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Fermenting Cell Walls of Processed Sugarcane Pith by Ruminal Bacteria, Protozoa and Fungi.

Byline: MORTEZA CHAJI, TAHEREH MOHAMMADABADI AND ALI AGHAEI

ABSTRACT

The objectives of this study were to determine contribution of ruminal bacteria, protozoa and fungi to the fermentation of cell walls of sugarcane pith treated with low temperature steam (121oC, 120 min.) and or sulfuric acid (0.0, 6, 12 and 18 g/kg DM), and effectiveness of this method for increasing the feed value of sugarcane pith and negative effects of this method on rumen microorganisms.

Gas production parameters were determined and cumulative gas production data were fitted to the exponential equation. The results showed that processing with low temperature steam resulted in significant increase in rate (C) and potential gas production (B) by all microbial fractions, as the highest (B) (112.9 mL) was for condition 18 g/kg of dry matter (DM) acid. The highest gas produced by total microorganism and rumen isolated bacteria was followed by fungi and protozoa fractions. In vitro cell wall degradation, organic matter digestibility (OMD) and metabolisable energy (ME) of sugarcane pith treated with steam and 18 g/kg acid was the highest and microbial biomass production (MB) was the lowest for all microbial groups.

Therefore, it appears that the gas production parameters and degradability of sugarcane pith by rumen microbial fractions are influenced by low temperature steam and sulfuric acid treatment, bacterial group generates a larger volume of gas production than fungal or protozoan group and this method had no negative effect on rumen microorganisms. (c) 2011 Friends Science Publishers

Key Words: Low temperature steam; Rumen microbial groups; Sugarcane pith; Sulfuric acid

INTRODUCTION

Rumen microorganisms; bacteria, protozoa and fungi have a key role in the degradation of polysaccharides of plant cell walls (Lee et al., 2000), but cellulolytic bacteria are a major microorganisms responsible for ruminal digestion of plant cell walls due to their metabolic diversity and numerical predominance (Cheng et al., 1991; Krause et al., 2003). Williams and Withers (1991) found that although the addition of protozoa to previously defaunated sheep reduced the bacterial population, it appears that protozoa may stimulate fiber digestion in the rumen either directly or by stimulation of the cellulolytic bacteria and digest fiber in the rumen by 5-21% (Dijkstra and Tamminga, 1995; Lee et al., 2000). Lee et al. (2000) concluded that fungal activity was mostly responsible for cell wall degradation. The production of gas during the microbial fermentation of plant material by rumen contents has been used as an indirect measure of substrate digestibility (Menke and Steingass, 1988).

Much amount of sugarcane pith is produced annually in the world including Iran. However, the low digestibility, high lignin and very low nitrogen content, are considered as the main reasons for unsatisfactory performance of animals fed these roughages (Osorio and Cruz, 1990). Many methods have proven successful in disrupting cell wall material e.g., using acids (Shambe and Kennedy, 1984) and high-pressure steam (Castro and Machado, 1990). Under steam conditions, acetyl groups are released from the hemicellulose matrix and suitable levels of cell wall disruption are achieved (Muzzy et al., 1983). Steam and pressure treatments alone or allied with chemical treatments are known to disrupt lignocellulosics in a way, which allows improved utilization of cell wall polysaccharides by cell-free enzymes (Grohmann et al., 1985) and rumen microbes (Castro and Machado, 1990).

The objective of these experiments was to estimate the relative roles of bacteria, protozoa and fungi in plant cell wall digestion of sugarcane pith processed with low temperature steam and sulphuric acid by in vitro gas production and investigation effectiveness of this method for increasing the feed value of sugarcane pith, and probably negative effects of this processing method on rumen microorganisms.

MATERIALS AND METHODS

Preparation of samples and inoculums: Sulphuric acid solution was added to sugarcane pith (USP) (100 g, about 92% DM) to obtain samples of approximately 30% dry To cite this paper: Chaji, M., T. Mohammadabadi and A. Aghaei, 2011. Fermenting cell walls of processed sugarcane pith by ruminal bacteria, protozoa and fungi. Int. J. Agric. Biol., 13: 283-286 mater (DM) content of 0.0, 6, 12 and 18 g/kg H2SO4 on a DM basis.

Then, a part of samples were autoclaved (at 121degC for 120 min, 2.2 bar) and then oven-dried at 55degC. Rumen fluid was collected from two fistulated sheep prior to the morning feeding. Animals fed 250 g concentrate and 750 g forage once at day.

Collected rumen contents were strained through four layers of cheesecloth; different microbial fractions for evaluating ruminal fermentation were prepared according to the method described by Zhang et al. (2006) as: the strained rumen fluid (total microorganism, TM), protozoa was isolated using centrifuged procedure (1000 RPM, 5 min), fungi (F) was prepared from non protozoa strained rumen fluid using antibacterial agent (streptomycin sulphate, penicillin G, potassium and chloramphenicol [0.1 mg/mL each]) and bacteria was isolated from non protozoa strained rumen fluid using antifungal agent [benomyle (500 mg/L) and metalaxyle (10 mg/L)]. The antibiotics and other chemicals were used 100 uL/mL of mixed culture solution was injected into the syringes to give desired concentrations before the mixed culture solution (30 mL) was added to the syringes.

