). Despite the fact that bulking agents represent a large proportion of

the composting mixture, relatively little remains known about the influence of their microbial

and biochemical characteristics although previous study (Yanez et al., 2009; Huet et al., 2012)

have also demonstrated the effectiveness of adding supplementary waste materials (wheat straw,
rice straw, cotton waste, sawdust and chips) as bulking agents, to improve the composting

process. However from long time several earlier researcher (Alburquerque et al., 2006; Karak et

al., 2013a, 2014) various combination of OFMSW with bulking agents for different composting

processes have been examined to determine the most suitable OFMSW and bulking agent

rational and their influence on microbial enzymatic activity during composting. But adding of

bulking waste material combined with raw substrate also advantages to adjusting initial C/N

ratios, the reduction of the NH3 emissions, and control the odorous compounds were mainly

emitted in the mesophilic and thermophilic phases (Liu et al., 2011; Karak et al., 2014).

Therefore, the inoculation of microbial consortium with lignocellulolytic white-rot

fungus is a most active strategy that could potentially enhance rate of release a wide range of

hydrolytic enzymes in particular, cellulases, xylanases, proteases, lipases, and phosphatases.

Other species are involved in microbial consortium also able to break down all components

composting mass (Raut et al., 2008; Zeng et al., 2010; Echeverria et al., 2012), which is

responsible for depolymerize various complex organic waste molecules into simple compounds

(Castaldi et al., 2008; Rashad et al., 2010). Cellulases, Xylanse and dehydrogenase are related

to carbon mineralization, proteases and urease are involved in Nitrogen cycle and phosphatases

and arylsulphatases are implicated in P and S cycles, respectively (Mondini et al., 2004; Zeng et

al., 2010; He et al., 2012).

Although, the variations in the dynamics of enzymatic activity and microbial community

have been reported (Ryckeboer et al., 2003; Vargas-Gracia et al., 2010), and contradictory

results have been described in relation to the total number of microorganisms and its enzymatic

activity at each phase. Nevertheless, most of studies in composting of OFMSW have mainly

focused on physico-chemical parameters to evaluate both process evolution and compost quality.

But characterization and quantification of intracellular and extracellular enzymes during

composting were reflect the real dynamics of composting process in terms of the decomposition

of organic matter, nitrogen transformation and also provide the information about the maturity of

compost (Tiquia, 2002; Vargas-Garcia et al., 2010; Liu et al., 2011).

The present investigation aims to studying the effects of various bulking waste (BW) on

efficiency of inoculated microbial consortium and its enzymatic dynamics to improve the

composting process. It also provided valuable information regarding the physicochemical

evolution of the composting process, most effective BW for rapid composting and finally

production of quality compost from OFMSW.

2. Materials and methods

2.1. Composting procedure and sampling

Three open windrow composting trials were carried out using shredded OFMSW as

including vegetables, food, garden and office waste. The OFMSW used as feed stock was

collected and processed as per the Awasthi et al. (2014). Selected chemical and physical

characteristics of the raw mixture were presented in Table 1. A typical turned windrow

composting was employed for 35 days with identical starting material, shaped and size was

design according to Awasthi et al. (2014). The initial C:N ratio (25:1) and bulk density of ~0.5

kg/L of 5 tone OFMSW composting mass was adjusted by adding the three different bulking

waste in the following proportions (dry weight basis):

Pile 1: OFMSW (75%) + Wood shaving waste (25%)

Pile 2: OFMSW (75%) + Agricultural waste (25%)

Pile 3: OFMSW (75%) + Yard trimming waste (25%)

All piles was inoculated with microbial consortium and mechanically turned weekly using a

loading shovel, because it rapidly enhances the microbial activities through adequate oxygen

supply and maintain uniform degradation for better compost quality. The moisture content of
composting materials was adjusted between 55 to 60% at the beginning of composting, if

needed; water was added on dry weight basis during turning. The pile temperatures (at 125 cm

top from the base of the pile and 85 cm middle from the base of the pile) were monitored daily

using Raytek infrared digital thermometer, respectively. The mean of the pile temperature at the

two monitoring points was reported. Sampling was scheduled on the installation day and after

each turning. Representative compost samples approximately 1 kg from ten different places of

the piles core were drawn at various stages of composting (0, 3, 7, 10, 14, 21, 28 and 35 days).

Then, each collected samples were finely divided into two equal parts, one was preserved at 4ºC,

while the other part was air drying, mixed well and properly grounded (<5 mm). All analysis was

performed in triplicates.

2.2. Microbial inoculum preparation

A pure culture of two lignocellulolytic fungal species Phanerochaete chrysosporium,

Microbial Type Culture Collection and Gene Bank (MTCC), MTCC787 (P. chrysosporium),

Trichoderma viride (T. viride) MTCC793 and one bacterial species Pseudomonas aeruginosa

MTCC2295 (P. aeroginosa) mixed together was used as the inoculum. Selection of the strains

for this study was made on the basis of mutual compatibility for enzymatic activity and substrate

specific OFMSW degradation at laboratory scale experiment (data not shown) and the

performance of consortium was also studied and compared with each individual strains.

