Nov 11, 2011 - gliding net and then with a drag net. Two reactors of the same shape and construction are used. One reactor is used for substrate pretreatment ...
Journal of Applied Animal Research
ISSN: 0971-2119 (Print) 0974-1844 (Online) Journal homepage: http://www.tandfonline.com/loi/taar20
Bioconversion of Lignocellulose into Ruminant Feed with White Rot Fungi—Review of Work Done at the FAL, Braunschweig F. Zadrazil , D. N. Kamra , O. S. Isikhuemhen , F. Schuchardt & G. Flachowsky To cite this article: F. Zadrazil , D. N. Kamra , O. S. Isikhuemhen , F. Schuchardt & G. Flachowsky (1996) Bioconversion of Lignocellulose into Ruminant Feed with White Rot Fungi—Review of Work Done at the FAL, Braunschweig, Journal of Applied Animal Research, 10:2, 105-124, DOI: 10.1080/09712119.1996.9706139 To link to this article: http://dx.doi.org/10.1080/09712119.1996.9706139
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Date: 14 November 2016, At: 17:45
J. Appl. Anim. Res. 10 (1996) : 105-124
Bioconversion of Lignocellulose into Ruminant Feed with White Rot Fungi - Review of Work Done at the FAL, Braunschweig F. Zadrazill, D.N. K a m r a * l , 0 3 . I s i k h u e m h e n * * l , F. S c h u c h a r d t 2 , G. Flachowsky3 1
Institut fur Bodenbiologie Institute fur Technologie 3 Institut fur Tierernahrung Bundesforschungsanstalt fur Landwirtschaft Bundesallee 50, 38116 Braunschweig, Germany 2
(Received February 24, 1996; accepted September 10, 1996)
Abstract Zadrazil, F., Kamra, D.N., Isikhuemhen, O.S., Schuchardt, F. and Flachowsky, G. 1996. Bioconversion of lignocellulose into ruminant feed with white rot fungi -- Review of work done at the FAL, Braunschweig. J. Appl. Anim. Res.,lO: 105-124.
The work on bioconversion of lignocellulosic agricultural by products for the production of feed and food has been going on for the last two decades at the Institut fiir Bodenbiologie, FAL Brartnschweig, Germany. Aborst 300 strains of basidionzycetes have been screened for their ability to degrade lignin and cause a change in in vitro dry matter digestibility of various lignocellrilosic agricultural by products. Aniong the fungal strains tmted, some species of Pleurotus, Ganoderma, Stropharia, Polyporus, Abortiporus, Dichomitus, Lentinus, Sporotrichum and Trametes have been forcnd to degrade lignin selectively and also *Animal Nutrition Division, Indian Vi>twinnry Research Institute, Izatnagar **Dept. of Botmy, University of Benin, T h i n , Nigeria.
105 J. Appl. Anim. Res. 0971-2119/96/$05.00 0 GSP, India
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249 122, India.
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increase the in vitro digestibility significantly. The fermentation parameters for cultivation of Pleurotus for food and feed have been studied in detail and a 1.5 tonne capacity solid state fermentation reactor has been constructed. Several experiments for bioconversion of wheat straw with Pleurotus sajorc@ have been conducted in this reactor and the results of increase in digestibility have been found to be comparable with wheat straw treated with chemicals like sodirinz hydroxide, urea or ammonia. Key words: Bioconversion, lignocellulose, feed, ruminant, white rot fungi, review, delignification, straw.
1.
Introduction
Cereal straws and other agricultural by products contain 60-70% of carbohydrates (Jackson, 1977) and are comparable to good quality hay in chemical composition. The lignin complexed with cellulose and hemicellulose makes the carbohydrates of these lignocellulosics less accessible to microbial attack in the rumen. Although these lignocellulosic by-products are used as staple ruminant feed in many countries, but there are some problems associated with this practice e.g. poor palatability, low digestibility and insufficient nutrient availability to the animals. I n order to increase the digestibility of lignocellulose, physical, chemical and biological methods of delignification can be used as summerized by Sundstol and Owen (1984)) Flachowsky (1987) and Zadrazil et al. (1995). I n this review fungal activities on lignocellulosics are briefly summarized. The main problem of biological upgrading of lignocellulosics into feed is to find suitable microorganisms, with metabolic patterns different from those of the rumen flora and fauna for use in cheap large scale process of delignification and upgrading. Proposed processes for upgrading lignocellulosics must be characterized by strong lignin decomposition with contemporary accumulation of digestible substances. Lignolytic microorganisms are mainly wood inhabiting fungi. They are able to colonize different plant residues (Zadrazil, 1976, 1979) and increase the digestibility of the substrate (Schanel et al., 1966; Kirk and Moore, 1972). "Ideal microorganisms" for upgrading lignocellulosics into animal feed should have a strong lignin metabolism with a low degradation of cellulose and hemicellulose so that in the treated product, carbohydrates are conserved for their utilization by the rumen microbes and the animal as energy source.