Gas production method: In vitro fermentation by various microbial groups of rumen was estimated by gas production (GP) method as described by Menke and Steingass (1988). Rumen fluid (total microorganism) and the isolated microbial groups (P, B and F) were added to the anaerobic buffer before transfer to the syringes (three parallel syringes of each treatment). The glass syringes were prewarmed to 39degC, then 30 mL of mixed culture medium (consisting of ruminal microbial groups and buffer; ratio 1:2) were pipetted into each glass syringe followed by incubation in a water bath at 39degC for 2, 4, 6, 8, 10, 12, 16, 24, 48, 72 and 96 h.

Syringes were used to measure the gas production and DM degradation of experimental samples within 96 h. The syringes were taken out of the incubator at 96 h. culture fluid of each sample carefully removed and residues in the syringes washed into a tube, carefully with distilled water separately. Then the residues were dried at 105degC for 12 h and used to calculate the degradation of samples. Cumulative gas production data were fitted to the exponential equation Y = B (1[?]e[?]Ct), where B is the gas production from the fermentable fraction (mL), C is the gas production rate constant C (mL/h), t is the incubation time (h) and Y is the gas produced at time t. The values of organic matter digestibility (OMD) and metabolisable energy (ME) of samples were calculated by the equation of (Menke and Steingass, 1988), OMD (g/kg OM) = 148.8 + 8.89 GP + 4.5 CP + 0. 651A and ME (MJ/kg DM) = 2.20 + 0.136 GP + 0.057 CP + 0.0029 CP2. Microbial biomass production (MB) was estimated by method of Blummel et al. (1997).

Data of gas production, cell wall degradability, OMD, ME and MB were subjected to analysis as a completely randomized design using the General Linear Model procedures of SAS institute (1996). Duncan's multiple range test was used to compare the means at Pless than0.05.

RESULTS AND DISCUSSION

In vitro gas production parameters (B and C) of experimental samples by total rumen microorganism (TM) and other microbial groups over 96 h incubation are shown in Table I. Processing with steam associated with acid caused to increase gas production by microbial fractions, and increased in amount of acid, resulted in a significant increase of gas production parameters (Pless than0.05).

The most amount of gas production by all microbial groups was for 18 g acid/kg DM (Pless than0.05). The highest gas production for all the samples was observed in TM group, followed by the group of fungi and protozoa. Steam treatment may be caused to partial or complete hydrolysis of hemicellulose fraction of raw pith and decrease of NDF, changing it into more soluble components (Toussaint et al., 1991), partial depolymerization of lignin (Morjanoff and Gray, 1987) and following increasing digestibility (Chaji et al., 2010). Using lower temperatures with an acid can achieve comparable cell wall disruption to steam treatment at high temperatures (Castro et al., 1994). Steam and pressure treatments alone or allied with chemical treatments are known to disrupt lignocellulosics in a way, which allows improved utilization of cell wall polysaccharides by cell-free enzymes (Grohmann et al., 1985) and rumen microbes (Castro and Machado, 1990).

The result of this study showed that treating acidified sugarcane pith with low temperature (121degC for 120 min. and 18 g/kg DM acid) had the highest cell wall degradability, OMD and ME and the lowest MB of both samples by microbial groups (Table II).

The researchers reported under these steam conditions hemicellulose fraction hydrolysed and acetyl groups are released from the hemicellulose matrix and suitable levels of cell wall disruption are achieved (Grohmann et al., 1985). By applying the steam explosion process to sugarcane bagasse, the susceptibility of cellulose to enzymatic hydrolysis was increased (Kling et al., 1987), also cellulose will be more accessible for rumen microbial enzymes (Castro and Machado, 1990) and cell-free enzymes (Liu and Orskov, 2000). Chaji and Naserian (2006) reported increased in vitro dry matter digestibility for sugarcane pith treated with steam by about 14%. Liu et al. (1999) reported any improvement in digestibility of bagasse resulting from steam treatment was due to the formation of water soluble substances.

