Biodegradation of agroindustrial wastes by Pleurotus spp for its use as ruminant feed
support: Project INIA-LIA 057, CSIC 308 and by the Maestría en
Biotecnología, Facultad de Ciencias, Universidad de
Keywords: agroindustry, basidiomycetes, ruminant feed.
The increasing expansion
of agro-industrial activity has led to the accumulation of a large
quantity of lignocellulosic residues all over the world. In particular,
large quantities of rice straw (300.000 t) and citric bagasse (50.000
t) are annually produced in
the production, processing and consumption of agricultural products,
there are a great variety of remainders, which create increasing problems
of elimination. In
Other authors have shown that some fungi, particularly some species of Pleurotus are able to colonize different types of vegetable wastes, increasing their digestibility (Platt et al. 1984; Commanday and Macy, 1985; Rajarathnam and Bano, 1989; Villas-Boas et al. 2002; Zhang et al. 2002; Mukherjee and Nandi, 2004; Salmones et al. 2005). Previous studies have shown the feasibility of using these kind of wastes to produce animal feed (Calzada et al. 1987; Adamovic et al. 1998), and as substrate for mushroom production (Breene, 1990; Sermanni et al. 1994; Kakkar and Dañad, 1998; Yildiz et al. 2002).
In the present work, we study the biodegradation of these wastes by P. ostreatus 814 for its use as ruminant feed.
strains used in this study (P. ostreatus 814, P. ostreatus
816, P. cornucopiae and P. djmour) were provided by Trinidad
Mushrooms. They were grown in malt agar (1,25% malt extract, 1,5%
agar, Oxoid) at
From these experiments, P. ostreatus 814 was the most promising strain.
1. Determination of the microbiological quality was performed using Petrifilm (3M). Total aerobes, total coliforms and E. coli were determined on the substrates without inoculation and after 44 days of fermentation with P. ostreatus 814.
2. Analysis of the chemical composition of the fermentation product consisted in the analysis of dry weight (AOAC, 1990), proteins (AOAC, 1990) and neutral and acid detergent fiber (AOAC, 1996) of the different substrates without inoculating and on different days of fermentation with P. ostreatus 814. The results were analyzed using t-Student test with P < 0.001.
The significance of the differences were estimated by using the Mann-Whitney U test (Mann and Whitney, 1947), with the limit of significance set at P < 0,05. Statistical analyses were performed on SPSS 9.0 Windows.
DNA extraction. Genomic DNA was obtained from pure cultures of fungi belonging to the mushroom producers “Trinidad Mushrooms” (Pleurotus ostreatus 814, Pleurotus ostreatus 816, Pleurotus cornucopiae y Pleurotus djmour) or from the fermentation product after inoculation of wheat seeds or citrus bagasse. Fresh mycelia or the corresponding fermentation product was grounded under liquid nitrogen in a sterile mortar, to obtain a fine powder. The extraction technique performed followed (Jasalavich et al. 2000), and is based in the classical extraction with cetyltrimethylammonium bromide (CTAB) in the presence of β-mercaptoethanol, followed by organic extractions and isopropanol precipitation of the DNA. DNA was quantified by spectrophotometrical measurement at 260 and 280 nm and its integrity was evaluated in 0,8% agarose gels, using ethidium bromide (EtBr). DNA bands were visualized by the fluorescence of the intercalated EtBr under UV light.
amplification. The ITS region was amplified by PCR from DNA isolated
from pure cultures of each of the fungi under study and from the corresponding
fermentation product. Primers ITS1-F (CTT GGT CAT TTA GAG GAA GTA
A) which is specific for the higher fungi, and ITS4 (TCC TCC GCT TAT
TGA TAT GC) the universal primer, were used together to amplify the
ITS region from higher fungi. The primer pair ITS1-F and ITS4-B (CAG
GAG ACT TGT ACA CGG TCC AG), which is specific for basidiomycetes,
were used to specifically amplify the ITS region from only basidiomycetes.
Amplification were performed in 50 μl reactions of PCR buffer
digestion of PCR products. PCR reaction products were digested
directly without further purification with restriction endonucleases
to obtain RFLPs; each sample was digested with AluI (Biolabs), HaeIII (Biolabs) and TaqI (Biolabs) in single-enzyme digests.
10 μl of amplified PCR reaction was mixed with the appropriate
restriction reaction buffer and 10 U of the appropriate enzyme and
then incubated for 6 hrs at
At 7 days of fermentation fungal growth is already appreciated on all the substrates under study, with all the strains assayed. A greater colonization is achieved at day 21 with P. ostreatus 814 (Figure 1).
Although an increase in the total aerobic count is seen for citrus bagasse with respect to day 0, no appreciable increase in total coliforms or E. coli is appreciated (Table 1).
