Local Feed Workshop, Oceanic Institute of Hawaii Pacific University, Nov 21, 2014, Honolulu, HI

Utilization of Local Agri-processing By-products to

Produce Fungal Protein for Aquatic Feed
Production

Samir Kumar Khanal, Ph.D.

Associate Professor of Biological Engineering

Department of Molecular Biosciences and Bioengineering

University of Hawai’i at Mānoa

Nov 21, 2014

Aquaculture Production

7.1%

Development of world capture fisheries and aquaculture

production
Direction of global production of major nutrient sources

NUTRIENT SOURCES FROM FISHING ACTIVITIES

AQUATIC ANIMAL
PROTEIN MEALS
& LIPIDS
OILSEED LAND
MEALS, PULSES ANIMAL
PROTEIN
& OILS
40 MEALS &
essential FATS
nutrients

CEREALS
INCLUDING MICROBIAL
BY-PRODUCT FEED
MEALS INGREDIENTS
& OILS

NUTRIENT SOURCES FROM AGRICULTURAL FARMING ACTIVITIES

Source: Albert G.J. Tacon
 MICROBIAL
INGREDIENT
SOURCES

Microbial-derived feed ingredient sources/insect larvae include the

use of mass produced harvested/extracted:
- Insect-larvae: BSF larvae, etc..
- Algae: Chlorella/ Spirulina/ Crypthecodium/ Nannochloropsis spp.
- Yeasts: Saccharomyces/ Candida/ Rhodotorula spp. etc
- Bacteria: Pseudomonas/ Methylophilus/ Methylococcus/ Bacillus
spp..
- Mixed SCP: activated sludges, flocs
- Filamentous fungi: Aspergillus spp., Rhizopus etc. Source: Albert G.J. Tacon
 Agri-processing By-products
 Biofuel- industries by-products
 Stillage
 Vinasse
 Glycerine
 Algal cake
 Cassava leaves
 Food processing industries
 Solid residues
 High strength wastewater
 Hawaii
 Molasses, Coffee processing wastewater,
Taro waste, Damaged papaya Glycerine,
What is fungi

200 µm
 Why Fungi-Rhizopus microsporus var. oligosporus?

 Food-grade filamentous fungal species belongs to the

phylum Zygomycota
 Not associated with the production of harmful
products/metabolites (Jennessen et al., 2008)
 GRAS status (Generally-recognized as safe)
 Known for making Tempeh
(an Indonesian delicacy)

Source: Jennessen, J., Schürer, J., Olsson, J., Samson, R. A., and Dijksterhuis, J. 2008. Morphological characteristics of sporangiospores of the tempe fungus
Rhizopus oligosporus differentiate it from other taxa of the R. microsporus group. Mycological Research. 112, 547–563.
Fungal fermentation– biofuel residues

Dewatered product

Filtered water

~45% protein

2d growth Settling Dried product

 Vinasse generation
Parameters Vinasse

pH 4.25 ± 0.32

Total solids (TS) (g/L) 22.30 ± 2.80

Volatile solids (VS) (g/L) 14.60 ± 0.60

Total suspended solids (TSS) (g/L) 7.60 ± 0.50

Volatile suspended solids (VSS) (g/L) 4.60 ± 0.40

Soluble chemical oxygen demand

55.55 ± 4.15
(SCOD) (g/L)

Total chemical oxygen demand (TCOD)

64.48 ± 2.31
(g/L)

Total Kjeldahl nitrogen (TKN) (g/L) 0.37 ± 0.08

Around 8-15 L vinasse per liter of ethanol is produced

Vinasse disposal: Fertirrigation
Demerits of land application

Risk of soil salinization

Nutrient leaching to surface
water and groundwater
Odor nuisance
Fungal fermentation on vinasse

