Recent Advances In Substrate Preparation

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Lignocellulosics account for about 60% of the total plant biomass and are the most abundant natural raw material present on the Earth. These are produced through photosynthetic reactions operating inside the cells of green plants and the net dry biomass production by plants is estimated to be 155 billion tons per year [187]. Lignocellulosics contain mainly cellulose, hemicellulose and lignin. This inexhaustible and renewable natural source can be utilized through microbes for the production of industrial chemicals, liquid fuel, protein rich food (mushroom or single cell protein) and feeds.

Mushrooms, particularly white button mushrooms A. bisporus and A. bitorquis, draw the nutrients from a specially fermented materials of agricultural origin. All the necessary nutrient components like carbon, nitrogen, phosphorus, iron, sulphur, potassium and vitamins required for mushroom growth are assimilated from this specially fermented substrate called compost and the process of its artificial preparation by adding all the requisite ingredients is called composting. It is a multistep process guided by a consortium of microorganisms including fungi, bacteria and actinomycetes. Role of thermophilic fungi in composting has been well-elucidated and the reports have also proved their role in obtaining desired selectivity in the substrate [188]. Some of these fungi not only help in obtaining the desired selectivity in the mushroom substrate but also contribute themselves to the Agaricus nutrition [189]. During the process of composting, also called substrate preparation, the readily available nutrients like carbohydrates and nitrogen are assimilated by the microorganisms present in compost ingredients and organisms multiply with the process of composting and as a result large fraction of nutrients is locked up inside the microbial cells present in the substrate and mushrooms have the ability to utilize these nutrients with the aid of their cell hydrolyzing enzymes [190]. The lignin-humus complex formed after fermentation can only be attacked by mushrooms because of the specific degradative enzymes producing capacity and thus selectivity in the mushroom substrate is achieved.

The fermentation process operating in the mushroom substrate preparation is different from other fermentation processes because of the defined attributes of the mushroom substrate which help in obtaining highest possible yield of mushrooms with little chance of failure because of undesired moulds, insect and pest infestation. It has been reported that the end product obtained in mushroom substrate fermentative-process must have a desired bulk density of about 550 to 600 kg m3 [191], selective only for A. bisporus [192], sufficient water holding capacity [193], sufficient air space for the growth of aerobic mycelium [194], specific nitrogen level of about 1.75% [195] and suitable pH ranging from 7.2 to 7.8 [195],

4.1 Factors Affecting Conversion of Lignocellulose into Fungal Biomass

Important factors affecting lignin degradation are temperature, pH, water content [196], carbon dioxide and oxygen levels [197]. Carbon dioxide, in general, is taken as inhibitory and oxygen as stimulatory in such processes [196,198]. The addition of other nutrient sources like nitrogen promotes the bioconversion of non-lignin fractions and inhibit lignin bioconversion [196,199]. But the most important factor which affects compost quality is the microbial population stimulating the fermentative process. Therefore, for the improvement in methods of mushroom substrate preparation, the knowledge of basics underlying this process is very important. The degradative process in the composting passes through phases like mesophilic in the beginning, followed by thermophilic in the middle and again mesophilic in the end. All these phases ultimately lead to the production of CO2, water, minerals and a stabilized organic matter called compost. The process the composting finally results in 15-35% weight loss due to the release of metabolic by products like CO2, H2O and other gaseous materials [200].

4.2 Methods of Composting

With due consideration to the quality of the product expected and parameters involved in composting, following three methods of compost preparation were evolved over the time as a process of improvement.

Long method of composting. This is the oldest method and now exists only in few pockets of the world because of the low mushroom yields, proneness to attack by pathogens, and more time as well as labour consuming process [194], This method is a completely outdoor activity and takes about 28 days, though production of this compost in lesser duration has also been reported [201]. The biomass loss in this method is also very high [30-35%] and also creates more environmental problems like release of unwanted harmful gases. The microbiological and chemical reactions operating inside the substrate to convert it in a suitable medium for mushroom growth remain unsystematic under uncontrolled environmental conditions of composting. The quality of compost is poor and often it causes heavy crop losses under unhygienic conditions of cropping rooms due to which it has presently limited relevance.

