The worldwide dairy industry generates over 80 million tones of whey each year. This byproduct of cheese manufacture has a high pollution load with a chemical oxygen demand (COD, see Chapter 15) of 60g oxygen per litre. Consequently, it usually has to be disposed of at a high capital cost to the dairy industry. Whey contains approximately 45 g/L lactose and 10 g/L protein. It is particularly suitable for the production of SCP using lactose-utilizing yeast, although attempts have also been made to grow other organisms, including Penicillium cyc/opium. Several processes have been developed for the utilization of lactose in milk whey. Some of the more successful have been those operated by Bel Industries in France. The Bel process was developed with the aim of reducing the pollution load of dairy industry waste, while simultaneously producing a marketable protein product. A number of plants are operated using
Kluyveromyces lactis or K. marxianus (formerly K. fragilis) to
produce a protein, Protibel, which is used for both human and animal consumption.
These processes initially involve whey pasteurization, during which 75% of whey proteins are precipitated. The lactose concentration is adjusted to 34 g/L and mineral salts are also added. This supplemented whey is introduced into a 22 m3 continuous fermentor, maintained at 38°C and pH 3.5, with an aeration rate of 1700 m3/h. The yeasts utilize the lactose and attain biomass concentrations of 25 g/L, with a biomass yield of 0.45-0.55 g/g lactose. Yeast cells are recovered by
centrifugation, and then resuspended in water, recentrifuged and finally roller-dried to 95% solids. Levels of residual sugar remaining in the spent medium are less than 1g/L.
• Ok, what other processes are available for SCP production?
The SYMBA process is another famous process. It was developed in Sweden to produce SCP for animal feed from potato processing wastes. It is not economically attractive as a stand-alone operation. However, alternative routes for the purification of these waste-waters are difficult and expensive, as
they contain up to 3% solids and have COD values of over 20g oxygen per litre. A high proportion of the available substrate is starch, which many microbes cannot directly utilize. To
overcome this problem the process was developed with two microorganisms that grow in a symbiotic association. They are the yeasts Saccharomycopsis fibuligera, which produces the hydrolytic enzymes necessary for starch degradation, and Candida utilis. The process is operated in two stages. In the first stage, S. fibuligera is grown in a small reactor on the sterilized waste, supplemented with a nitrogen source and phosphate. At this point, the starch is hydrolysed, which is the rate-limiting step of the whole process. The resulting broth is then pumped into a second larger fermentor of 300 m3 capacity where both organisms are present. However, C. utilis comes to dominate the second stage and constitutes up to 90% of the final product. The Symba process operates continuously and after 10 days the pollution load of the waste is reduced by 90%. Resultant protein-rich biomass (45% proteins concentrated by centrifugation and finally spray drum dried.
THe Symba Process
• What is the next process we should see?
Then there is the Pekilo process.This process began operating in 1975 and was the first commercial continuously operating process for production of a filamentous fungus. It had to overcome the special problems caused by the pseudoplastic rheological behaviour of submerged cultures of fungal mycelium, which particularly affect oxygen transfer rates. The process was developed in Finland for the utilization of spent sulphite liquor, derived ‘ from wood processing that contains monosaccharides and acetic acid. Supplements of other carbon source usually molasses, whey and hydrolysed plant was may also be added prior to inoculation with Paecilomyces variotii. This continuous process is operated aseptically and produces over 10000tonnes of SCP year from two 360 m3 fermentors. Resulting dried Pekilo protein containing up to 59% crude protein, is used in the preparation of compound animal feed. Methanol has several advantages over methane and many other carbon sources, particularly as it is completely miscible with
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water and is available in a very pure form. Consequently, the resultant protein does not have to undergo purification. As methane is readily converted to methanol, several oil and gas companies developed processes based on this attractive carbon source in the 1970s. However, there are some problems associated with methanol as a substrate. Only relatively low concentrations, 0.1-1.0% (v/v), are tolerated by the
microorganisms that utilize it, and some methylotrophic yeasts form pseudo-mycelium while growing on methanol. During the fermentation, the oxygen requirement is high, as is the heat of fermentation. Nevertheless, the oxygen demands are somewhat lower than when using methane or other hydrocarbons.
