Production of Mushrooms Using Agro-Industrial Residues as Substrates
Residue-Based Substrates and their Solid-State Fermentation by Mushroom Fungi. Types, Availability and Chemical Composition of Raw Materials. Nutritional and Environmental Aspects of Mushroom Growing. Output, Stages and results of Mushroom Cultivation.
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Ministry of education and science of Ukraine
National aviation university
Institute of ecological safety
Department of biotechnology
Discipline "Theoretical basis of biotechnology"
Theme: "Production of Mushrooms Using Agro-Industrial Residues as Substrates"
Student : Yurchenko Yuriy,
Instructor: Drazhnikova A.V.
Mushroom cultivation as a prominent biotechnological process for the valorization of agro-industrial residues generated as a result of agro-forestry and agro-industrial production. A huge amount of lignocellulosic agricultural crop residues and agro-industrial by-products are annually generated, rich in organic compounds that are worthy of being recovered and transformed. A number of these residues have been employed as feedstocks in solid state fermentation (SSF) processes using higher basidiomycetus fungi for the production of mushroom food, animal feed, enzymes and medicinal compounds.
Likewise, the above-mentioned microorganisms have been successfully employed in processes related with the bioremediation of hazardous compounds and waste detoxification. Mushroom cultivation presents a worldwide expanded and economically important biotechnological industry that uses efficient solid-state-fermentation process of food protein recovery from lignocellulosic materials. impacts of different environmental and nutritional conditions on mycelium growth and fruiting bodies production are highlined.
Moreover, cultivation technologies of Agaricus bisporus, Pleurotus spp and Lentinula edodes, comprising spawn (inoculum) production, substrate preparation and mushroom growing process i.e. inoculation, substrate colonization by the cultivated fungus, fruiting, harvesting and processing of the fruiting bodies, are outlined. Finally, the efficiency of residues conversion into fruiting bodies are outlined in two medicinal mushroom genera, Pleurotus and Lentinula, widely cultivated for their nutritional value and extensively researched for their biodegradation capabilities. Experimental data concerning residue-substrates used, as well as biological efficiencies obtained during their cultivation were considered and discussed.
1. Residue-Based Substrates and their Solid-State Fermentation by Mushroom Fungi
1.1 Types, Availability and Chemical Composition of Raw Materials
2. Nutritional and Environmental Aspects of Mushroom Growing
3. Output and Stages of Mushroom Cultivation
On the surface of our planet, around 200 billion tons per year of organic matter are produced through the photosynthetic process. However, the majority of this organic matter is not directly edible by humans and animals and, in many cases, becomes a source of environmental problem. Moreover, today's society, in which there is a great demand for appropriate nutritional standards, is characterized by rising costs and often decreasing availability of raw materials together with much concern about environmental pollution. Consequently, there is a considerable emphasis on recovery, recycling and upgrading of wastes. This is particularly valid for the agro-food industry, which furnishes large volumes of solid wastes, residues and by-products, produced either in the primary agro-forestry sector or by secondary processing industries, posing serious and continuously increasing environmental pollution problems. It is worth mentioning that only crop residues production is estimated to be about 4 billion tons per year, 75% originating from cereals.
Nevertheless, residues such us cereals straw, corn cobs, cotton stalks, various grasses and reed stems, maize and sorghum stover, vine prunings, sugarcane and tequila bagasse, coconut and banana residues, corn husks, coffee pulp and coffee husk, cottonseed and sunflower seed hulls, peanut shells, rice husks, sunflower seed hulls, waste paper, wood sawdust and chips, are some examples of residues and by-products that can be recovered and upgraded to higher value and useful products by chemical or biological processes. In fact, the chemical properties of such lignocellulosic agricultural residues make them a substrate of enormous biotechnological value. They can be converted by solid state fermentation (SSF) into various different value-added products including mushrooms, animal feed enriched with microbial biomass, compost to be used as biofertilizer or biopesticide, enzymes, organic acids, ethanol, flavours, biologically active secondary metabolites and also for bioremediation of hazardous compounds, biological detoxification of agro-industrial residues, biopulping etc. Among applications of SSF, mushroom cultivation has proved its economic strength and ecological importance for efficient utilization, value-addition and biotransformation of agro-industrial residues. Current literature shows that lignocellulose degrading mushroom species are used in various SSF applications such as bioremediation and biodegradation of hazardous compounds, biological detoxification of toxic agro-industrial residues, biotransformation of agroindustrial residues to mushroom food and animal feed, compost and product developments such as biologically active metabolites, enzymes, and food flavour compounds. Moreover, recent research work indicates medicinal attributes in several species, such as antiviral, antibacterial, antiparasitic, antitumor, antihypertension, antiatherosclerosis, hepatoprotective, antidiabetic, anti-inflammatory, and immune modulating effects.