To obtain final compost the microbial consortium of above selected fungal strains were

cultivated on potato dextrose agar and spore suspensions was prepared as previous described by

(Awasthi et al., 2014), while bacterial strains grown in Luria broth medium (DifcoTM). The

microbial consortium suspension grown on air dried OFMSW with a particle size of <2 cm3 at

37±2ºC for 1 days to a concentration of 5.7 X 108 colony-forming units (CFU) g-1 (P. aeroginosa)

and at 28±2ºC for 6 days to a 6.8 X 106 CFU g-1 (P. chrysosporium and T. viride). The above
bacterial and fungal strains grown in OFMSW mixed 1:1 ratio and inoculated into the all piles

composting feed stock and turned fully after inoculation to properly spread the microbial

consortium as described by Echeverria et al. (2012).

2.3. Physico-Chemical analyses

The pH and electrical conductivity (EC) were measured in 1:5 water soluble extract ratio on

dry weight basis using digital pH meter (PB-10, Sartorius) and conductivity electrode (LF91,

Wiss. Techn. Werkstatten), while moisture content of samples was determined based on weight

loss at 105oC in an oven and total organic matter after ignition at 550oC in muffle furnace for 24

hrs. Extractable ammonia (NH+4) by the indophenol blue spectrophotometric method, total

organic carbon (TOC), total Kjeldhal nitrogen (TKN), total phosphorus (TP) was determined

following the methods of (TMECC, 2002). The cress seed germination test was performed to

assess the compost maturity as procedure describe by (Zucconi et al., 1981).

2.4. Microbial enzyme activities

The selected enzymatic activities were analyzed using aqueous extracts of fresh compost

samples in relation to amylase, cellulase, protease, phosphatase, dehydrogenase and xylanase

activity is expressed in as mg product g-1 h-1 on a dry weight basis. The protease activity was

evaluated by determining the amount of tyrosine released after incubation of the aqueous

compost extracts with casein for 1 h at 37ºC and the subsequent reaction with Folin Ciocalteau

reagent according to the method of Ladd and Butler (1972). The same aqueous compost extracts

were used for the analysis of amylase and cellulases activity, which is quantified from the

reducing sugars generated from incubation of compost extracts with 0.1M acetate buffer (pH 6.5)

for 1 h at 37ºC and the subsequent reaction with the addition of 3, 5-dinitrosalicylic acid (DNSA)

solution as per the standard method (Miller, 1959). The estimation of phosphatase activity was

carried out by the hydrolysis by p-nitrophenol phosphate (PNPP) as suggested by Alef et al.
(1995). Dehydrogenase activity (DA) was determined according to the standard method of

Barrena et al. (2008) by detecting triphenyl formazan in a triphenyltetrazolium chloride (TTC)

solution treated with aqueous compost extracts and the values were expresses as mg TPF g-1 dw

h-1. The xylanase activity was estimated according to the method of Schinner and Von-Mersi

(1990) using aqueous extracts compost and the enzyme activity was expressed as mg sugar g-1

dw-1 h-1 under the assay conditions.

2.5 Statistical analysis

All the physico-chemical properties of samples were carried out in triplicate and the

mean values with standard deviation are presented. One-way analysis of variance (ANOVA) and

multiple comparison tests were performed with SPSS 13.0 (SPSS for Windows, Version 13.0,

USA) to compare the mean values for the different levels of sampling time (P < 0.05) during

composting.

3. Result and discussion

3.1. Changes of temperature and pH

The temperature is one of the most ominous factors used to monitoring the progress of

composting process transformation. Changes in temperature could be used to know the microbial

activity along the entire process and to determine the organic material stability (Goyal et al.,

2005; Liu et al., 2011). The thermophilic phase (>60ºC) was reached within one day and lasted

for about three weeks in pile 1 and two weeks in pile 3, while in pile 2 started from day 5 for

very short duration (Fig. 1a). The temperature pattern was not similar for all piles with respective

of bulking agent and microbial efficiency. The temperature of pile 1 and pile 3 sharply increased

up to 60ºC from the beginning of the process and started decreasing from about 53ºC after 23

day and the peak temperature of pile 1 was obtained at 4 day and pile 3 on day 8, except in pile 2

in which the temperature increased gradually and reached its peak on day 14. The temperature
within the pile influences by the microbial activity and aeration, since it directly affects rate of

organic matter degradation, whether determining a mature and stable product (Cayuela et al.,

2008; Zeng et al., 2010). The results indicated that there was microbial enzymatic activity in pile

1 and 3, where OFMSW was combined with WSW and YTW bulking waste was higher.