Attempts to develop methanol-based processes were made in Europe, the former Soviet Union, Japan and the USA. They involved bacteria (Hyphomicrobium species, Methylococcus species and Methylophilus methylotrophus), yeasts (Candida boidinii, Pichia angusta and Pichia pastoris) and even filamentous fungi (Gliocladium deliquescens, Paecilomyces variotii and Trichoderma lignorum).
A typical example of this kind of processes is the Pruteen process.
• Let’s find out more about the Pruteen process. This was the most technically adventurous was the process developed by ICI in the UK, which started production in 1980. This process used the methylotrophic bacterium, M.
methylotrophus, to produce a feed protein for chickens, pigs and veal calves, called Pruteen. Production ceased in 1987 for economic reasons, due to the rise in price of methanol, which constituted 59% of the production costs, and a fall in the price of competing soya meal. Nevertheless, this process is worthy of examination due to the advances made in fermentation design and technology during its development. This was, apart from certain systems for wastewater treatment, the world’s largest continuous aerobic bioprocess system. It consisted of a 3000 m3 pressure cycle airlift fermentor with inner loop and a working fluid volume of 1.5 x 106 L, capable of producing up to 50000tonnes of Pruteen per annum. The fermentor weighs in excess of 600tonnes, is over 60m high, with a 5 atm pressure difference from the top to the bottom and cost US$80 million in 1979.
Filter-sterilized compressed air was used for both oxygenation and agitation, and all streams into the fermentor were sterilized. The fermentation was performed at pH6.5-6.9 and 34-37°C with entirely inorganic commercial-grade nutrients. It was operated as a methanol-limited chemostat, the methanol being supplied through numerous distribution points within the fermentor. Bacterial cells were recovered by a novel separation technique, involving initial concentration from 3% (w/w) to 12% (w/w) by flocculation, which was promoted by acid and heat shock. This was followed by centrifugal dewatering, with recycle of water, and air drying. The dried unprocessed product contained 16% nucleic acids and over 70% crude protein. Strain development of M. methylotrophus led to several improvements in its composition and fermentation
performance. Protein content of the product was increased by
5%. The cell concentration achieved during fermentation rose from 4 g/L to 30 g/L.
• Are mushrooms SCP too? Yes they are.
Certain mushrooms and other fruiting bodies of filamentous fungi are edible and provide a good source of protein, whereas others have narcotic effects and some are highly toxic. A wide range has been traditionally used for food, but relatively few are grown commercially. In fact, of the hundreds of species that are edible, only about 10 are produced in any quantity. Mushroom production involves controlled non-axenic solid substrate fermentation. It is currently the only economically viable product from lignocellulose fermentation.
Exploitation of such fruiting fungi for the generation of edible biomass has several advantages:
1 they represent examples of the most efficient conversion of plant wastes into edible food;
2 unlike many other single cell proteins, they are directly edible and many are considered to be food delicacies because of their characteristic texture and flavour;
3 harvesting of fruiting bodies is the easiest possible method of separating edible biomass from the substrate in a solid- state fermentation; and
4 compared with animal sources of protein, many have far superior protein conversion efficiency per unit of land and per unit of time.
Agaricus Bisporus
In Europe and the USA, Agaricus bisporus (button mushroom) accounts for over 90% of total mushroom production value. Agarics are decomposers of cellulosic materials and are naturally found in meadows and woodlands, where they degrade plant debris. They are grown commercially in temperate regions using a substrate of com posted straw. A crop is produced within 6 weeks, whereas other mushrooms may take several months or even years to fruit. A closely related species, Agaricus bitorquis, is also grown in some areas. It is less prone to certain viruses and the bacterial blotch disease of mushrooms, caused by
Pseudomonas tolaasii. The Agaricus production regime involves
the following stages.