Commercial mushroom production, carried out in a large or small scale, is an efficient and relatively short biological process of food protein recovery from negative-value lignocellulosic materials, utilizing the degrading capabilities of mushroom fungi. Among mushroom fungi, L. edodes and Pleurotus species reveal high efficiency in degradation of a wide range of lignocellulosic residues, such as wheat straw, cotton wastes, coffee pulp, corn cobs, sunflower seed hulls wood chips and sawdust, peanut shells, vine prunings and others into mushroom protein, the productivity of the conversion being expressed by biological efficiency. Their mycelium can produce significant quantities of a plethora of enzymes, which can degrade lignocellulosic residues and use them as nutrients for their growth and fructification. However, the nature and the nutrient composition of the substrate affect mycelium growth, mushroom quality and crop yield of this value-added biotransformation process.
The focus of this work is to highlight significant aspects of utilization of low- or negative-value agro-industrial residues in mushroom biotechnology, emphasizing on their biotransformation to fruiting bodies that are nutritious human foodstuff regarded also as functional food. Aspects to be reviewed in this article include: an overview of availability, sources and types as well as chemical composition of solid lignocellulosic agro-residues suitable for mushroom cultivation, some background on mushroom degrading abilities and of their nutritional and environmental demands,an outline of commercial production technologies of A.bisporus,Pleurotus spp. and L. edodes mushrooms, and finally a consideration and discussion of experimental data regarding productivity (biological efficiency) on various agro-industrial residues during cultivation ofPleurotus spp. and L. edodes.
1. Residue-Based Substrates and their Solid-State Fermentation by Mushroom Fungi
As a result of agro-forestry and agro-industrial production, a huge amount of live- stock waste, agricultural crop residues and agro-industrial by-products are annually generated, the major part being lignocellulosic biomass. Although agro-industrial residues contain beneficial materials, their apparent value is smaller than the cost of collection, transportation and processing for beneficial use. However, if residues are utilized, such as to enhance food production, they are not considered as wastes but new resources. A number of agro-industrial residues have been employed as feed stocks in SSF processes, using high basidiomycetus fungi for the production of valuable metabolites. However, mushroom production is one of the areas with great potential for exploitation of forest and agricultural residues.
1.1 Types, Availability and Chemical Composition of Raw Materials
Reddy and Yang (2005) and very recently, Zhang (2008), reviewing the global world information about lignocellulose availability, estimated the production of lignocellulosic biomass to be more than 200 Ч 109tons per year. Especially, the amount of crop residues produced annually in the world from 27 food crops is estimated at about 4 Ч 109tons, from which 3 billion tons account per annum for lignocellulosic residues of cereals. Cereals, accounting 75% of global world production, furnish these outstanding amounts of waste products as wheat residues, rice straw and hulls, barley residue, maize stalks and leaves, millet and sorghum stalks. Sugar cane provides the next sizeable residue with two major crop wastes, leaves and stalk, and bagasse, which is the crop processing residue. The cotton crop also provides significant residue in the form of stalks and husks, while no negligible are the residues furnished by minor crops as sunflower, oil palm, coconut, banana, vines, groundnut and coffee. In fact, this generation of residues is a result of the limited portions of the crops that are actually used. To give the order of magnitude, 95% of the total biomass produced in palm and coconut oil plantations is discarded as a waste material; the respective values for sisal plant and sugar cane biomass are 98% and 83%. Moreover, in the flax industry only 2% of the produced biomass is effectively used, less than 9% in the palm oil industry and only 8% in the brewing industry. Biomass availability is a primary factor for bio-based industrial production. Indeed, the available resource potential (the amount of residues used for various purposes) is smaller than the one generated.
The quantities of crop residues that can be available for bioprocesses are estimated using total grain production, residue to product ratio (RPR), moisture content, and taking into consideration the amount of residue left on the field to maintain soil quality (i.e. maintain organic matter and prevent erosion), grazing and other agricultural activities. Concerning cereal straw, the RPRs for rice, barley, wheat and corn are 1.4, 1.2, 1.3 and 1 respectively.
Assuming that one quarter of the residues can be harvested and that roughly one third of the harvested straw is used in animal husbandry, 0.22 tons straw per ton cereal grain and 0.25 ton residues per ton maize are available biomass for other uses, as energy, enzyme production or mushroom growing. As agro-industrial residues accumulate in fields and factories, availability issue tends to become a regional and local matter. Geographical distribution of crop residues is skewed by large crop productions in India and China, where increased quantities of crop residues and agro-industrial by-products are generated because of expanding agricultural production.