Influences of bulking waste and microbial consortium for OFMSW degradation and pH

during composting process are shown in Fig 1b. The initial pH of pile 1 and 3 was rapidly

decreased during the first week of composting was observed, while in pile 2 it decreased until

day 21 and thereafter slightly increased (Fig 1b). In the first weeks pH decrease in pile 1 and 3

might be due to the intense microbial activity and organic matter degradation led to the

formation of organic acid and afterwards increased pH in pile 1 and 3 followed was attributed to

the production of ammonia associated with protein degradation in the samples and to the

decomposition of organic acids. At the end of maturation, pH values of 7.38 and 7.19 were

observed for pile 1 and 2 respectively. Previous studies also reported that pH dropped below 6

during the initial phase of composting because of organic acids produced from the breakdown of

sugars and fats by the microbes (Goyal et al., 2005; Liu et al., 2011; Awasthi et al., 2014).

Among all three bulking agent mixed with OFMSW, WSW and YTW from pile 1 and 3 were

greater degradation rates of organic matter, while pile 2 mixed with AW were lower than pile 1

and 3, which was also indicated with its lowest temperature profile as shown in Fig 1a. The

result suggests that the microbial activity in pile 1 and 3 was the highest comparatively the pile 2,

might be the organic matter degradation in pile 2 was inhibited due to its insufficient free air

space between OFMSW.

3.2. Changes of TOC, extractable ammonia and TKN

Total organic carbon (TOC) gradually decreased with the time and reached its lowest

values at the end of the process in pile 1 (33.56%), pile 2 (38.28%) and pile 3 (36.47%), because
of the net loss of dry mass and organic carbon as CO2 during composting. At the end of the

process, the lowest TOC was observed in pile 1, while highest in pile 2. TOC loss has been

reported as an important parameter that may serve as an indirect indicator of the degree of

compost maturity (Vargas-Gracia et al., 2010; Karak et al., 2014). These results (Fig. 2b)

suggested that inoculated microbial consortium feed organic carbon for their metabolic

processes, due to this region heat, CO2, and water vapor are produced during this process. The

high TOC mainly came from the slow degradation of organic substrate by the inoculated

microbial consortium in pile 2 compared to other two piles.

The maximum concentration of ammoniacal-N was generated during the thermophilic

phases of composting (Fig. 2a and Table 2), when the organic matter degradation was most

intense and ammonium was produced through the mineralization of organic nitrogen. In this

experiment, the NH+4 levels were relatively high in pile 1 and 3 at the beginning of the

composting as a result of the digestate which was rich in ammonia/ammonium, and decreased

after day 10 in pile 1 and from day 21 in pile 3, due to the conversion of NH+4 into NH3 and

subsequently by its volatilization under high pH and temperature condition. However at the end

of the composting process, the NH+4 in piles 1 and 3 under the permissible limit of compost

application, while concentration of pile 2 increased up to day 21 and then slightly decrease;

however, the concentrations were significantly higher at the end of composting, mainly due to

the late organic decomposition.

Changes in TKN content of all piles is presented in Fig. 2c. From start of the composting

process TKN content gradually decreases in all piles, but after day 10 significantly increase in

pile 1 and 3, except in pile 2 where there was no obvious change over the time. The initial

decline in TKN was perhaps due to the volatilization of ammonia, which in turn, depended upon

the type of materials used for composting (Goyal et al., 2005; Liu et al., 2011). Later stages of
the composting process TKN content gradually increased due to the bacterial nitrogen fixation

(Vargas-García et al., 2010; Raut et al., 2008). The TKN values decreased in pile 1 from (1.84%)

to (1.18%), pile 2 from (1.81%) to (1.32%) and pile 3 from (1.92%) to (1.52%) respectively. The

result indicated that maximum TKN content was observed in pile 1 (1.98%) at the end of

composting process, while in pile 3 (1.75%) and pile 2 (1.48%) slightly lower.

3.3. Microbial enzymatic activities and compost maturity properties

Most of the modifications that organic matter undergoes during composting are mediated

by enzymes. Various hydrolytic enzymes are thought to control the degradation rates of different

substrates, and they are the main mediators of various degradation processes (Tiquia et al., 2002;

Liu et al., 2011). Thus, the monitoring of enzymatic activities throughout the process gives

valuable information related to the dynamics of important nutritional elements like C, N or P and

contributes to a better understanding of the transformations that take place during composting

(Vargas-García et al., 2010; Raut et al., 2008). Thus, variation of concentrations of some key

enzymes like amylase, cellulase, protease, phosphatase, dehydrogenase and xylanase were

studied to understand how inoculated microbial consortium combine with bulking waste

degraded various organic wastes.