Bitxlottuersion.
2.
of
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ligg,roirlltdose into nciirinuz,it fiecl w i t h white rot fiingi
Ecological background of conversion of lignocellulosics into animal feed
White rot fungi (Ganodernta applanatuna and Armillariella sp.) decompose and upgrade wood in South Chile into Palo podrido (a decomposed wood, used as animal feed). Feeding of Palo podrido was described for the first time by Phillippi (1893). Digestiblity, content of water soluhle substances and lignin were estimated by Zadrazil et. al. (1982). Non decomposed wood showed very low in uitro digestibility (ca. 3 units) in contrast to modified wood by fungi (30-60 units).The lowest lignin content was estimated in white rot degraded wood (1%)and highest in brown rot degraded wood (77%). This degradation of wood in nature was used a s a model for following laboratory- and pilot scale-studies. 3.
Factors influencing in vitro digestibility
3.1. Fungal species The influence of fungal species on the decomposition of wheat straw and in uitro digestibility (IVDMD) and decomposition of lignin has been comprehensively discussed by Zadrazil (1985). The different physiological behaviours shown by these fungi have been used to divide them into four groups (Table 1) and this is in agreement with the classification of Rypacek (1966). Table 1 Characteristics of lignocellulose degrading fungi Groups Characteristics
a
Decomposition of nutrients
Cell contents, cellulose and hemicellulose
Influence on IVDMD Typical fungi
Decrease (-)
Agrocybe aegerita Flam.rn.ri1in.a uelutipes Voluariella. uol uacea
b Selectively lignin, other substrates only partially Increase (t)
Uich.omi!us sqiialen,s Aborfiporus biennis Pleurotus spp. Stropharia rrigosoannu la t a
C
Lignin, cellulose, hemicellulose
d
Lignin and other substrates rapidly Decrease (-) or Not much no change change (t) Some strains Sporotriczm. of I1olypoi-r~.s, pulueixlentuin. Gan.oclerina and Plerimliis spp.
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F. Zadratil a i d rouiorkers
3.2. Substrate composition and quality Different substrates, including .beech sawdust, rape straw, reed stems, sunflower stalks, rice husk etc. have been tested with different fungi for the rate of decomposition of organic matter, lignin and changes in in vitro dry matter digestibility (Table 2). Table 2 Loss of organic matter (OM) and lignin and changes in in uztro dry matter degradability (IVDMD) of different agricultural by-products treated with higher fungi.* -
Fungus
Loss of (76)
Substrate -
OM Stropharia rugosoannulata Beech sawdust Reed stems Rape straw Sunflower stalks Rice husk Pleurotus sp. Florida Beech sawdust Reed stems Rape straw Sunflower stalks Rice husk Pleurotus comiicopiae Beech sawdust Reed stems Rape straw Suiiflower stalks Rice husk Beech sawdust &roc-ybe aegeiita. Reed stenis Rape straw Sunflower stalks Rice husk ___
18.9 22.8 28.0 24.4 19.9 17.2 29.5 30.1 27.4 9.1 12.0 26.2 44.5 43.2 ND 3.9 8.4 24.0 17.7 6.6
.-
Ligiiin 46.5 33.9 58.2 46.8 42.1 34.7 55.8 67.6 60.3 21.3 20.1 35.5 68.1 59.6 ND 5 .6 2.0 36.0 13.4 12.6
Changes in rVDMD** (%) 12,.4 16.1 48. 2 -2.3 -5.4 28.8 15. 0 37.1 20. 8 -9.4 11.5 32.6 27.8 22.9 ND -3.1 -13. 7 -6.7 -8.7 -7.9