Furthermore, Asia along with Europe, North America and Australia are world leader mushroom producing regions and consequently the major residue demanding for this bio-based industrial activity. Among countries in the Asian and Pacific Region, China produces the largest quantities of agricultural and forest residues, mainly by-products of rice, corn and wheat. China's quantities, estimated to reach about 1 billion tons/year, are followed by India's yielding at least 200 million tons/year of agricultural residues according to Das and Singh, while according to Mande (2005) India's total amount of agro-industrial residues reaches 600 mil- lion tons. This quantity comprises 480 tons of crop residues (rice, wheat, millet, sorghum, pulses, oilseed crops, maize stalks and cobs, cotton stalks, sugarcane trash etc.) and 120 tons of processing-based residues (mainly groundnut shells, rice husk, sugarcane bagasse, cotton waste, coconut shell and coir pith). Rice and sugar are Asia's rest southeast countries dominant crops. Moghtaderi et al. (2006) report that Australian agro-industrial biomass reaches 100 million tons/year, including bagasse, cane trash, wood residues, energy crops etc.
As far as Africa is concerned, wheat and barley predominate in the north, millet and sorghum are the main crops in sub-Saharan Africa, while farther south maize is the dominant crop. Kim and Dale (2004), estimated Africa's annual lignocellulosic biomass from rice straw, wheat straw and sugar cane bagasse to be about 40 million tons, indicating that the fraction of most crop residues collectable is less than 30% because of low yields. In the same work, Central and South America's lignocellulosic residues were estimated to be about 140 million tons from rice and wheat straw, corn stover and sugar cane bagasse, not taking into account coffee, banana and other agricultural residues.
Concerning North America, according to USDA-US DOE report (2005), USA is able to produce 1.3 billion tons of dry residues per year, including agricultural (933 million tons) and forest resources (368 million tons). Main lignocellulosic by-products in considerable quantity are corn stover, the most abundant agricultural residue in USA, wheat, rice, barley straw, sorghum stalks, coconut husks, sugarcane bagasse, pineapple and banana leaves. Canada, the second largest supplier of wood lignocellulosic biomass, supplies more than 200 million m3 of lignocellulose annually through commercial operations. Finally, Europe is not only a great wheat straw producer, but also outstanding quantities of lignocellulosic residues from barley, maize, sunflower, rapeseed, cotton, olive trees and vines, summarized as 120 million tons/ year.
Regarding the types of wastes, according to Mande (2005), agricultural residues can be divided into two groups: crop-based residues (generated in the field) and processing-based residues (generated during wood and industrial processing). Crop- based residues, which are plant materials left behind in the field or farm after removal of the main crop produce, are consisted of different sizes, shapes, forms, and densities like straw, stalks sticks, leaves, haulms, fibrous materials, roots, branches, and twigs.
Crop-based residues are produced from various sources such as field and seed crops (including straw or stubble from barley, beans, oats, rice, rye, and wheat, stalks or stovers from corn, cotton, sorghum, grasses and reeds, soybeans and alfalfa), fruit, nut, vegetable or energy crops (brushes and orchard prunings, e.g. vine shoots or leaves that remain on the ground after harvesting), and livestock manure. Processing-based agro-industrial residues are by-products of the post-harvest processes of crops such as cleaning, threshing, linting, sieving, and crushing. They are in the form of husk, dust, stalks etc. Food processing wastes that come from plant materials are culls, rinds, seeds, pits, pulp, press cakes, marc, malts, hops and a variety of other by-products from mass food production processes. Some examples of these materials are coffee processing by-products, sugarcane bagasse, hulls and husks, wheat middlings, corncobs, seed meals etc.
Moreover, this category comprises wood residues produced either from the primary processing or from secondary manufacturers (producing bark, chips, sawdust, coarse residues, and planer shavings). During the sawing of a log at a typical sawmill, approximately 50% of the initial log volume is converted into wood products and 50% is converted into wood residues. In general, solid agro-industrial residues are heterogeneous water insoluble materials having a common feature, their basic macromolecular structure being cellulose, hemicellulose and lignin and to a lesser extend pectin, starch and other polysaccharides (Thomsen 2005). Cellulose, the most abundant renewable organic resource comprising about 45% of dry wood weight, is a linear homo polymer of glucose units linked with ? 1,4-glucosidic bonds. Hemicelluloses, hetero polysaccharides containing two to four different types of sugars, are divided in three major groups: xylans, mannans and galactans. They consist of short-branched chains of hexoses, e.g. mannose units in mannans and pentoses such as xylose units in xylans. After cellulose, lignin is the second most abundant renewable biopolymer in nature. Lignin, representing between 26 to 29% of lignocellulose, is strongly bounded to cellulose and hemi- cellulose, imparting rigidity and protecting the easily degradable cellulose from the hydrolase attack.