3.3.1. Changes of amylase and cellulase activity

Amylase played a key role for the hydrolysis of various starchy substrates of alpha-1, 4-

glycosidic linkages of polysaccharides to yield dextrins, oligosaccharides, maltose and D-

glucose. The amylase enzymes activity gradually increase as shown in (Fig. 3a) with the

composting process in all piles and maximum amylase activity was detected in pile 1 (3.19 mg

glucose g-1 dw h-1) and 3 (2.16 mg glucose g-1 dw h-1) at day 14, while in pile 2 (1.15 mg glucose

g-1 dw h-1) at day 21. The results of the present investigation indicated that a maximum

degradation of starch substrates happened first 14 to 21 days, respectively in pile 1 and 3, while
pile 2 found very low amylase activity through composting. High content of degradable organic

compounds in the initial mixture may have stimulated microbial growth and enzyme synthesis in

early stage of composting, as available substrate decreased, the enzyme activity decreased as

well (Cunha-Queda et al., 2007; Liu et al., 2011). The production of enzyme depends on

microbial biomass, which implies that OFMSW is utilized by microbes degraded by enzymatic

activity decreases (López et al., 2007; Echeverria et al., 2012). The results further indicated that

the activity of amylase was high in the pile 1 and 3 where WSW and YTW was added as bulking

waste, while in pile 2 showed very low amylase activity. Mixing of WSW and YTW could have

favored the microbial growth by providing carbon source and good aeration during the early

phase of composting. There some earlier researcher has been reported that bulking waste to

stimulate the enzymatic activity during the composting (Liu et al., 2011; Karak et al., 2013).

The perusal of the results showed that the cellulase activity had a similar trend like

amylase, showing gradual increase in its activity at the beginning of composting in all piles (Fig.

3b). The cellulase activities in pile 1 (1.68 mg glucose g-1 dw h-1) and 3 (2.56 mg glucose g-1 dw

h-1) reached the highest value on day 10 and 14, while the activities in pile 2 (1.16 mg glucose g-1

dw h-1) mixed with agricultural waste as bulking waste reached the peak on day 21, and the

activity were lower than those in pile 1 and 3. A similar trend was also observed by Raut et al.

(2008). In the present investigation the highest enzyme activity in pile 1 and 3 were might be due

to the rapid degradation of cellulolytic waste by inoculated microbial consortium combine with

WSW and YTW as bulking waste. The cellulase activities were not much different among the

four runs during the second fermentation phase. This result suggests that mixing of different

bulking waste responsible for the change of cellulase activities in each pile, while the similar

consortium was inoculated in all piles. Previous studies (Goyal et al., 2005; Gabhane et al., 2012)

also found that bulking agent availability could increase growth of microbial biomass and
stimulate the secreting of cellulase enzymes, so the reason for the later decrease in cellulase

activities might be the reduction in C/N ratio in the later stage of composting.

3.3.2. Changes of protease and phosphatase activity

Proteases activity is appropriate indicators of decomposition of organic matter, are

important enzymes during the composting process. From the beginning of composting,

maximum protease activity was recorded at 7th day in pile 1 (6.85 mg Tyr g-1 dw h-1) followed by

10th day in pile 3 (6.73 mg Tyr g-1 dw h-1), pile 2 (5.10 mg Tyr g-1 dw h-1) on day 28 and then

gradually started decreasing until the end of the composting. Similar trend of protease activity

was reported by Liu et al. (2011) when composting horse and poultry manures with wheat straw

and Raut et al. (2008) for the composting of OFMSW mixed with some additives. Protease

activity in the different piles closely related to the nitrogen cycle and breakdown of complex

nitrogen compounds to ammonia or more simple short chain poly peptide fractions, which

occurred during the active phase of composting (Cunha-Queda et al., 2007). Castaldi et al.

(2008) observed that protease activity was strongly correlated with the water-soluble nitrogenous

forms and generation of ammonia.

Moreover, in case of pile 1 and 3 were relatively higher protease activity observed than

pile 2 until the end of composting. The result indicated that WSW and YTW had more actively

enhanced the protease activity in pile 1 and 3, except pile 2 compost after 35 days was not

mature and further evidenced by the obnoxious smell emitted from the pile 2 till the last day of

the experiment due to extractable ammonia content more than permissible limit.

Phosphatase activity is involved in the hydrolysis of organic phosphorus compounds to

different inorganic forms which plants can metabolize. This enzymatic activity plays critical

roles in P cycles and it is considered a general microbial indicator, although some phosphatases

are synthesized not only by microorganisms but also by plants (Raut et al., 2008). As shown in
(Fig.4b), the maximum activity was observed at the maturation period of composting, which

might be due to the abundance of organic phosphorus compounds that characterize these

biosolids (Goyal et al., 2005), derived from detergents, dish-washing powders and even urine

and faeces (Mondini et al., 2004). Among all three piles, the maximum phosphates activity was

observed in pile 1 (4.38 mg PNP g-1 dw h-1) followed by pile 3 (4.13 mg PNP g-1 dw h-1) and 2

(1.74 mg PNP g-1 dw h-1) at the end of composting. Similar results have been reported by

Pramanik et al. (2007), who described minimal phosphatase activity in a MSW vermin-compost

in relation to final products obtained from three different types of wastes. Phosphatase activity

seems to be influenced by the microorganisms, since their activity modifies P availability and

even some diazotrophic bacteria are recognized as phosphatase producers (Raut et al., 2008).