'Zadrazil (198O).**IVDMD of untrealctl substrates (%): Beech sawdust (6.1), Reed stvms (29. 9) Rape straw (34.1),Sunflower stalks (41.4) and Rice husk (14.8). NL) not rletermintd
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Bitwonversion of Iignocellulose into ruminant feed with white rot fimgi
Table 3 Effect of fungal species and temperature on loss of organic matter,*ligiiiii and changes in in vitro digestibility (IVDMD) o f wheat straw
Fungus 1 Abortiporus biennis
Agrocybe aegerita Dichomitus squalens
Flaminulina velutipes
Ganoderina apptanatuin
Ganoderna lucidum
Hymnochaete tabacina
Lentinus edodes
Plewotus cortrcatus
Pleurotus eiyngii
Pleurotus Jlabellatns Pleurotus osiieatus
PIeu rot us sajo rcaju
Temp.oC 2 25 30 25 30 22 25 30 22 25 30 22 25 30 22 25 30 22 25 30 22 25 30 22 25 30 22 25 30 25 22 25 30 22 25 30
Loss of (%)
OM 3 24.2 25.4 9.6 6.2 29.7 43.2 51.1 9.9 11.3 10.5 29.4 29.3 16.7 20.8 25.2 25.4 10.5 7.9 0.0 13.9 17.4 10.2 9.6
16.3 21.0 11.0 14.0 14.3 30.8 15.4 17.8 18.3 20.0 17.6 20.9
Lianin 4 7.1 12.3 0.0 0.0 11.3 13.5 17.2 0.9 1.7 0.4 15.4 15.4 6.4 11.2 10.8 8.3 5.6 0.3 0.0 4.3 3.0 2.3 2.0 3.5 7.1 ND ND ND 14.2 7.0 6.5 4.5 13.9 14.4 14.6
Changes in
IVDMD 5 23.4 18.1 -29.0 -32.1 32.2 28.3 24.2 -15.1 -18.8 -5.0 13.3 6.8 -20.4 22.5 25.7 13.2 23.8 -15.2 0.0 18.1 24.6 -13.8 -3.8 3.1 14.5 15.3 22.9 23.2 21.2 22.4 15.6 -1.2 20.6 18.6 20.1
(970)
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F. Zadmril and coworkers
Table 3 Contd ............
1 Polyporus versicolor
Poiia expansa Sporotricuin pulveiulentuin Stropharia rugosoannulata Trainetes hirsuta Volva.reilla volvaceae
2 22 25 30 22 25 30 22 25 30 22 25 30 22 25 30 22 25 30
3 23.6 24.0 24.6 19.4 25.2 45.8 55.8 62.4 64.1 22.0 26.2 30.6 25.6 23.4 52.0 14.5 17.9 23.4
4 7.4 7.3 7.4 4.0 6.1 9.5 22.0 25.2 24.4 6.3 9.1 11.7 9.0 10.3 12.5
ND ND ND
5 1.4 -6.7 -3.6 13.7 21.1 16.2 15.9 8.2 7.0 9.5 13.4 25.7 20.0 11.5 6.5 -21.0 -25.4 -30.3
*Zndrnzil (1985)IVDMD % ’ of untrented wheat struw 40.0.
Pleurotus sp. Florida, Pleiwotus cornucopiae and Stropharia rugosoannulata and other species showed good lignin decomposition and increase the in uitro digestibility of all substrates except rice husk. Agrocybe aegerita decomposed lignin only to a small extent and decreased the in uitro digestibility of all the substrates tested in this experiment. But the digestibility of rice husk was reduced with all the fungal strains which might probably be caused by the high incrustation of rice husks with Si02 (15-2596 of dry matter). Pleurotus SajorcajzL, Polyporus hirsutus, Stropharia rugosoannulata and Ganodernza lucidunz degrade lignin of sugarcane bagasse selectively and cause a significant increase in in uitro digestibility (Kewalramani et al., 1988 and Kamra et al., 1993). 3.3. Tenzperature The temperature of incubation influences the decomposition speed of the organic matter and the sequence of decomposition of the substrate components (Table 3). With all fungi tested, a n increase of temperature from 22 to 30C, the decomposition rate of organic matter also increases (Zadrazil 1977; 1985). Large differences in the rates of straw decomposition and positive correlation between increases in temperature and lignin decomposition or in uitro digestibility have been estimated €or
Bivcoriversioii. of ligirocellulose into riinrinnii.t feed with white rot fiiiigi
111
Stropharia rrigosoannrdata.