Lignin is an aromatic polyphenol macromolecule, 3-dimensional and amorphous. Additionally, crop residues contain, on a dry weight basis, approximately 0.5-1.5%N, 0.15-0.2%P, 1%K, 1%Ca, 0.5%Mg, 0.2%S, 30mgKg-1Mn, 100mgKg-1Fe, 30mgKg-1Zn, 5mgKg-1Cu, 20mgKg-1B and about 1mgKg-1Mo(MillsandJones1996).However, these values differ with crop, plant part, season, soil moisture as well as other factors that affect plant growth. Substrates used in mushroom cultivation include both field-based residues and processing based-residues.
However, as the nutrient composition of the substrate is one of the factors limiting colonization as well as quantitative and qualitative yield of cultivated mushrooms, supplements containing sugars and starch (easily available carbohydrates) and fats (slower degraded and time-lasting nutrient sources) are added to the basal ingredient. Supplements are used to increase nutritional content, speed-up growth and increase mushroom yield, especially in the cultivation of the white-rot mushroom fungi L. edodes and Pleurotus spp. The various organic supplements used in mushroom cultivation comprise molasses, brewer's grain, grasses and waste paper, cotton and coffee wastes etc. However, soybeans and cereal grains or their milling by-products are the most commonly used supplements, as they are generated in considerable amounts and contain increased levels of protein, fats and easily metabolized carbohydrates: soybeans (carbohydrates 21.5%, N 6.3%), wheat bran (carbohydrates 49.8%, N 2.4%), rice bran(carbohydrates 37.0%, N 2.0%) and millet (carbohydrates 57.3%, N 1.9%)
2. Nutritional and Environmental Aspects of Mushroom Growing
From about 14000 mushroom-forming fungal species, at least 2000 are edible, of which 80 species are grown experimentally and around 20 are cultivated commercially. The most cultivated worldwide species are A. bisporus, P. ostreatus and L. edodes, followed by Auricularia auricula, Flammulina velutipes and Volvariella volvacea. Other mushroom species produced successfully on various substrates include Agrocybe aegerita, Ganoderma spp., Grifola frondosa, Hericium erinaceus, Hypsizygus marmoreus, Lepista nuda, Coprinus comatus, Pholiota nameko and Stropharia spp. Although the mentioned mushroom species have the ability to degrade lignocellulosic residues in their original or composted form, they exhibit differences regarding production of enzymes necessary to degrade lignocellulosic substrates and thus different abilities to grow and fruit on residue-substrates.
In plant residues, cellulose and hemicellulose are the main sources of carbohydrates, often incrusted with lignin, which forms a physical seal around these two components. Lignocellulose is physically hard, dense and recalcitrant, the degradation of which is a complex process requiring a battery of hydrolytic or oxidative enzymes. Taking into consideration that the substrates are insoluble, degradation occurs extracellularly, by two types of extracellular enzymatic systems: the hydrolytic system, which produces hydrolases and is responsible for cellulose and hemicellulose degradation; and a unique oxidative lignolytic system, which depolymerizes lignin. The hydrolytic breakdown of cellulose by fungi is catalyzed by extracellular cellobiohydrolases, endoglucanases and glucosidases, which hydrolyze the long chains of cellulose, liberating cellobiose and finally glucose, while the major hemicellulose-degrading enzymes are endoxylanases and endomannanases.
Most of these enzymes have been detected in both wood-degrading mushroom fungi (WDF), like P. ostreatus and L. edodes and litter-decomposing mushroom fungi (LDF), such as A. bisporus or V. volvacea. Due to its complicated structure, lignin is more difficult to break down than cellulose or hemicellulose.
The main extracellular enzymes participating in lignin degradation are lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase, with MnP, prooving to be the most common lignin-modifying peroxidase produced by almost all wood-degrading basidiomycetes. In addition, litter-decomposing basidiomycetes can degrade lignin e.g. A. bisporus produces at least two lignolytic enzymes, laccase and MnP, however, the overall lignin degradation rate by these fungi is lower compared to that of white-rot fungi. Besides the lignocellulosic enzyme complex, lignocellulolytic fungi also produce other enzymes, such as pectinases, proteases, lipases and phytases on lignocellulosic substrates. Basidiomycetes fungi comprise diverse ecological groups ,i.e. WDF (white rots, brown rots) and LDF, which may insure their nutrition in different ways. White-rot fungi are able of a simultaneous degradation of all wood components (cellulose, hemicellulose and lignin), while brown-rot fungi, a relatively small group of Basidiomycetes, degrade only cellulose and hemicellulose.