These results indicate that the cellulolytic activity of whole ruminal groups was highest, and there was a complementary synergism on the degradation of plant cell walls among ruminal bacteria, protozoa and fungi. The highest gas production for all the samples was observed in TM group, whereas a less gas volume was produced by the group of fungi and protozoa. The gas production and

Table I: Gas production coefficients (mean +- SE, mL/200 mg DM) of sugarcane pith treated with low temperature and acid by rumen microbial fractions

Temp (oC)###Time (min.)###Acid (g/kg DM)###Microbial fractions

###TM###B###F###P

0.0###0.0###0.0###B###64.2+-2.8e###54.2+-1.3d###28.5+-0.3e###10.1+-0.1d

121###120###0.0###B###76.4+-2.3d###67.5+-1.2c###37.7+-0.8d###15.5+-0.4c

121###120###6###B###80.4+-1.6c###71.1+-1.1b###43.3+-0.7c###19.5+-0.6b

121###120###12###B 105.3+-2.6b###89.6+-2.7a###52.3+-1.0b###23.3+-0.6a

121###120###18###B 112.9+-3.5a###92.6+-1.1a###54.7+-0.9a###23.5+-0.5a

###SEM###2.06###0.98###0.34###0.08

0.0###0.0###0.0###C 0.05+-0.003a###0.03+-0.002a###0.01+-0.002b###0.02+-0.004b

121###120###0.0###C 0.06+-0.001b###0.01+-0.002b###0.01+-0.004b###0.02+-0.001b

121###120###6###C 0.03+-0.001c###0.01+-0.003b###0.02+-0.003a###0.03+-0.005a

121###120###12###C 0.04+-0.002d###0.03+-0.004a###0.02+-0.004a###0.03+-0.005a

121###120###18###C 0.04+-0.008d###0.01+-0.005b###0.01+-0.004b###0.02+-0.002b

###SEM###0.002###0.0022###0.0032###0.0024

B: Gas production from the fermentable fraction (mL for 96h) ; C: Gas production rate constant (mL/h)

Table II: Estimated parameters of sugarcane pith treated with low temperature and acid by rumen microbial fractions

Temp (oC)###Time (min.)###Acid (g/kg DM)###Microbial fractions

###TM###B###F###P

0.0###0.0###0.0###CEL###42.1e###30.3e###11.7e###4.6e

121###120###0.0###56.1d###40.2d###15.7d###6.5d

121###120###6###63.4c###45.2c###17.8c###7.3c

121###120###12###72.5b###51.6b###20.3b###8.3b

121###120###18###78.5a###56.2a###22.1a###10.4a

###SEM###0.2###0.1###0.2###0.3

###P###less than0.05 less than0.05###less than0.05 less than0.05

0.0###0.0###0.0###OMD###425.20e###347.32e###196.10e###79.81e

121###120###0.0###535.62d###433.21d###243.70d###95.51d

121###120###6###550.46c###454.12c###261.20c###110.83c

121###120###12###733.26b###600.61b###341.91b###135.80b

121###120###18###822.16a###676.90a###382.83a###155.31a

###SEM###1.5###2.2###3.2###2.4

###P###less than0.05 less than0.05###less than0.05 less than0.05

0.0###0.0###0.0###ME###6.60e###5.45e###3.50e###1.20e

121###120###0.0###7.50d###6.59d###3.76d###1.44d

121###120###6###8.23c###6.83c###3.90c###1.56c

121###120###12###11.30b###9.18b###5.19b###2.14b

121###120###18###12.36a###10.75a###5.82a###2.22a

###SEM###0.02###0.01###0.02###0.01

###P###less than0.05 less than0.05###less than0.05 less than0.05

0.0###0.0###0.0###MB###24.08a###110.40a###122.30a###134.21a

121###120###0.0###22.80b###101.29b###109.40b###120.10b

121###120###6###20.70c###96.35c###104.91c###115.01c

121###120###12###19.12d###84.80d###92.30d###104.02d

121###120###18###16.70e###77.80e###85.20e###96.10e

###SEM###0.4###1.1###1.2###0.4

###P###less than0.05 less than0.05###less than0.05 less than0.05

CEL: cell wall degradability; OMD: organic matter digestibility; ME: metabolisable energy; MB: microbial biomass production TM: total rumen microorganism; P: protozoa; B: bacterial; F: fungal If means within each column have different letters are significantly different (Pless than0.05)

degradability by B group was higher than fungi and protozoa. Although, the rumen cellulolytic bacteria are believed to be responsible for most of the feed digestion in the rumen (Cheng et al., 1991; Flint and Bayer, 2008), the anaerobic fungi to penetrate deeply into plant tissues that are not normally accessible to bacteria suggests that they have a special role in fiber digestion (Thareja et al., 2006). The quantitative estimation revealed that rumen protozoa cause

CONCLUSION

Ruminal bacteria, protozoa and fungi together contribute to ruminal microbial fermentation of plant cell walls. Within the protozoal, bacterial or fungal group, bacterial group generates a larger amount of gas than fungal or protozoal group and this method had no negative effect on rumen microorganisms. Low temperature steam (121degC, 120 min, 2.2 bar) associated H2SO4 (18 g acid/kg DM) increased gas production parameters, cell wall degradability, OMD and ME, and decreased MB by all rumen microbial fractions and therefore, improved nutritional values of sugarcane pith.

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Department of Animal Science, Ramin Agriculture and Natural Resource University, Molasani, Ahvaz, Iran
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Author:Chaji, Morteza; Mohammadabadi, Tahereh; Aghaei, Ali
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:7IRAN
Date:Apr 30, 2011
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