3.4 Incubation time In the life cycle of a white rot fungus while growing on lignocellulosic substrate the following phases of growth have been observed : * colonization of the substrate * maturation of fungal mycelium * induction of fruiting bodies * autolyses. During the first phase of growth, the easily digestible soluble carbohydrates are utilized for mycelial growth and therefore, the i n uitro dry matter digestibility decreases in comparison to untreated wheat straw (Zadrazil, 1977). After colonization was completed, the in uitro digestibility of fungal treated substrate starts increasing, but it decreases thereafter in older substrates which have a relatively high content of accumulated minerals. Under favourable conditions, some fungi can totally mineralise cereal straw in 80-100 days of fermentation (Zadrazil, 1985).
3.5. Nitrogen stipplementation Supplementation of the substrate with ammonium nitrate changes the decomposition rate and also the sequence of decomposition of different substrate components. Some fungi such as Stropharia rzLgosoannuZata, Lentinus edodes and Plezwotzts sp. Florida are stimulated during decomposition of the substrate by the addition of lower levels of NH,NO,, while Pleurotus eryngii was inhibited by this nitrogen source. The lignin decompositon rate is relatively unchanged by NH,NO, supplementation, but it is also stimulated in Stropharia rogosoannulata a t 0.25% level of ammonium nitrate (Fig. 1). The decrease of in uitro digestibility of substrate is observed with all fungi when substrate received higher concentrations of NH,NO,. It is evident, that fungi mineralize only the substrate a t a faster rate with nitrogen supplementation without any accumulation of easily digestible components in the treated substrate. Ammonium nitrate supplementation to wheat straw does not affect significantly the yield of fruit bodies of Pleurotus sajorcaju, but the yield is significantly higher when soybean meal or alfalfa meal are added to the substrate (Zadrazil, 1980).
3.6 Ratio of solid to liqzLid in substrate With increasing water content in a constant substrate volume, the air content of the substrate decreases. This results in an increased water tension and an increased swelling of the substrate. All fungi investigated show good growth on substrates with varying water contents (from 25 t o
112
F. Zadruzil arul coworkers
Fig. 1 Effect of N H f l o 3 supplementation on dry matter loss (DML), lignin deg-radation (lig D) and changes in in vitro dry matter digestibility (IVDMD) of wheat straw treated with white rot fungi. (GA = Ganodmma applanatum, LE = Lentinus edodes, SR = Stropharia rugosoannulata and PF = Pleurotus sp. Florida, Zadrazil and Brunnert, 1980).
150 ml waterl25g straw). At both the lowest and the highest water contents, the decomposition rate of the total organic matter decreases as does decomposition of lignin and accumulation of digestible subst,ances. The fungi tested show specific growth optima for various air and water contents of the substrate (Zadrazil and Brunnert 1981; 1982). For Pleurofus growth on ground wheat straw a solid : liquid ratio of 1:3 has been found optimum. The unground wheat straw will be able t o hold a little lower level of water in the substrate.
3.7. Composition of gas phase Gaseous metabolites of fungal degradation of straw have strong influence on mineralization of organic matter, loss of lignin and in uitro digestibility
113
Bioconuerswn of lignocellulose into ruminant feed with white rot fungi
Table 4 Effect of gas phase on loss of organic matter and lignin and changes in in vitro digestibility of wheat straw treated with Pleurotus eryngii (25'C, 40 days)* Treatment
0, 100
Gas mixture %
co,
5
0 1
5
5
20
1
20
5
20
10
20
20 30
20
N, 0 94 90 79 75 70 60 50
Loss of (%)
O.M. 8.4
Lignin
Changes in in uitro digestibilty*
27.3
21.4
4.5
12.0
3.8 6.5 6.8 6.3 6.8 7.5
14.6 23.0 23.6 23.6 23.9 17.3
-5.6 -5.6 9.3 11.7 14.6 12.1
9.4
*Kamra and Zadrazil (1986). **IVDMD of untreated wheat straw was 40.0%.
(Zadrazil et al., 1991). For large scale process the composition of gaseous phase is the key-factor. The influence of gas phase composition on degradation of substrate and lignin and changes in in uitro digestibility of wheat straw has been studied in detail in Pleurotus sajorcaju, P. eryngii, Phanerochaete chrysosporium and Stropharia rugosoannulata by Kamra and Zadrazil (1985, 1986); Zadrazil and Kamra (1989);Zadrazil et al. (1994)and Puniya et al. (1995).Some of the results are summerized i n Table 4. The influence on composition of substrate after different treatment with gaseous metabolites is summarized by Buta et al. (1989) and Chiavari et al. (1989). 4.