Short method of composting. With many shortcomings associated with the long method of composting and with the realization of importance of temperature and microbes in composting process, improved method of substrate preparation with scope of temperature modulation came in existence near 1950's. The original thinking behind this process was to have a temperature of 50 to 60°C for obtaining a selective substrate with very less chances of infestation by problematic moulds, insects and pests. This was based upon the work of an American Scientist, Lambert [202], who studied the compost from different zones having different temperature and oxygen conditions and found that productive compost came from regions having temperature between 50-60°C and adequate supply of 02 Based upon the findings of Lambert, Sinden and Hauser [203,204] came with a new concept of substrate preparation having pasteurization as the integral part of composting process. This process of composting was later on called as the short method of composting because it took comparatively less time than the long method of composting. The short method of composting mainly consists of two phases: outdoor composting for 10-15 days (Phase-1), followed by pasteurization and conditions inside an insulated well built structure called as the pasteurization tunnel taking around another seven days. In this method, the remixing/turning of substrate ingredients is carried out almost on every alternate day so that alteast 5 to 6 turnings can be achieved in 10 days of outdoor composting (Phase-I). Another important parameter is the maintenance of aerobic condition by blowing oxygen @1 to 15 m3/ton of wet compost/hour which facilitates high temperature, production of more homogenous substrate in comparatively 30% less time than long method of composting [205]. The desired quantity of air can be inserted either by making compost stacks on the GI pipes with holes and connected to an air blower or by ventilating the stacks by inserting vertical ducts in the stack [206]. During phase-I of composting the temperature can rise up to 80°C into the core of stack resulting into charring of the ingredients giving a dark brown colour of the compost. However, the outer surface having a temperature around 50-60°C facilitates the growth of thermophilic microorganisms. The compost after completion of Phase-I of composting should be of blackish colour having moisture around 72-75%, very heavy smell of ammonia, pH around 8-8.5, nitrogen content between 1.5- 2.0 with ammonia concentration around 800-1000 ppm [207]. Based upon the temperature conditions maintained inside the pasteurization tunnel (phase-II of composting) it can be divided into two sections i.e. pasteurization and conditioning.

  1. During phase-I of composting, different zones of the stack possessdifferent levels of temperature and different types of microorganisms both qualitatively as well as quantitatively. However, during pasteurization a homogenous environmental conditions is achieved and temperature is maintained between 57 to 60°C for 6-8 h. Temperature beyond 60 °C is not recommended because it leads to elimination of some beneficial thermophilic microorganisms having role later in compost conditioning. The temperature of 60°C can be achieved either by self-heating of the compost or by injecting steam; in either of the cases this temperature is sufficient to kill the problematic insect/pests of the mushroom crop. The time and duration of pasteurization has less bearing on the crop yield and it can be effected immediately after tunnel filling or later on [208].
  2. During conditioning the temperature of the bulk is maintained in the range of 45 to 52°C suited for the growth of desirable thermophilic microorganisms. The excess of ammonia released during this stage is utilized by the growing microorganisms and gets entrapped as microbial protein/or biological nitrogen. The high population of thermophiles achieved at this stage is helpful for whole of the composting process because majority of them perish during the pasteurization process and some of the left outs will help in building up the requisite population during post-pasteurization conditioning process [209], Besides the temperature, another important factor affecting the outcome of the conditioning process is the sufficient supply of oxygen. Oxygen level of about 10% during this process is essential to build up requisite number of thermophiles and assimilation of ammonia in the form of microbial protein. Both pasteurization and conditioning are essential for achieving the selectivity in the compost at the expense of harmful organisms. There are many advantages of short method of composting: (i) more compost per unit weight of the ingredients is produced, (ii) more mushroom yield per unit area of cropping room or per unit weight of compost, (iii) less chances of infestation from insects and infection from competitor or pathogenic moulds, (iv) shorter duration of substrate preparation and cropping cycle, and (v) less environmental pollution.

Indoor composting. The problems of environmental pollution associated with the long method and to a lesser extent with short method of composting drew the attention of researchers to evolve alternative methods where production of stinking gases like ammonia, methane, hydrogen sulfide and other methylated sulfur compounds can be brought to their minimum level [210-213]. The gases are usually produced under high temperature and anaerobicity inside the compost mass [214], The production of these gases also depends upon the type and quantity of the basal ingredients used for substrate preparation [215]. In Europe and Australia, the strict environment safety related legislations forced many of the companies to close their composting units. So a 'clean' process was in demand which could produce the high quality mushroom substrate without any adverse effect on the environment [216], Because first time the work on such composting system started using completely indoor system for compost preparation, it is also called as indoor composting [217], or environmentally controlled composting [218], rapid indoor composting and aerated rapid composting [219], Though the work initially started with omitting the phase-I completely [220] which resulted in sharp decrease in mushroom yield [221] but, later on, the phase-I of composting was tried inside tunnel with proper arrangement of forced air and satisfactory results were obtained [222,223]. On the basis of temperature conditions maintained inside the tunnel, the indoor composting can be divided into two categories i.e. INRA methods and Anglo-Dutch method.