3.3.3. Changes of dehydrogenase and xylanase activity

Dehydrogenase activity (DA) is generally considered a reliable index of overall microbial

activity and its metabolic reactions for organic waste degradation because it is directly involved

in the respiratory chain (Castaldi et al., 2008). During the thermophilic phases maximum DA

was observed and then gradually decreased towards the end of the composting which is

indicating that there was insignificant active degradation. The highest DA was obtained in pile 1

(28.35 mg TPF g-1 dw h-1) and pile 3 (26.04 mg TPF g-1 dw h-1) on 14 days, while pile 2 (18.29

mg TPF g-1 dw h-1) at day 28 (Fig. 5a). However, the change in DA can be used to monitor the

state of composting and to provide information of compost maturity (Liu et al., 2011; Castaldi et

al., 2008). The results of this study indicated that the addition of WSW and YTW with OFMSW

had direct correlation between DA and efficacy of inoculated microbial consortium, might be

due to proper aeration would help to increase the DA. These results confirmed the studies of

several earlier researchers who found a positive correlation between DA and temperature

(Barrena et al., 2008; Gabhane et al., 2012).

 Xylanase like cellulase involved in the hydrolysis of hemicelluloses to xylose and

gloucose, which is one of the major components of plant cell wall and OFMSW (Goyal et al.,

2005). The highest xylanase activity was obtained within 14 days in pile 1 (8.50 mg reducing

sugar g-1 dw h-1), and pile 3 (7.42 mg reducing sugar g-1 dw h-1), while pile 2 (4.80 mg reducing

sugar g-1 dw h-1) showed the peak activity on day 28. Hemicellulose is the main substrate for the

secretion of xylanase, therefore the increased activity of xylanase was consistent with the

degradation of hemicellulose as reported by Liu et al. (2011). The results indicate during the

whole thermophilic phases, xylanase activity was higher in pile 1 and 3 which is combined with

WTS and YTW, while pile 2 with AW showed relatively low activity, might be due to the

presence of more air space and carbon compounds derived from the cellulolytic and

hemicellulolytic activities, that benefited for the growth of xylanase-producing microorganisms

(Raut et al., 2008). Similar observation were also obtained by Castaldi et al. (2008) who

observed on xylanase activity throughout the composting of OFMSW with plant wastes, Liu et

al. (2011) on dairy manure with rice chaff and Zeng et al. (2010) during agricultural waste

composting.

3.3.4. Changes of C/N ratio

C:N ratio precisely correlates with the maturity of end product (Liu et al., 2011). In this

experiment C:N ratio was slightly increased during thermophilic phase due to the loss of nitrogen

as ammonia and comparatively reduced decomposition and then gradually decreased until the

end of composting process in all piles (Fig. 6a and Table 2). In the pile 1 and 3 up to day 10 C/N

ratio slightly increase, while in pile 2 on day 21 increased from the beginning and then decreased

until the end of composting process, respectively. The result of several earlier reports also

suggested that as the composting started, due to the loss of carbon dioxide, the carbon content of
the raw material decreased and total nitrogen content increased, then as conclusion C/N ratio

decrease (Rashad et al., 2010; Karak et al., 2013a).

3.3.5. Seed germination index

Germination index (GI) is one of the important indicator to evaluate the compost maturity

(Zucconi et al., 1981; CCME, 2005). The GI values was slightly decreased on day 3 and then

gradually increased until the end of composting process in all piles (Fig. 6b and Table 2). Several

researchers already prove that a compost is well mature compost if the GI value is >90% (Liu et

al., 2011). In pile 1 GI was 94.60% after 28 days and pile 3 93.50% after 35 days, while pile 2

was showed the lowest GI value 68.46% at the end of composting respectively. The final GI

result was confirmed that the pile 1 and pile 3 compost was free of phytotoxins, which also

indicated that the compost will not have a toxic effect on plant growth. In previous studies,

highest GI was obtained after a longer period of composting, such as after 84 days (Rashad et al.,

2010) and 70 days (Karak et al., 2014).

3. Conclusion

The novelty of this work is that we explored the influence of bulking waste combine with

OFMSW as useful tool to accelerate the microbial enzymatic activity during composting. From

this study, major differences is found between pile 1 and 2 in relation to physicochemical and

microbial enzyme activities, while maturity parameters showed that pile 1 rate of organic matter

degradation is very fast and end product is well mature within 28 days. However, OFMSW

combined with bulking waste WSW and YTW seems to be promising cost effective approach to

provide valuable information of enzymatic activity and compost maturity.

Acknowledgements

The author gratefully acknowledges to Ministry of Environment and Forests, New Delhi

for financial support. Our special thanks go to Madhya Pradesh Pollution Control Board Bhopal
and Mycological Research Laboratory, Department of Biological Sciences, Rani Durgavati

University, Jabalpur for technical and laboratory support. Also thanks to Municipal Corporation

of Jabalpur for continuously provide the segregated solid waste.

References

1. Adam, J.D.M., Frostick, L.E., 2009. Analysis of bacterial activity, biomass and diversity

during windrow composting. Waste Manage. (Oxford) 29, 598-605.