Scale up of solid state fermentation
Above examples and the analysis of "Palo podrido" samples (Phillippi, 1893;Knoche et al., 1929;Zadrazil et al., 1982) clearly show that the use of white rot fungi for upgrading lignocellulosics into animal feed is possible, at least on a laboratory scale and in the natural conditions. On the other hand, only a little is known about the large scale processing of lignocellulosic feeds. 4.1. Definition of solid state fermentation
Solid state fermentation (SSF) can be defined as a process, in which solid substrates are decomposed by known mono- or mixed-cultures of
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microorganisms (mainly fungi, which can grow on and through the substrate) under controlled environmental conditions, with the aim of producing a high quality product. The substrate (mixture of different particles) is characterized by a relatively low water content. Since much of the water is chemically or physically bound to the substrate, physical properties, e.g. porosity and density are uniform. The substrate is not mixed or moved during the process (Zadrazil et al., 1990a and 1990b). Recently many different reactors for solid state fermentation have. been designed, developed and constructed. Some are used in Koji process for production of soybean sauce or in the production of substrate for the cultivation of edible fungi: Aguricus bisporus (white mushroom) (Francescutti, 1972; Gerrits, 1988) and Pleurotus spp. (Schuchardt and Zadrazil, 1982; 1988). Many factors influence the course of solid state fermentation, but only some factors such as temperature, humidity and composition of gas phase can be monitored and manipulated.
E g . 2a :Solid state fermentor of 3521 mpacity made of PVC
b :Principle of gassing through the solid substrate
Biocoriversion of ligiiocellulose into rutiiiruzrit feed with white rot fungi
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4.2. Reactors for solid state fermentatzon
4.2.1 Small scale reactor This reactor was constructed to scale up the treatment process of straw with Pleurotus spp. (Schuchardt and Zadrazil, 19821, from laboratory scale in Erlenmeyer flasks and small batch cultivations in sacks. The fermentor consisted of PVC and had a filling height of 2 m, diameter of 0.5 m with a net volume of 352 1 (Fig. 2a and b). The fermentor was hermetically sealed by two lids with openings for gas circulation pipes. For gassing a pump was used with a capacity of 40 m3/h with a 212 way value, gassing of substrate was alternated between bottom and the top, changing the direction of flow for definite periods. The electronic control system kept constant temperature, humidity, CO, and 0, concentration in the gas phase passing through the substrate in the fermentor. The temperature in the fermentor was measured with PT,,, thermocouples and the gas concentration of CO, with an infrared detecter and 0, with a paramagnetic measuring instrument. On the basis of experiments conducted with this fermentor, we observed that it was difficult to control temperature of substrate during the initial stages of fermentation and with fungal growth resistance of the substrate increased, which makes the further circulation of gases through the substrate more difficult.
4.2.2. Large scale reactor The reactor is constructed of polyurethane foam sandwich panels which are covered on both sides with polyester board (Fig. 3a and b). The filling height is approximately 2.0 m, the internal width 2.3 m and the depth 2.0 m. This results in a net filling volume of 9.2 m3 equivalent to 1.5 t of straw or 3.0 t wood chip substrate. T w o similarly insulated swing doors are situated at the front of the reactor. The width of the doors is equal to the width of the reactor itself. Inside the reactor there is a raised slatted floor covered firstly with a gliding net and then with a drag net. Two reactors of the same shape and construction are used. One reactor is used for substrate pretreatment and the second one for substrate colonisation (Zadrazil et al., 1990a,b). On moving the substrate from one reactor to the other, the substrate is inoculated (Fig. 3a,b). The substrate is filled into the reactor using a specially designed machine which deposits it on the drag net. A removable front panel keeps the substrate from falling out of the container during filling. The substrate is removed from the container by attaching the drag net to a
116
F. Zadmril and coworkrrs
Fig. 3a Solid state ferinen,tation reactor b. cross section of reactor 1. SSF reactor 2. Net for emptying the substrate 3. Panels for aerating the substrate 4. Equipment for emptying th.e substrate 5. Euipmen.t for fragmen.ting th.e substrate 6. Equi1jmen.t for filling the substrate 7. conveyor to bring substrate to spawning machine* 8. Conveyor for fi11in.g th.e reactor with. substratc 9. Carrier for moving th.e conueyor 10. Spawning m.achin.e
Bioconversion of lignocellirlose into rum.in.ant feed with white rot fungi
117
winch. The substrate is loosened as it is pulled through a set of toothed bars before falling onto the elevator. Then it is filled into another reactor either for incubation or for treatment of the fully colonized substrate. This translocation from one reactor into the other decreases the bulk density of the substrate and reduces the stream resistance of the gas phase by the substrate, which is a parameter indicating fungal growth activity.
