Utilization of Local Agri-processing By-products to Produce Fungal Protein for Aquatic Feed Production

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1 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

2 Aquaculture Production 7.1% Development of world capture fisheries and aquaculture production

3 Direction of global production of major nutrient sources NUTRIENT SOURCES FROM FISHING ACTIVITIES OILSEED MEALS, PULSES & OILS AQUATIC ANIMAL PROTEIN MEALS & LIPIDS 40 essential nutrients LAND ANIMAL PROTEIN MEALS & FATS CEREALS INCLUDING BY-PRODUCT MEALS & OILS MICROBIAL FEED INGREDIENTS NUTRIENT SOURCES FROM AGRICULTURAL FARMING ACTIVITIES Source: Albert G.J. Tacon

4 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

5 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,

6 What is fungi 200 µm

7 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 Morphological characteristics of sporangiospores of the tempe fungus Rhizopus oligosporus differentiate it from other taxa of the R. microsporus group. Mycological Research. 112,

8 Fungal fermentation biofuel residues Filtered water Dewatered product ~45% protein 2d growth Settling Dried product

9 Vinasse generation Parameters Vinasse ph 4.25 ± 0.32 Total solids (TS) (g/l) ± 2.80 Volatile solids (VS) (g/l) ± 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 (SCOD) (g/l) ± 4.15 Total chemical oxygen demand (TCOD) (g/l) ± 2.31 Around 8-15 L vinasse per liter of ethanol is produced Total Kjeldahl nitrogen (TKN) (g/l) 0.37 ± 0.08

10 Vinasse disposal: Fertirrigation Demerits of land application Risk of soil salinization Nutrient leaching to surface water and groundwater Odor nuisance

11 Fungal fermentation on vinasse Feed and food production

12 Fungal cultivation in airlift bioreactor ph: 5.0 NaOH ph 5.0 ph controller H 2 SO Foam trap Temperature: 37 o C Nutrient supplementation: COD:N:P = 100:5:1 Aeration rates: 0.5, 1.0, 1.5, and 2.0 volume air /volume liq /min 1. Sampling port 2. Acid inlet 3. Base inlet 4. ph probe 5. Temperature probe Air pump Air filter Rotameter

13 Fungal biomass yield 9 c 8 Fungal biomass yield (g biomass increase /g initial biomass ) a b b Aeration rate (vvm)

14 Fungal morphology 16 h 24 h 48 h 72 h Fungal cultivation at 1.5 vvm Fungal cultivation at 2.0 vvm (72 h)

15 Airlift vs. bubble column Airlift reactor Bubble column reactor Air pump Air pump Air filter Rotameter Air filter Rotameter

16 Fungal biomass yield: airlift vs. bubble column 12.0 Airlift reactor Bubble column reactor b Fungal biomass yield (g biomass increase /g initial biomass ) a b a b a a a Aeration rate (vvm)

17 Fungal cultivation for 72 h 1.5 vvm 2.0 vvm Fungal morphology (bubble column reactor)

18 Fungal cultivation on commercially-produced vinasse Sugarcane-ethanol derived vinasse obtained from a commercial caneethanol facility in Brazil (Brazil vinasse) without dilution ~ 60% SCOD reduction a Fungal biomass yield (g biomass increase /g initial biomass ) a With nutrients supplementation

19 Fungal biomass characterization Compositions (on dry weight basis) Fungal biomass Fishmeal Soybean meal Crude protein Crude lipid Ash % in vitro protein digestibility comparable to soybean meal and fishmeal with overall digestibility of about 85%

20 Amino acid profiles of fungal biomass Percentage of essential amino acids (based on % protein) Fishmeal Soybean meal Vinasse-derived fungal biomass 0 Essential fatty acids Values (% dry weight) Linoleic acid 1.35 Linolenic acid 0.32 Eicosapentaenoic acid (EPA) 0.12 Docosahexaenoic acid (DHA) 0.11

21 Soluble chemical oxygen demand (SCOD) reduction SCOD (g/l) vvm SCOD (g/l) vvm Cultivation time (day) Cultivation time (day) SCOD (g/l) vvm SCOD (g/l) vvm Cultivation time (day) Cultivation time (day) Microbial contamination during fungal fermentation

22 Specific Oxygen Uptake Rate (SOUR) SOUR and SCOD concentrations during fermentation without fungal inoculation under 0.5 vvm (airlift reactor) Specific oxygen uptake rate (SOUR) (mg O 2 /g VSS-hr) SCOD SOUR Cultivation time (h) SCOD (g/l)

23 Pilot-scale study Molasses, Taro wastes and damages Papaya bioconversion into fungal protein Fungal protein into aquatic feed. Feeding trials at OI

24 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.

25 Acknowledgements Saoharit Nitayavardhana, Ph.D. Lee Jakeway

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