Given that the majority of cultivated higher basidiomycetes is WDF, while few of them are LDF, emphasis is given here to the nutritional behaviour and degradation potentials of these two groups, represented by the most cultivated species A. bisporus, Pleurotus spp. and L. edodes. White rot mushroom-forming fungi, comprising cultivated species like Pleurotus spp., L. edodes, Ganoderma spp. etc., are the most efficient degraders, due to their capability to synthesize relevant hydrolytic (cellulases and hemicellulases) and unique oxidative (lignolytic) extracellular enzymes. Their general strategy is to decompose the lignin in wood, so that they can gain access to the cellulose and hemicelluloses embedded in the lignin matrix. However, laccase expression in fungi is influenced by culture conditions, such as nature and con- centration of carbon and nitrogen sources, media composition, pH, temperature, presence of inducers and lignocellulosic materials, etc.
A wide variety of lignin degradation efficiency and selectivity abilities, enzyme patterns and substrates enhancing lignin degradation are reported from white-rot fungi. An interesting category of white-rot fungi are selective degraders that degrade lignin rather than cellulose, like Pleurotus spp., which are used in a wide range of biotechnological applications. Lignin degradation by these fungi is thought to occur during secondary metabolism and typically under nitrogen starvation. Non-composted, chopped and water-soaked straw is sufficient for the cultivation of Pleurotus spp., while L. edodes is cultivated on logs or in bags on moisturized sawdust supplemented with cereal bran.
Although not necessarily optimal, since they are low in readily accessible nutrients, these commercially used substrates satisfy the needs of the fungi for growth and fruiting, and most importantly, help to withstand microbial competitors. In basidiomycetes LDF, comprising cultivated mushroom species like Agaricus spp., Agrocybe spp., Coprinus spp., Stropharia spp. and V. volvacea, degradation involves a succession of biodegradative activities that precede attack by lignocellulose degraders.
However, the ability to break down lignin and cellulose enables some of the LDF to function as typical "white-rot fungi" in soil. Well known mushroom forming LDF are A. bisporus and V. volvacea, both grown commercially on composted lignocellulose. As A. bisporus contains lignolytic enzymes, degrades both cellulose and lignin, the former more rapidly. Compost prepared from straw, horse or chicken manure, calcium sulphate (gypsum), water and some nutritional supplements is a cheap cultural substrate for A. bisporus and some other saprophytic basidiomycetes. Manure in the compost serves as N source, straw as C source. It must be pointed out that after the initial medium preparation stage, little control can be exerted over the composition of the solid substrate medium.
In composted substrates this is particularly crucial since the nutrient composition of the initial medium ingredients has to allow both a successful composting process and good fungal colonization and fruiting. Mushrooms have a two-phase life cycle, the mycelium (vegetative or colonization phase) and the fruiting body (reproductive phase that bears the spores). The mycelium grows through the substrate, biodegrades its components and supports the formation of fruiting bodies. Mushroom growers call the switch from mycelial extension to the production of mushroom primordia "pinning", the successive development of primordia into mushrooms "fruiting". While growth of mycelium lasts for several days, weeks or months, production of fruiting bodies is short lived, and the phenomenon is called `fructification'. However, both vegetative and reproductive phases are very much influenced by the physiological condition and nutritional state of the mycelium.
Since the carbon sources utilized by basidiomycetes are usually of a lignocellulosic character, fungi during vegetative growth produce a wide range of enzymes to degrade the lignocellulosic substrates. Data obtained in various studies demon- strate that the type and composition of lignocellulosic substrate appear to determine the type and amount of enzyme produced by basidiomycetes fungi during vegetative growth.
Moreover, cellulose/lignin ratios of wheat straw and cotton waste substrates were positively correlated to mycelial growth rates and mushroom yields of P. ostreatus and P. pulmonarius and with the yield of V. volvacea (Philippoussis et al. 2001a). According to KЁ ues and Liu (2000), considerable changes in enzyme activities occur during fruiting, indicating a connection to the regulation of fruiting body develop- ment. For example, in A. bisporus and L. edodes, laccase activities are highest just before fruiting body initiation and decline rapidly with primordia formation. Cellulase activities are highest when fruiting body develops. Regarding the influence of nitrogen availability, recent studies revealed a positive correlation between the C/N ratio and P. eryngii mushroom yield. They also demonstrated that mycelium growth rates of Pleurotus spp. and L. edodes were positively correlated to C/N ratio. Similar conclusion was drawn by Silva et al., indicating that L. edodes extension rate is related to bioavailability of nitrogen and is enhanced by supplementation with cereal bran.