4.3. Air-conditioning Fungal growth and heat exchange from the substrate is controlled by recirculating the air within the reactor. For the cultivation of Plerwotrrs spp. and other wood decaying fungi, the gaseous phase is recycled and gas composition is controlled by monitoring CO, and 0, concentrations (Schuchardt and Zadrazil, 1982 and 1988). To control and reduce the growth of competitive microorganisms, CO, could be added at the commencement of the fermentation process as this will not allow the aerobic microbes to grow (Zadrazil and Peerally, 1986). The reactor has an air-conditioning system installed on the roof or in the gas-tubes. A system of aluminium air ducts, with supply duct starting from below and ending in the roof, ventilates the substrate. The total quantity of circulated air can be varied by changing the speed of electronically controlled fans. A centrifugal type fan was chosen to ensure that various processes would be controlled adequately. It has a capacity of 800m3/h at a static pressure of 1200 Pa or approximately 200-500 m3 per ton substrate per hour (depending on its specific volume weight). The most suitable fans for this purpose are of the centrifugal type, with backward curved blades, as their capacity varies only slightly with decreasing resistance.
In order to humidify the air and raise the substrate temperature for pretreatment of substrate (e.g. pasteurisation), a steam injection pipe is installed under the slatted floor. Steam injection is controlled by a 2-way valve operated by a servomotor. Heat exchange takes place by cooled refrigeration in the air-circulation system controlled by a servomotor driven 4-way valve. The lowest temperature of cooling liquid can be -15C. Fresh air, oxygen or carbon dioxide can be added after gas analysis by computer-controlled valves. Exhaust gases leave the fermentor through an over-pressure valve located above the substrate.
4.4. Gas humidity The humidity of gaseous phase can be controlled by hygrometers (Hygrotest 720) situated in different parts of the reactor. The air
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F. Zadrazil and coworkers
humidity fluctuates between 95 and 100%. Commercially available hygrometers are not sufficiently sensitive enough in this region to give more precise control.
4.5. Water evaporation The gaseous phase has a lower temperature than the substrate and water evaporates. From the circulating gases, water condenses on the cooling equipment and within cold areas of substrate and reaches 100 % relative humidity again. Evaporation of water in circulating gases was measured by water loss from a 20 cm2 ceramic disc (Czeratzki, 1968) placed in the space above the substrate. Evaporation of water from the substrate and condensation on the colder parts of reactors and cooling unit is undesirable but cannot be eliminated. The translocation of water could be a measure of the technical standard of the reactor and the efficiency of the control system.
4.6. Temperature For scientific studies the ducts must be insulated to eliminate the uncontrolled heat loss from the reactor. The rate of heat liberation during the fermentation process was measured by a flow meter installed in the cooling system of the reactor. The temperature of the gas phase was monitored by placing 4 PTioo resistance thermometer sensors at the inlet and outlet ducts. Temperature of substrate is measured in 4 different layers with 8 thermometer sensors and registered in 2 independent computers. Minimum and maximum limits for temperature can be adjusted. If the temperature of the air rises above or falls below the temperature limit, the heating or cooling system is activated and an alarm could be calibrated for critical temperature areas. By operating temperature controls, temperature differences in different substrate layers can be estimated. Different possibilities and mathematical and physical models for temperature control are discussed by Teifke and Bohnet (1990).
4.7. Air supply and control of circulated air The amount of air circulated in the reactor through the substrate is measured on the supply side of the fan by measuring the difference in pressure across a gauge ring, The volume of circulated gas is indicated in m3. The required fresh air is added with an air pump and measured with a flowmeter.