1 Inoculum preparation: growth of spawn (inoculum) on sterilized cereal grains.
2 Solid-substrate preparations: compo sting of straw, manure and fertilizers at 60-70°C for 2 weeks.
3 Substrate ‘sterilization’, so-called ‘peak heating’ of compost for 5-7 days.
4. Spawn inoculation into ‘sterilized’ compost and growth, referred to as a ‘run’ at 25°C for 2-3 weeks
5. Application of a casing (covering) layer of peat and chalk over the substrate.
6. Fruiting body production, fructification, in about four flushes (successive crops) over a period of 4-6 weeks.
Specialty Mushrooms
Mushroom production worldwide has increased in last 35 years from about 350 000 tonnes in 1965 to now over 500000 tonnes.
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The majority of this increase has occurred during the last 15 years and a major occurred in the range of genera cultivated on a commercial scale.
Now, exotic mushrooms are becoming increasingly popular. China is the major producer of specialty mushrooms. The main speciality genera cultivated are Lentinula ( Shiitake), Flammulina (Enokitake), Pleurotus (Oyster mushshroom), Hypsizygus (Bunashimeji), , Volvariella (Paddy Straw mushroom) and
Grifolla (Maitake).
Now there is a demand for the t of improved technology to cultivate these species more efficiently, as traditional practices are not very productive. Some very valuable fungi are obtained only from wild sources and have found to be very difficult to cultivate.
Cultivation of Shiitake mushroom , Lentinula edodes, started in China almost a thousand years ago and was then introduced into Japan. They are becoming popular in the west, and are now grown in Europe and the USA. Worldwide production is approaching 200,000 tonnes/annum. These mushrooms may be used fresh or dried, and apart from culinary use, several medicinal properties have been attributed to Shiitake.
Components detected include antihistamines, antitumour and antiviral agents, anticholesterol substances and compounds that inhibit platelet agglutination.
A problem with traditional methods of cultivation on natural logs is the time required before fruiting, which may be several years. In Japan, logs of the shii tree have been used, thus the derivation of the name Shiitake, but most production is now on species of oak. Logs of about 7-15 cm diameter are cut into lengths of about 1 m and drilled with holes spaced at one hole per 500cm2. The holes are inoculated with wood piece spawn or
sawdust spawn and then usually covered with hot wax to prevent excessive drying. Spawn run, the development of fungal mycelium within the log, takes 6-9 months, after which the logs are transferred to a cooler and more moist ‘raising’ yard. This change in conditions provides an optimum environment for the growth and development of the mushrooms. The first crop is normally produced in the following year.
Modern production on synthetic logs is much quicker, taking about 4 months. The synthetic logs are prepared from sawdust, straw and corn cobs, along with supplements of wheat bran, rice bran, millet, rye and corn. Water is added to raise the moisture content to around 60% (w/w). The mixture is placed into bags and autoclaved for 2 h at 121°C. After cooling they are inoculated with Shiitake spawn. The inoculum is allowed to develop mycelium for 20-25 days and then the covering bags are removed. After about 4 weeks, exposed substrate blocks begin to form fruiting body about 2mm under the surface. The stimulation of their maturation is promoted by soaking these synthetic logs in water at 12°C for 3-4h. The first crop or flush of mushrooms is ready to harvest about 10 days after soaking.
• Exercise: study and carry out the cultivation of oyster mushrooms on rice paddy and other agricultural waste products. Compare the relative yield on different substrates:
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Learning Objectives
In this lecture, you will learn• Introduction
• General aspect of enzyme production
• Amylase & Protease, Immobilized enzyme
• Industrial application of microbial enzyme
Enzymes are used for a variety of purposes. They are employed in three major fields: (a) laboratory, (b) industrial and (c) clinical. In some cases, they may be used in their crude forms. But at other times, they are used in purified states (e.g. urease for urea estimation). Microbial enzymes are listed in Table
Table – Microbial enzymes
What are the various aspects of enzyme production? Enzymes arc commercially produced by two methods: (1) Semisolid culture and
(2) Submerged culture.
There is great competition among enzymes’ manufacturers. Therefore, manufacturers are reluctant to reveal the process details. It is not possible to undertake a survey of the production methods being used. But, it is evident that the submerged culture method has been gaining ground during the past three to four decades. These two methods have been briefly dealt with here.