Moreover, both nature and concentration of nitrogen sources are factors regulating enzyme production by wood rotting basidiomycetes, e.g. in L. edodes cultivation on wheat straw, nitrogen supplementation represses MnP and enhances laccase activity. According to KЁ ues and Liu (2000), for fruiting body induction it is of importance to keep a balance between C and N sources, e.g. in A. bisporus compost, the optimal C/N ratio for fruiting has been determined to lie between 80:1 and 10:1. In addition, substrate supplementation with protein-rich materials proved to enhance yield of Agaricus, Pleurotus and Lentinula strains.
Apart from nutrition, mycelial growth and fruiting of basidiomycetes fungi are also regulated by temperature, gaseous environment, water activity and in certain cases by light. During substrate colonization, the effect of environmental parameters plays an essential role on mycelium growth, and hence confers significantly to the success of the entire cultivation process. In addition, the duration of the substrate colonization phase is of direct economic importance, since media that are non-thoroughly impregnated with the hyphae, are sensitive to fungal and bacterial infections resulting in reduced yields. Production of the vegetative mycelium usually occurs over a wide range of temperatures.
Zervakis et al. (2001) examined the influence of temperature on mycelium linear growth of P. ostreatus, P. eryngii, P. pulmonarius, A. aegerita, L. edodes, V. volvacea and A. auricula-judae. Their temperature optima were found to be 35?C for V. volvacea strains, while P. eryngii grew faster at 25?C, P. ostreatus and P. pulmonarius at 30?C. Moreover, A. aegerita grew faster at 25?C or 30?C and A. auricula-judae at 20?C or 25?C depending on the nutrient medium used, and L. edodes at 20?C or 30?C depending on the strain examined. It is generally believed that basidiomycetes tolerate relatively high levels of salts for growth, but fruiting body development can be more sensitive. Likewise, mycelial growth is less affected by pH but fruiting body development of several species occurs best at neutral or slightly acidic pH values around 6-7 (Wood and Smith 1987) or, in L. edodes, at a pH 4.0 (Ohga 1999). On lignocellulosic substrates, Pleurotus and Lentinula species are growing with a linear rate (Philippoussis et al. 2001a, Diamantopoulou and Philippoussis 2001), which is influenced by substrate salinity and porosity.
Measurements of electric conductivity through the entire colonization process of three residue-substrates by L. edodes strains revealed an increase of salinity values until mycelium colonized 60 to 75% of the substrate, and then it slightly declined or remained constant until the end of incubation, presenting the highest and lowest values in the wheat straw and oak sawdust media respectively. In addition, a negative correlation was established between final salt content of the substrates and mycelium extension rates. Furthermore, monitoring of CO2 concentrations in pilot-scale cultivation of L. edodes on synthetic blocks, revealed higher respiration rates on oak sawdust and corncobs than on wheat straw, which are further correlated with substrate colonization rates. Following colonization of the substrate, fruiting is induced by environmental and/or cultural manipulation. The optimal environmental parameters for mycelial growth and the subsequent fruiting are usually very distinct. Depending on the species and the degree of investment in environmental control technology, temperature is normally manipulated by heating or cooling systems to maintain the optima for vegetative growth or fruiting.
However, fruiting body development is often induced after drastically altering the environmental parameters, usually favored by reducing the temperature by at least five ?C compared to mycelium growth. In fact, fruiting is typically induced, after vegetative growth, e.g. in A. bisporus to 16-18?C in P. ostreatus to 15?C, and in L. edodes to 10-16?C for the cold temperature strains and 16-21?C for the warm temperature strains. Other parameters of fruiting body initiation and matura- tion include CO2 concentration, humidity, salinity and pH. High humidity (90-95%) is favorable for pinning and fruiting but the moisture content of the substrate might be even more critical. The optimal water content for wooden substrates is 35-60% and for other substrates 60-80%. The lower values reflect the oxygen demand of the fungi in the substratum, balanced against their requirement for water. Carbon dioxide (CO2) level is also critical for efficient mycelial growth, fruit body initiation and fruit body development. Higher CO2 concentrations (e.g. 1% v/v in air) may stimulate mycelial growth and inhibit fruiting. Increased aeration is used to reduce CO2 levels, which otherwise produces increased elongation of stipe growth and abnormality of cap development. Light has been implicated in the fruiting of several mushroom genera e.g. Lentinula and especially Pleurotus species have an obligate requirement for light for fruiting induction. Brief exposure of the culture to daylight or suitable artificial light is sufficient.