Bioconversion of lignocellulose into ruminant feed with white rot fungi
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4.8. Digestibility and homogenity of product
The in vitro digestibility of wheat straw (Tilly and Terry, 1963) on fermentation increases on average by 13.8 digestibility units. The highest increases (18.7 and 18.3 units) are found in the two layers near the surface and the lowest (7.0 units) on the bottom layer. The observed increases in the digestibility of wheat straw after fungal treatment on a large-scale are comparable with results obtained by sodium hydroxide or ammonia treatment (Sundstol and Owen, 1984). After incubation, the substrate may also be used for production of edible fungi. Colonized substrate was placed into a container for fructification. The yield of fruit bodies is satisfactory and has been found comparable to that obtained with other cultivation systems. The final product (fungal substrate) differs in water content and digestibility. One may assume that differences between the water contents of different layers had an influence on the digestibility of substrate. We hope that this phenomenon can be eliminated by better control of the gas phase passing through the substrate during substrate treatment. 4.9. Infection by competitive microorganisms
The proposed system of SSF is based on the use of non-sterile culture conditions. Selective propagation of thermophilic and mesophilic microorganisms during substrate pretreatment supports the saprophytic colonization by the cultivated fungus. Infection of substrate was not observed during colonization of substrate. With the increase in digestibility of substrate, the risk of contamination increases. Colonies of Trichoderma sp. were observed on the surface of the substrate at the end of fermentation. Infection was frequently observed, when condition for the growth of Pleurotus sp. was suboptimal (e.g. temperature being too high). 6.
Feeding of fungal treated lignocellulosic feeds to ruminants
Majority of the experiments conducted on evaluation of fungal treated straw are performed by either in vitro digestibility or in sacco degradability estimations. The work conducted at Braunschweig is based mainly on in uitro dry matter digestibility of fungal treated straw. The results of experiment conducted in Guatemala on feeding of spent wheat straw after harvesting of Pleurotus sajorcuju to lambs showed that digestibility co-eficients of various nutrients were slightly lighter than the untreated wheat straw (Calzada et ul., 1987). Similarly paddy straw left after harvest of P. sujorcaju, when replaced at a level of 50% of untreated straw intake in buffalo, did not affect the digestibility of
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nutrients, but 100% replacement had a negative influence of nutrient availability to animals (Dhanda e f al., 1994). In one of the authors laboratory at Indian Veterinary Research Institute, India; the digestibilities of nutrients were significantly improved when straw treated with Pleurotus ostreatus was fed to sheep (Pal, 1996). The experiments conducted for in uiuo evaluation of fungal treated straw are very few and of short duration. To estimate the influence of feeding fungal treated straw on physiology and performance of animals, long term feeding experiments on a larger number of animals are needed, before reaching any conclusion on the feasibility of fungal bioconversion of lignocellulosic feeds for the feeding of ruminants. 6.
Conclusions
The described SSF-pilot reactor, utilized a t the Institute of Soil Biology, FAL Braunschweig since 1985, can be used after some technical modifications as a model for preparation of substrate for mushroom cultivation and large-scale technology of straw delignification. More basic and applied research must be done, before this "mushroom" technology could be transferred to another area of biotechnology. For each organism, special strategies for the design of reactor and for the control of the process must be developed. For the application in developing countries, cheap and easily controlled reactors must, therefore, be developed. Based on the studies reported here and elsewhere, one may conclude that there is a need for further research on several aspects of the SSF process. These include the following:
6.1. Development of new designs and construction of SSF reactors.
* *
Requirements: Homogenous conditions during processing. Minimal differences between process parameters (growth conditions) in different layers of substrates.
6.2. Development of equipment and sensors for the control of SSF process.
* Control of the air speed in different parts of reactors.
* *
Control of air humidity (95-100%relative humidity). Control of water evaporation from substrate. * Control of water translocation.
6.3. Development of strategy for process control. 6.4. Development of mathematical models of SSF process.
Bioconrrersion of lignorelluiose irito rzimzriant feed with udiite rot fungi
121
6.5. Investigations on in uiuo digestibility and nutritive value of variously processed feeds.
6.6. Verifications of results in laboratory and pilot-scale reactors.
6.7. Feeding experiments with growing and lactating ruminants to compare fungal treated straw with untreated material and chemically treated substrate. 6.8. Comparative economic studies with other lignocellulose-upgrading processes.
Acknowledgements These investigations in the Institute of Soil Biology, FAL, Braunschweig were supported by the Deutsche Forschungsgemeinschaft, European Community Project, COST 84 bis and Federal Ministry of Agriculture, Forestry and Fishery, Germany.
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