Mushroom Production through Solid-State Fermentation

Experiment 6 Solid-state fermentation – Mushroom production Date: Solid-State Fermentation (SSF) is defined as any fermentation process allowing the growth of microorganisms on moist solid materials in the absence of free-flowing water. The low moisture content means that fermentation can only be carried out by a limited number of microorganisms, mainly yeasts, and fungi; some bacteria also been used. Solidstate fermentation uses culture substrates with low water levels (reduced water activity), which is particularly appropriate for mold. SSF promoted higher yields and better product characteristics than cultivation in the liquid medium. In addition, costs are much lower due to the efficient utilization and value addition of wastes. The main drawback of this type of cultivation concerns the scale-up of the process, largely due to heat transfer and culture homogeneity problems. SSF processes generally employ a natural raw material as carbon and energy source; the nature of the solid substrate is one of the most important factors affecting SSF processes, and its selection depends on several factors mainly related to cost and availability. The substrates usually used are cellulose, sugars, and starch material nitrogen sources (ammonia, urea, peptides) and mineral salts. Paddy straw is lignocellulosic waste material, and the major constituents of lignocellulose are cellulose, hemicelluloses, and lignin. Lignocellulosic waste material yields a variety of products viz., enzymes, flavors, organic acids, single-cell protein, and bioactive compounds. Objective: Solid-state fermentation using lingo-cellulosic waste material (paddy straw) for SCP (Mushroom) production using Pleurotus sajor-caju. This process consists of depositing a solid culture substrate, such as paddy straw, on flatbeds after seeding it with Pleurotus; the substrate is then left in a temperature-controlled room for fifteen days. Production of Mushroom by solid substrate fermentation Paddy straw is a lignocellulosic material comprising roughly about 35-55% cellulose, 20-40% hemicellulose, and 10-25% lignin. This by-product, rich in cellulose, can be effectively utilized for the production of value-added products by the action of certain microorganisms possessing the enzyme cellulase. One good example is the fermentation of cellulosic materials present in paddy straw by the fungus Pleurotus resulting in the production of protein-rich mushroom. Paddy straw serves as a suitable substrate for the growth of this mushroom fungus. AGM 312 Microbial Biotechnology (1+1) Page 27 Base materials required   Pure culture of the mushroom fungus, Pleurotus sp. Potato Dextrose Agar medium (PDA) Potato juice - 250g Dextrose - 20g Agar - 20g Distilled water - 1000 ml pH - 7.0   Sorghum or wheat grains High-density polypropylene bags (30x15cm size and 150 gauge thick) / saline bottles (for spawn production) Calcium carbonate Non-absorbent cotton Polythene bags (60x30cm or 75x45cm) (for mushroom production) Processed paddy straw     Spawn production i. Select healthy and whole sorghum grains ii. Put the grains in water. Remove the chaffy and damaged grains which float on the top. iii. Half cook the grains for 30 minutes in boiling water to soften them. Grains should not be sticky but should slightly break iv. Drain the excess water, spread the grains over a clean sac or cloth to dry them (approx. 30 min) v. For every one kg of grains, mix 20g of calcium carbonate thoroughly (This is to adjust the pH of the grains, to remove excess moisture and to prevent the grains from sticking together) vi. Fill the grains in clean glucose/saline bottles or in poly bags @ 250-300g, plug them with non-absorbant cotton, cover with aluminum foil vii. Autoclave the bottles/bags at 1.42 kg/cm2 pressure for 90 minutes AGM 312 Microbial Biotechnology (1+1) Page 28 viii. Cool them to room temperature and inoculate with a pure culture of Pleurotus fungus aseptically ix. Incubate the bags for 10-15 days to obtain complete growth of white mycelium covering the entire spawn bag. This is known as the “Mother Spawn” x. Use the mother spawn within 25-35 days for making further generations known as the “Bed Spawn.” From each mother spawn bag (18-30 days old), 25-30 bed spawn bags can be produced by transferring 10g of spawn from mother spawn bag to bed spawn bag. Preparation of mushroom beds 1. Processing of substrate i. Cut the raw paddy straw into bits of 3-5cm ii. Soak them in water for 4-6 hours iii. Drain the water and boil them at 800c for one hour (or) treat the straw with steam at 1.2 Kg/cm2 for 30-45 min iv. Drain the water and spread the straw on a clean sac previously soaked in Carbendazim solution or potassium permanganate solution @ 1g/lt v. Shade dry to 65-70% moisture (water should not drip when squeezed by hand) vi. One kg of straw when boiled and shade dried weighs about 4-5 Kg. 2. Spawning i. Take a polythene bag of the given specification, tie it at the bottom to provide a flat circular bottom ii. Divide the spawn present in the spawn bag into four equal portions by spreading them evenly in a clean disinfected plastic tray iii. Spread the paddy straw bits uniformly into the bag to a height of 5 cm. Sprinkle one portion of spawn over this, evenly covering the entire surface. Gently press with fingers iv. Spread a second layer of straw to a height of about 10 cm and sprinkle the second portion of the spawn evenly. Give a mild pressing v. Similarly, spread the third layer of straw to 10 cm height and sprinkle the third portion of spawn vi. Spread the fourth layer of straw to 10cm height and sprinkle the fourth portion of spawn vii. Finally, spread paddy straw to 5 cm height. Gently press with fingers and tie the bag with a thread or rope viii. For aeration, make 2-4 vents of one cm diameter at different places of the bag. AGM 312 Microbial Biotechnology (1+1) Page 29 . Filling mother spawn during mushroom bed preparation Cellulosic paddy straw under SSF Mushroom fruiting bodies 3. Spawn running & cropping      After preparation, arrange the cylindrical beds in racks in spawn running room maintained at a humidity of around 80% Mycelium from the spawn slowly grows and covers the entire bed surface within 1525 days depending upon the species used. Until this stage, the water spray is not needed. At the end of spawn run, small pinheads appear through the vents which is the indication of fruiting Now transfer the beds to the cropping room with more ventilation and aeration. Here open the beds carefully either fully or partly or in splits. Water spraying daily as the fine mist is necessary for the growing beds to develop into fruiting bodies (18–45 days). The buds turn into mature mushrooms within 24-48 hours. Harvest the mushrooms before the margins roll upward or downwards The average yield of oyster mushroom would be approx. 635g/bed (500g of paddy straw) with a good shelf life of up to 72 h Activity  Observe the bed for the emergence of fruiting bodies.  Calculate the average yield per bed  Estimate the product cost AGM 312 Microbial Biotechnology (1+1) Page 30 Review Questions 1. Mention some cellulose-rich substrates amenable for microbial fermentation. 2. Name a few microorganisms capable of degrading cellulose. 3. Give a list of some value-added products produced by submerged and solid substrate fermentation of cellulose. 4. Why are fungi more efficient in SSF than other yeast/bacteria? Substantiate. 5. Mention the list of products and microorganisms obtained by SSF. 6. Differentiate SSF vs. Submerged fermentation. Reference Luiz A. Junior Letti L.A.J., F. M. Destéfanis Vítola, G. V. Pereira, S. G. Karp, A.B. P. Medeiros, E.S. Ferreira da Costa, L. Bissoqui, C.R. Soccol. 2018. Solid-State Fermentation for the Production of Mushrooms. (Eds. A.Pandey, C.Larroche, C.R. Soccol. In: Current Developments in Biotechnology and Bioengineering. Elsevier. AGM 312 Microbial Biotechnology (1+1) Page 31