After fermentation, a decrease in the dry weight, increase in the levels of proteins and a decrease in the values of neutral detergent fiber (hemicellulose, cellulose and lignin) and acid detergent fiber (lignin and cellulose) were detected (Table 2). These last determinations could be indicative of the degradation of the cell wall components of the substrates produced by the extracellular enzymes of P. ostreatus. Previous authors concluded that lignification of structural polysaccharides not only inhibited ruminal microbial digestion of polysaccharides by forming 3-D matrix, but also that the presence of highly lignified tissues formed a physical barrier preventing the accessibility of the otherwise highly digestible tissues to the action of hydrolytic enzyme of the rumen microorganisms (Karunanandaa et al. 1995), and have shown that increased digestibility was associated with the degradation of structural carbohydrates (Mukherjee and Nandi, 2004).
No nitrogen source was added, as previous work showed that the in vitro digestibility of the fermented substrate was decreased as compared with fermentation substrate not amended with nitrogen.
When using primers ITS1F and ITS4B, specific for basidiomycetes, an interspecies polymorphism is observed in the band profile obtained, whilst there is no difference observed within a same species (Figure 2a). When making the amplification with primers ITS1F and ITS4, general for higher fungi, the same restriction profile for fungi of the same species (P. ostreatus 814 and 816) is obtained. This same profile is obtained when DNA is extracted from the substrates under study colonized by P. ostreatus 814. It is important to emphasize that when using these general primers no additional bands, that could correspond to other fungi, were obtained (Figure 2b).
These results show not only that strain in use in the mushroom producing industry can be identified to species level using molecular techniques, but also that it is plausible to carry out a molecular characterization of the fermentation products (Villas-Boas et al. 2002).
From the results obtained, we can conclude that P. ostreatus 814 is an appropriate strain to use as inocula of the agroindustrial wastes under study. Higher protein levels, better conservation of the substrate and an increase in in vitro digestibility is observed. A scale up process, using rice straw bales in the open, is under way.
M.; GRUBIC, G.; MILENKOVIC,
CALZADA, J.F.; FRANCO, L.F.; DE ARRIOLA, M.C.; ROLZ, C. and ORTIZ, M.A. Acceptability, body weight changes and digestibility of spent wheat straw after harvesting of Pleurotus sajor-caju. Biological Wastes, 1987, vol. 22, no. 4, p. 303-309. [CrossRef]
COMMANDAY, F. and MACY, J.M. Effect of substrate nitrogen on lignin degradation by Pleurotus ostreatus. Archives of Microbiology, June 1985, vol. 142, no. 1, p. 61-65. [CrossRef]
JASALAVICH, C.; OSTROFSKY, A. and JELLISON, J. Detection and identification of decay fungi in spruce wood by restriction fragment length polymorphism analysis of amplified genes encoding rRNA. Applied and Environmental Microbiology, November 2000, vol. 66, no. 11, p. 4725-4734.
KAKKAR, V.K. and DAÑAD, S. Comparative evaluation of wheat and paddy straws for mushroom production and feeding residual straws to ruminants. Bioresource Technology, November 1998, vol. 66, no. 2, p. 175-177.[CrossRef]
KARUNANANDAA, K.; VARGA, G.A.; AKIN, D.E.; RIGSBY L.L. and ROYSE, D.J. Botanical fractions of rice straw colonized by white-rot fungi: changes in chemical composition and structure. Animal Feed Science and Technology, October 1995, vol. 55, no. 3-4, p. 179-199. [CrossRef]
MUKHERJEE, R. and NANDI, B. Improvement of in vitro digestibility through biological treatment of water hyacinth biomass by two Pleurotus species. International Biodeterioration and Biodegradation, January 2004, vol. 53, no. 1, p. 7-12. [CrossRef]
PLATT, W.M.; HADER, Y. and CHET, I. Fungal activities involved in lignocellulose degradation by Pleurotus. Applied Microbiology and Biotechnology, August 1984, vol. 20, no. 2, p. 150-154. [CrossRef]
RAJARATHNAM, S. and BANO, Z. Pleurotus mushrooms III. Biotransformation of natural lignocellulosic wastes: commercial applications and implications. Critical Reviews in Food Sciences and Nutrition, 1989, vol. 28, no. 1, p. 31-113.
SALMONES, D.; MATA, G. and WALISZEWSKI, K.N. Comparative culturing of Pleurotus spp. on coffee pulp and wheat straw: biomass production and substrate biodegradation. Bioresourse Technology, March 2005, vol. 96, no. 5, p. 537-544. [CrossRef]
SERMANNI, G.G.; D'ANNIBALE, A.; DI LENA, G.; VITALE, N.S.; DI MATTIA, E. and MINELLI, V. The production of exo-enzymes by Lentinus edodes and Pleurotus ostreatus and their use for upgrading corn straw. Bioresource Technology, 1994, vol. 48, no. 2, p. 173-178.[CrossRef]
VILLAS-BOAS, S.G.; ESPOSITO, E. and MITCHELL, D.A. Microbial conversion of lignocellulosic residues for production of animal feeds. Animal Feed Science and Technology, July 2002, vol. 98, no. 1-2, p. 1-12.[CrossRef]
S.; YILDIZ, U.C.;
ZHANG, R.; LI, X. and FADEL, J.G. Oyster mushroom cultivation with rice and wheat straw. Bioresource Technology, May 2002, vol. 82, no. 3, p. 277-284.[CrossRef]
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