INRA method is mainly followed in France, Italy, Belgium and Austria and large quantity of compost is being prepared by this method [224]. In this process the phase-I is carried out at a constant temperature of 80°C for 2-3 days under indoor conditions followed by phase-II in which the temperature is kept at 50°C for 5-7 days [225]. As very high temperature attained during phase-I results in killing of all desirable microbes also [226] reinoculation with mature compost or thermophilic fungi becomes necessary in this process [227,228,229], In situations where proper ventilation in the tunnel during phase-I is maintained or the phase-I is being carried out in the bunker system, reinoculation with thermophiles can be dispensed with because comparatively cooler regions in the stack support the existence of thermophiles [193,230], Work of evolving a suitable formula for inoculation of Scytalidium thermophilum, desirable for this important thermophilic fungus for obtaining compost of desired quality has also been carried out [231],

The another method called as Anglo-Dutch method was the result of continuous research in several European countries and Australia [216,232,233,234] which basically involves the maintenance of an optimum temperature for the growth of thermophilic micro-organisms particularly Scytalidium thermophilum and two species of Humicola [235], In this method, a short pasteurization phase of 4-6 hours at 60°C is followed by a week-long conditioning phase at 41°C. This method has been reported to provide excellent selectivity with excellent outdoor control and substantial saving of raw material [191,193]. However, on the contrary some problems like light texture of finished compost which poses problems during compressing [236] and low yield with lower level of selectivity have also been reported [210]. The importance of physical parameters like thorough mixing of ingredients, adequate moisture and good oxygen supply was studied in detail in two Dutch firms, Christiaens and Gieoam and all these factors were found to be important for minimising toxic compounds and malodorous gas production [237]. Some advatages of indoor composting over other methods are: (i) composting takes lesser time than the short and long methods [236], (ii) the mushroom yield per unit weight of compost is very high [30-35kg/100kg of compost [224], (iii) efficient control and management of environmental pollutants conforming to civic laws, (iv) lesser loss of raw materials [10-15%] and thus increased end-product [193], and (v) improved selectivity of mature compost for A. bisporus [191]

4.3 Role of Microbes and Their Metabolites in Composting

Compost being a rich source of nutrients harbours a diversified microbial flora which includes bacteria, fungi, actinomycetes, viruses, helminths and protozoa [238,239] and their qualitative as well as quantitative existence depends upon the ingredients and the geographical location [240]. Generally, a microbial progression from mesophilic to thermophilic and again to mesophilic is observed during composting. The factors which determine the dominance of a particular microbial flora during composting depend upon the temperature, moisture, aeration, hydrogen ion concentration (pH) and microbial interactions prevailing inside the compost substrate [241]. Recently molecular techniques like sequencing of rDNA genes have also been employed in monitoring the microbial diversity in mushroom compost [242]. In fact, the compost ingredients harbour varied types of microflora but the fungi have been studied by majority of the researchers. The fungi found growing saprophytically on the cereal straw can be divided into three major groups [207]: (i)rimary saprophytes: Alternaria alternata, Cladosporium cladosporoides, Doratomyces stemonitis, Stachybotrys atra, Stilbum nanum, (ii) fungi growing on stored materials: Aspergillus spp., Penicillium spp., Absidia ramosa etc., and (iii) opportunist fungi: Mucor hiemalis, Rhizomucor pusillus and Trichoderma viride etc.

Work [243,244] has also been carried out on mesophilic fungi of chicken manure, an important ingredient of modern day mushroom compost, but majority of these fungi disappear as soon as the temperature of compost pile attains the thermophilic range. Fungi flourish where growth conditions are sub-optimal for other microorganisms [bacteria and actinomycetes] for example lower moisture contents of substrate [245] and lower pH levels. In the initial stage, mesophilic organisms, mainly bacteria, begin to breakdown and utilize the easily available carbohydrates and proteins [246] and the heat generated leads to rise in the stack temperature which is further enhanced by the insulating properties of the compost mass [247]. This rise in temperature results in change in the microflora from mesophilic to thermophilic and less diverse [248,249], With enhanced metabolic activities, the temperature continues to rise and optimum degradation rates are reported at a temperature range between 45 to 55°C [250]. Excessive temperature is not conducive for proper composting because it inactivates most of the microbial population as well as their activities [211],

Several studies have been conducted to elucidate the role of thermophilic fungi in the composting, which led to the development of the latest method i.e. indoor composting. During the rapid multiplication of thermophilic fungi cellulose and hemicellulose are broken down to expose the available lignin for selectivity to the compost. The thermophilic fungi also utilize available proteins and other nitrogenous substances during their growth which results in production of ammonia which further raises the temperature and softens the straw. The available nitrogen and ammonia during indoor composting is 'locked' inside the microbial cells as this microbial protein is utilized by the mushroom mycelium for its nutrition [190]. The role of thermophilic fungi in composting and as nutrient of Agaricus bisporus has been studied by several workers [188, 251-254], Among different thermophilic fungi, the role of S. thermophilum and Humicola spp. has been highlighted in most of the cases [231,253-257], Specific studies like enhanced growth of A. bisporus mycelium on H. insolens encapsulated grains and subsequent better growth of such spawn in mushroom compost further confirmed the importance of thermophilic fungi during composting [258]. Based upon the findings with thermophiles Nair [259], Nair and Price [233], Savoie and Libmond [260] and Savoie et al. [261] used PRIT AN, an accelerator, bacterial polysaccharides and Express™ for further improving the composting techniques. In these studies several beneficial effects like enhanced process control, less time, better environment protection and good yields were obtained.

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