2. Alburquerque, J.A., Gonzalvez, J., García, D., Cegarra, J., 2006. Effect of bulking agent on

the composting of ‘‘alperujo’’, the solid by-product of the two-phase centrifugation method

for olive oil extraction. Process Biochem. 41, 127-132.

3. Alef, K., Nannipieri, P., Trasar-Cepeda, C., 1995. Phosphatase activity. In: Alef, K.,

Nannipieri, P. (Eds.), Methods in Applied Soil Microbiology and Biochemistry. Academic

Press, London, pp. 335-344.

4. Awasthi, M.K., Pandey, A.K., Khan, J., Bundela, P.S., Wong, J.W.C., Selvam, A., 2014.

Evaluation of thermophilic fungal consortium for organic municipal solid waste composting.

Bioresour. Technol. 168, 214-221.

5. Barrena, R., Vázquez, F., Sánchez, A., 2008. Dehydrogenase activity as a method for

monitoring the composting process. Bioresour. Technol. 99, 905-908.

6. Castaldi, P., Garau, G., Melis, P., 2008. Maturity assessment of compost from municipal solid

waste through the study of enzyme activities and water soluble fractions. Waste Manage. 28,

534-540.

7. Cayuela, M.L., Mondini, C., Sánchez-Monedero, M.A., Roig, A., 2008. Chemical properties

and hydrolytic enzyme activities for the characterization of two phase olive mill wastes

composting. Bioresour. Technol. 99, 4255-4262.

8. CCME, 2005. Canadian Council of the Ministers of the Environment, Guidelines for Compost

Quality, Ministry of Public Works and Government Services Canada. Cat. No. PN1341.

9.Cunha-Queda, A.C., Ribeiro, H.M., Ramos, A., Cabral, F., 2007. Study of biochemical and

microbiological parameters during composting of pine and eucalyptus bark. Bioresour.

Technol. 98, 3213-3220.

10. Echeverria, M.C., Cardelli, A., Bedini, S., Colombini, A., Incrocci, I., Castagna, A.,

Agnolucci, M., Cristani, C., Ranieri, A., Saviozzi, A., Nuti, M., 2012. Microbial enhanced

composting of wet olive husks. Bioresour. Technol. 104, 509-517.

11. Eftoda, G., Mc-Cartney, D., 2004. Determining the critical bulking agent requirement for

municipal biosolids composting. Compost Sci. Util. 12, 208-218.

12. FAI, 2007. The Fertilizer (Control) Order 1985. The Fertilizer Association of India 10.

13. Gabhane, J., Prince William, S.P.M., Bidyadhar, R., Bhilawe, P., Anand, D., Vaidya, A.N.,

Wate, S.R., 2012. Additives aided composting of green waste: Effects on organic matter

degradation, compost maturity and quality of the finished compost. Bioresour. Technol. 114,

382-388.

14. Goyal, S., Dhull, S.K., Kapoor, K.K., 2005. Chemical and biological changes during

composting of different organic wastes and assessment of compost maturity. Bioresour.

Technol. 96, 1584-1591.

15. He, Y., Xie, K., Xu, P., Gu, W., Zhang, F., Tang, S., 2012. Evolution of microbial

community diversity and enzymatic activity during composting. Res. Microbiol. 164, 189-

198.

16. Huet, J., Druilhe, C., Trémier, A., Benoist, J.C., Debenest, G., 2012. The impact of

compaction, moisture content, particle size and type of bulking agent on initial physical
 properties of sludge-bulking agent mixtures before composting. Bioresour. Technol. 114, 428-

436.

17. Karak, T., Bhattacharyya, P., Paul, R.K., Das, T., Saha, S.K., 2013a. Evaluation of composts

from agricultural wastes with fish pond sediment as bulking agent to improve compost

quality. Clean-Soil Air Water, 41, 711-723.

18. Karak, T., Sonar I., Paul, R.K., Das, S., Boruah, R.K., Dutta, A.K., Das, D.K., 2014.

Composting of cow dung and crop residues using termite mounds as bulking agent. Bioresour.

Technol. 169, 731-741.

19. Ladd, J.N., Butler, J.H.A., 1972. Short-term assays of soil proteolytic enzyme activities using

proteins and dipeptide derivatives as substrates. Soil Biol. Biochem. 4, 19-30.

20. Liu, D., Zhang, R., Wu, H., Xu, D., Tang, Z., Yu, G., Xu, Z., Shen, Q., 2011. Changes in

biochemical and microbiological parameters during the period of rapid composting of dairy

manure with rice chaff. Bioresour. Technol. 102, 9040-9049.

21. López, M.J., Vargas-García, M.C., Suárez-Estrella, F.F., Nancy, N.N., Bruce, S.D., Joaquín,

M., 2007. Lignocellulose-degrading enzymes produced by the ascomycete Coniochaeta

ligniaria and related species application for a lignocellulosic substrate treatment. Enzyme

Microbial. Technol. 40, 794-800.

22. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination if sugars. J. Anal.

Chem. 31, 426-428.

23. Mondini, C., Fornasier, F., Sinicco, T., 2004. Enzymatic activity as a parameter for the

characterization of the composting process. Soil Biol. Biochem. 36, 1587-1594.

24. Pramanik, P., Ghosh, G.K., Ghosal, P.K., Banik, P., 2007. Changes in organic-C, N, P and K

and enzyme activities in vermin-compost of biodegradable organic wastes under liming and

microbial inoculants. Bioresour. Technol. 98, 2485-2494.

25. Rashad, F.M., Saleh, W.D., Moselhy, M.A., 2010. Bioconversion of rice straw and certain

agro-industrial wastes to amendments for organic farming systems: 1. Composting, quality,

stability and maturity indices. Bioresour. Technol. 101, 5952-5960.

26. Raut, M.P., Prince William, S.P.M., Bhattacharyya, J.K., Chakrabarti, T., Devotta, S., 2008.

Microbial dynamics and enzyme activities during rapid composting of municipal solid waste -

a compost maturity analysis perspective. Bioresour. Technol. 99, 6512-6519.

27. Ryckeboer, J., Mergaert, J., Vaes, K., Klammer, S., De-Clerq, D., Coosemans, J., Insam, H.,

Swings, J., 2003. A survey of bacteria and fungi occurring during composting and self-heating

processes. Ann. Microbiol. 53, 349-410.

28. Schinner, F., Von-Mersi, W., 1990. Xylanase, CM-cellulase and invertase activity in soil: an

improved method. Soil Biol. Biochem. 22, 511-515.

29. Tiquia, S.M., 2002. Evolution of extracellular enzyme activities during manure composting.

Waste Manage. (Oxford) 92, 764-775.

30. Tiquia, S.M., Wan, J.H.C., Tam, N.F.Y., 2002. Microbial population dynamics and enzyme

activities during composting. Compost Sci. Utili. 10, 156-161.

31. TMECC, 2002. Test Methods for the Examination of Composts and Composting. In:

Thompson, W., Leege, P., Millner, P., Watson, M.E. (Eds.), The US Composting Council, US

Government Printing Office.

32. Vargas-Gracia, M.C., Surez-Estrella, F.F., Lopez, M.J., Moreno, J., 2010. Microbial

population dynamics and enzyme activities in composting processes with different starting

materials. Waste Manage. (Oxford) 30, 771-778.

33. Yañez R., Alonso, J.L., Díaz, M.J., 2009. Influence of bulking agent on sewage sludge

composting process. Bioresour. Technol. 100, 5827-5833.

34. Zeng, G., Yu, M., Chen, Y., Huang, D., Zhang, J., Huang, H., Jiang, R., Yu, Z., 2010. Effect

of inoculation with Phanerochaete chrysosporium at various time points on enzyme activities

during agricultural waste composting. Bioresour. Technol. 101, 222-227.

35. Zucconi, F., Forte, M., Monac, A., Bertodi, M., 1981. Biological evolution of compost

maturity. Biocycle. 22, 27-29.

Fig. 1. Changes of temperature (a) and pH (b) of different piles during composting process. Pile 1-

OFMSW with wood shaving waste, Pile 2- OFMSW with agricultural waste and Pile 3-

OFMSW with yard trimming waste and all piles inoculated with microbial consortium; Results

are the mean of three replicates and error bars indicates standard deviation.

Fig. 2. Changes of extractable ammonium (a), total organic carbon (b) and total Kjeldahl nitrogen (c)

of different piles during composting process. Pile 1- OFMSW with wood shaving waste, Pile 2 -

OFMSW with agricultural waste and Pile 3- OFMSW with yard trimming waste and all piles

inoculated with microbial consortium; Results are the mean of three replicates and bars indicate

standard deviation.

Fig. 3. Evolution of amylase (a) and cellulase (b) activities of different piles during composting

process. Pile1- OFMSW with wood shaving waste, Pile 2 - OFMSW with agricultural waste and

Pile3 - OFMSW with yard trimming waste and all piles inoculated with microbial consortium;

Results are the mean of three replicates and bars indicate standard deviation of three replicates.

Fig. 4. Evolution of protease (a) and phosphates (b) activities of different piles during composting

process. Pile 1- OFMSW with wood shaving waste, Pile 2- OFMSW with agricultural waste and

Pile 3- OFMSW with yard trimming waste and all piles inoculated with microbial consortium;

Results are the mean of three replicates and bars indicate standard deviation of three replicates.

Fig. 5. Evolution of dehydrogenase (a) and xylanase (b) activities of different piles during composting

process. Pile 1- OFMSW with wood shaving waste, Pile 2- OFMSW with agricultural waste and

Pile 3- OFMSW with yard trimming waste and all piles inoculated with microbial consortium;

Results are the mean of three replicates and bars indicate standard deviation of three replicates.