Usually, light positively influences hyphal aggregation and fruiting body maturation. However, light is not needed for the fruiting of A. bisporus.
3. Output and Stages of Mushroom Cultivation
Mushroom industry presents a worldwide expanded and economically important biotechnological application, which can be divided into three main categories: cultivated edible mushrooms, medicinal mushroom products and wild mushrooms, with an annual global market value in excess of $45 billion. The global annual mushroom output (including production and wild mushroom collection) surpass nowadays 10 million metric tons, with China being the top world producer (about 8.000.000 tons), followed by Europe and USA.
Commercial mushroom production is an efficient solid state fermentation process of food protein recovery from lignocellulosic materials carried out on a large or small scale. Taking into account the value and volume of the product, the number of people involved in the industry, or the geographical area over which the industry is practiced, mushroom cultivation is the greatest application of exploitation of filamentous fungi using SSF and the biggest (non-yeast) biotechnology industry in the world. The economic strength of mushroom cultivation derives from the successful use as feed stocks of a variety of low- or negative-value residues from agriculture, forestry or industry. These wastes are processed using relatively cheap microbial technology to produce human foodstuff, which could also be regarded as a functional food or as a source of drugs and pharmaceuticals.
Moreover, the effective exploitation of resources from agricultural solid wastes and by-products, rich in organic compounds that are worthy of being recovered and transformed, is a sound environmental protection strategy. There are three major stages involved in mushroom cultivation: (1) inoculum (spawn) production, (2) substrate preparation, and (3) mushroom growing i.e. inoculation of the substrate with propagules of the fungus, growth of the fungal mycelium to colonize the substrate, followed by fruiting, harvesting and processing of the fruiting bodies. Inoculum (spawn) production. In order to achieve reliable and vigorous fungal growth and fruiting bodies production of good quality, inoculum fungal cultures are necessary. Inoculum is produced by inoculation of sterilized cereal grains (usually wheat, rye or millet) from high quality stock mycelial cultures.
Essential prerequisite is the selection and breeding work to acquire suitable biological material for commercial cultivation, which ensures good yield and quality. The various mushroom inoculate are often the only micro biologically pure part of the whole technology. Spawn-making is a rather complex task, not feasible for the common mushroom grower, and is produced by specialist companies (spawn-makers) using large scale bulk autoclaving, clean air and other microbiological sterile techniques for vegetative mycelia cultures onto cereal grains, wood chips and plugs or other materials. The colonized cereal grain/mycelium mixture is called spawn and is grown under axenic conditions in autoclavable polyethylene bags, ensuring gas exchange, or rarely in jars.
Finally, after quality control to assure biological purity and vigor, spawn is distributed from the manufacturer to individual mushroom farms in the same aseptic containers used for spawn production. Substrate preparation. Fermentation process involves cultivation on specific substrates imitating the natural way of life of mushroom fungi. Regarding the litter-decomposer A. bisporus, this has come to mean cultivation of a mushroom crop on composted plant litter. On the other hand, the white-rot mushroom fungi Pleurotus spp and L. edodes are cultivated on non-composted lignocellulosic substrates, using methodologies that exploit their ability to produce enzymes capable of degrading all wood components.
The first stage of mushroom production has to do with assembly and treatment of the substrate to prepare a growth medium. The substrates used for mushroom production, varying according to cultivated species, are prepared from waste agricultural or forest product materials using ingredients such as manures, cereal straws or other crop residues, saw dusts etc. In certain cases the substrate is be directly inoculated and require very little pre-treatment, e.g. L. edodes production using logs. In other cases, the substrate is microbiologically or physically pretreated. Microbiological pre-treatment normally comprises some form of controlled bulk composting process. Physical pre-treatment could include steam treatment or sterilization by autoclaving. Substrates for fungal growth can be prepared as sterile materials, to produce an axenic growth medium, e.g. bottle cultures of F. velutipes, or be non-sterile, e.g. compost substrates to produce A. bisporus.
One of the aims of substrate preparation is to introduce sufficient water into the substrate to ensure that the water activity of the final medium is optimal for fungal growth. The scale of substrate preparation varies according to the type of species to be cultivated and the size of the production unit. For A. bisporus production, many tons of straw are processed per day to produce compost. Thus, large-scale bulk handling machinery is used for this process.