Feed and food production

Fungal cultivation in airlift bioreactor

 pH:5.0
4 5  Temperature: 37oC
1
NaOH
23
 Nutrient supplementation:
H2SO4
COD:N:P = 100:5:1
Aeration rates:
Foam trap

pH 5.0

pH controller
0.5, 1.0, 1.5, and 2.0
volumeair/volumeliq/min

1. Sampling port
2. Acid inlet
3. Base inlet
4. pH probe
5. Temperature probe

Air pump

Air filter
Rotameter
 Fungal biomass yield
c
9

7
b
6
(gbiomass increase/ginitial biomass)

b
Fungal biomass yield

5
a
4

0
0.5 1.0 1.5 2.0

Aeration rate (vvm)

 Fungal morphology
16 h 24 h 48 h 72 h

Fungal cultivation
at 1.5 vvm

Fungal cultivation
at 2.0 vvm (72 h)
 Airlift vs. bubble column

Airlift reactor Bubble column reactor

Air pump Air pump

Air filter Air filter

Rotameter Rotameter
Fungal biomass yield: airlift vs. bubble column

Airlift reactor Bubble column reactor

12.0 b
(gbiomass increase/ginitial biomass)

10.0 a
Fungal biomass yield

b
8.0
b
a a a
6.0
a
4.0

2.0

0.0
0.5 1.0 1.5 2.0
Aeration rate (vvm)
 Fungal cultivation for 72 h

1.5 vvm 2.0 vvm

Fungal morphology (bubble column reactor)

Fungal cultivation on commercially-produced vinasse

• Sugarcane-ethanol derived vinasse obtained from a commercial cane-

ethanol facility in Brazil (Brazil vinasse) without dilution

a
~ 60% SCOD reduction
(gbiomass increase/ginitial biomass)

a
Fungal biomass yield

With nutrients supplementation

Fungal biomass characterization

Compositions Fungal Fishmeal Soybean

(on dry weight basis) biomass meal

Crude protein 49.57 73.33 48.10

Crude lipid 5.72 10.51 1.43

Ash 3.46 17.23 6.21

 84% in vitro protein digestibility

 comparable to soybean meal and fishmeal
with overall digestibility of about 85%
 Amino acid profiles of fungal biomass
Percentage of essential amino acids

12
Fishmeal Soybean meal Vinasse-derived fungal biomass
(based on % protein)

10

Essential fatty acids Values (% dry weight)

Linoleic acid 1.35

Linolenic acid 0.32

Eicosapentaenoic acid (EPA) 0.12

Docosahexaenoic acid (DHA) 0.11

Soluble chemical oxygen demand (SCOD) reduction
0.5 vvm 1.0 vvm
40 40
35 35
30 30
25 25

SCOD (g/L)
SCOD (g/L)

20 20
15 15
10 10
5 5
0 0
0 1 2 3 0 1 2 3
Cultivation time (day) Cultivation time (day)

1.5 vvm 2.0 vvm

40 40
35 35
30 30
SCOD (g/L)

25 25

SCOD (g/L)
20 20
15 15
10 10
5 5
0 0
0 1 2 3 0 1 2 3
Cultivation time (day) Cultivation time (day)

Microbial contamination during fungal fermentation

 Specific Oxygen Uptake Rate (SOUR)
SOUR and SCOD concentrations during fermentation without fungal
inoculation under 0.5 vvm (airlift reactor)

50 40
45
Specific oxygen uptake rate

SCOD 35
(SOUR) (mg O2/g VSS-hr)

40
30
35

SCOD (g/L)
30 25
SOUR
25 20
20 15
15
10
10
5 5

0 0
0 8 16 24 48 72
Cultivation time (h)
 Pilot-scale study
• Molasses, Taro wastes and
damages Papaya
bioconversion into fungal
protein

• Fungal protein into aquatic

feed.
• Feeding trials at OI
 Conclusions
• R. oligosporus can utilize vinasse o support its growth with
concomitant wastewater reclamation.

• The fungal biomass yield was found to be dependent on

aeration rate and the optimal aeration rate was 1.5 vvm.

• The vinasse-derived fungal biomass could potentially serve as

a high quality protein-rich ingredient for animal feeds and
even human food supplement.

• The innovative fungal technology can be a sustainable

solution to meet the growing need of aquatic feed.
 Acknowledgements
Saoharit Nitayavardhana, Ph.D.

Lee Jakeway