Fig. 6. Changes of C/N ratio (a) and germination index (b) of different piles during composting

process. Pile 1- OFMSW with wood shaving waste, Pile 2 - OFMSW with agricultural waste
and Pile 3- OFMSW with yard trimming waste and all piles inoculated with microbial

consortium; Results are the mean of three replicates and bars indicate standard deviation.
 80
A
Pile 1
70 Pile 2
Pile 3
60
Temperature (°C)
50

40

30

20

10
0 10 20 30 40
9
B
B
8

7
pH

3
0 10 20 30 40
Time (days)

Figure 1
 6000
A Pile 1
5000 Pile 2
Pile 3

NH4+-N (mg/kg)
4000

3000

2000

1000

0
0 10 20 30 40
55 B

50
Total organic carbon (%)

45

40

35

30

25
0 10 20 30 40
2.2 CC

2.0
Total Kjeldahl nitrogen (%)

1.8

1.6

1.4

1.2

1.0
0 10 20 30 40
Time (days)

Figure 2
 10
A Pile 1
Pile 2
8

mg glucose g -1 dw h -1
Pile 3

0
0 10 20 30 40
5 B
B
4
mg glucose g -1 dw h -1

0
0 10 20 30 40
Time (days)

Figure 3
 8 Pile 1
A
Pile 2
Pile 3
6
mg Tyr g -1 dw h -1

0
0 10 20 30 40
5
B

4
-1
mg PNP g dw h

3
-1

0
0 10 20 30 40
Time (days)

Figure 4
 35
A Pile 1
30 Pile 2
Pile 3
mg TPF g -1 dw h -1 25

20

15

10

0
0 10 20 30 40
10
B
mg reducing sugar g -1 dw h -1

0
0 10 20 30 40
Time (days)

Figure 5
 40
A

30
C/N ratio

20

10 Pile 1
Pile 2
Pile 3
0
0 10 20 30 40
120
B

100
Germination index (%)

80

60

40

20

0
0 10 20 30 40
Time (days)

Figure 6
 Table 1 Selected physical and chemical properties of the raw materials used for the composting
feedstock’s.

Parameter Bulking waste

OFMSW
WSW AW YTW
Moisture content (%) 56.38±0.31 10.2±0.17 8.74±0.23 14.35±0.10
pH 7.27±0.16 6.54±0.12 7.21±0.15 7.39±0.19
Electrical conductivity (mS cm-1) 2.06±0.08 0.85±0.04 0.86±0.07 0.74±0.05
Total organic matter (% dw) 85.43± 4.37 94.15±3.18 95.24±5.02 93.14±4.21
Total organic carbon (% dw) 40.29±2.43 45.18±3.04 44.36±2.19 40.72±2.83
Total Kjeldahl nitrogen (% dw) 2.65±0.13 0.52±0.02 0.76±0.05 0.89±0.07
C:N ratio (dw) 15.20± 1.26 86.88±2.32 58.36±4.06 45.75±3.28
Results are the mean of three replicates ± standard deviation. *OFMSW- Organic fraction of
municipal solid waste, WSW- Wood shaving waste, AW- Agricultural waste, YTW- Yard
trimming waste.
 Table 2 Maturity properties of compost at the end of composting.

Parameters Pile 1 Pile 2 Pile 3 FAIa TMECCb/CCMEc

Moisture content (%) 37± 1.32 38± 2.92 43± 2.03 35-55 35-45
pH 7.38± 0.04 5.94± 0.10 7.19± 0.02 6.5-8.5 5.5-8.5
Electrical conductivity (mS cm-1) 2.64± 0.05 3.42± 0.04 2.51± 0.03 2-6 ≥4
Ammonium (mg/kg dw) 348.83 ± 23.19 2513.02 ± 47.80 533.35 ± 12.34 -- 75–500
Total organic carbon (% dw) 33.56± 1.92 38.28± 2.04 36.47± 1.45 ≥16 --
Total organic matter (% dw) 58.52± 1.64 83.95± 1.38 64.86± 2.52 >30 ≤ 40
Total phosphorus (% dw) 0.93± 0.03 0.84± 0.05 0.78± 0.04 0.4-1.1 --
Total Kjeldahl nitrogen (% dw) 1.98± 0.05 1.48± 0.07 1.75± 0.12 1.0-3.0 --
C:N ratio (dw) 16.94±1.28 25.86±1.04 20.84±1.13 < 25 ≤ 25
Seed germination index (%) 105.90±5.78 68.46±4.18 93.50±4.32 -- 80-90
a
FAI (2007): Indian Fertilizer Control Order, 1985, bTMECC (2002): Test Methods for the Examination of Composts and
Composting, cCCME: Canadian Council of the Ministers of the Environment (2005), *Results are the mean of three
replicates ± standard deviation.
Highlights:

1. The bulking wastes enhance the enzymatic activity and rate of composting.

2. Addition of WSW as bulking waste significantly improved compost quality.

3. Inoculation of microbial consortium with bulking agent reduces the composting period.

4. All three microbial strains are compatible and form a stable consortium.