After the initial preparation stage, little control can be exerted over the composition of the solid substrate medium. In composted substrates, this is particularly crucial since the nutrient composition of the initial medium ingredients has to allow both a successful composting process and good fungal colonization and fruiting. Although nutrient balances and status, e.g. for carbon, nitrogen, pH and other components, can be measured on the initial ingredients, little can be done to regulate the quantity or feed rate of these once the production processes are under way. Mushroom growing. This stage deals with the two phases of mushrooms life cycle i.e. the mycelium (vegetative phase) and the fruiting body formation (reproductive phase).
Following inoculation, the mycelium, grows through the substrate, biodegrades its ingredients and supports the formation of fruiting bodies. Mycelial growth and fruiting during this stage are regulated by temperature, gaseous environment, nutrient status, water activity and in certain cases by light e.g. Pleurotus spp. has an obligate requirement for light for fruiting induction, Agaricus spp. have no light requirement . The level of environment and cultural control used is determined by the type of production technology. In controlled environment growing system, temperature is manipulated by heating or cooling systems to maintain the optima for vegetative growth or fruiting. Carbon dioxide (CO2) level and humidity are also controlled. Basidiomata production on the culture medium surface occurs as a series of cycles (flushes). Depending on the fate of the harvested product as fresh or preserved material, the fruit bodies are harvested either by hand or mechanically and processed accordingly. After harvesting, mushrooms are normally cooled down to retard fruiting body metabolism, packed and sent to the fresh market, or processed further through freezing, canning, drying etc., depending on marketing strategies.
mushroom fung cultivation chemical
Current mushroom industry is based on both application of techniques for the production of mushroom fruiting bodies and the application of modern biotechnological techniques to produce medicinally beneficial compounds and nutraceutical products. The medicinal properties of bioactive substances, like polysaccharides with antitumor and immune-stimulating properties occurring in higher basidiomycetes have become a subject of numerous recent reviews.
Among them, the commercial polysaccharide of L. edodes, lentinan, has been researched extensively as it offers the most clinical evidence for antitumor activity. Recently, some mushroom polysaccharides have shown to exert a direct cytotoxic effect on cancer cells in vitro. Moreover, solid-state fermentations other than fruiting body production are suggested for upgrading and valorizing lignocellulosic residues using basidiomycetes cultures, either through protein enhancement and transformation of residues into animal feed, or for enzyme production. In the first case, agro-industrial residues such as rice straw, coffee pulp, sugarcane bagasse, banana leaves etc. have been fermented by white-rot basidiomycetes to improve the digestibility of the residues for use as ruminate feed supplement. In the second case, lignocellulose degrading mushroom species like Pleurotus sp, Lentinula edodes, Trametes versicolor, Flammulina velutipes are used for the production of enzymes of industrial importance, such as cellulases, xylanases and laccases, using as substrates agro-industrial residues, from which wheat straw and bagasse are the most commonly used substrates.
Most results, however, come from laboratory, or semi-pilot-scale experiments. Additionally, lignocellulolytic mushroom fungi like Pleurotus ostreatus and Trametes versicolor have been investigated for bioremediation and biodegradation of toxic and hazardous compounds like caffeinated residues as well as toxic chemicals such as pesticides, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), chlorinated ethenes (CIUs) etc., in polluted soils or contaminated ground water. In terms of food production process, the aim of mushroom growing should be to follow the holistic concept of production, according to Laufenberg et al. This approach tries to connect differing goals, such as highest product quality and safety, highest production efficiency and integration of environmental aspects into product development and food production.
An outstanding example of integrated crop management practice of mushroom cultivation is the use of spent substrate, that is the residual growth medium after cropping (1) as animal feed, since the mushroom mycelium boosts its protein content, (2) as soil conditioner and fertilizer as it is still rich in nutrients and with poly- meric components that enhance soil structure, (3) as a source of enzymes, (4) for the biological control of plant pathogens and even (5) used for bioremediation purposes as to digest pollutants on land-fill waste sites because it contains populations of microorganisms able to digest the natural phenolic components of lignin. In this concept, solid state fermentation processes are not only the methods of mushroom production for food and nutraceutical purposes but also examples of an organic system integrated with waste treatment that contributes to sustainability and benefits the human population, health and environment.
1. Albores S, Pianzzola MJ, Soubes Metal. (2006) Biodegradation of agroindustrial wastes by Pleurotus spp for its use as ruminant feed. Electron J Biotechnol 9(3): 215-220
2. Zervakis G, Philippoussis A, Ioannidou S et al. (2001) Mycelium growth kinetics and optimal temperature conditions for the cultivation of edible mushroom species on lignocellulosic substrates. Folia Microbiol 46(3): 231-234
3. Thomsen MH (2005) Complex media from processing of agricultural crops for microbial fermentation. Appl Microbiol Biotechnol 68: 598-606
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