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The effect of sodium hydroxide on in vitro disappearance of dry matter and neutral detergent fibre of rice straw by rumen anaerobic fungi.

Rumen fungi are as primary colonizers have polysaccharide degrading enzymes (eg. cellulase and xylanase) and degrade lignin-containing plant cell walls in pure cultures (Orpin, 1983). Encysted zoospores of rumen fungi produce a rhizoidal system capable of penetrating plant tissues, thereby allowing access by fungi to fermentable carbohydrates that are not accessible to other rumen micro-organisms (Bachuap, 1981). Although fungi preferentially colonize the lignocellulosic tissues (Bauchop, 1979), their ability to degrade and utilize lignin is not clear. Although it is recognised that the cellulolytic bacteria are the major contributors to plant cell wall digestion in the rumen (Chesson and Orskov, 1984), the role of anaerobic fungi has also been appreciated in the recent past (Dehority, 1995).

Available information on the effect of sodium hydroxide on degradation of NDF of rice straw by rumen fungi is scarce. It has been proposed that sodium hydroxide may breakdown hemicellulose, hydrolyse the ester bonds between lignin and hemicellulose, swell cellulose microfibrils (Canale et al., 1992), expose the cellulose to microbial attachment and carbohydrates more accessible to the action of rumen micro-organisms and improve organic matter digestibility (Goto et al., 1993), therefore cause to significant increase in dry matter intake by animals (Wadhwa and Makkar, 1995). The objective of this study was to evaluate the effect of different levels of NaOH on rumen fungi growth and the disappearance of dry matter (DM) and neutral detergent fiber (NDF) of rice straw using in vitro anaerobic rumen fungi culture.

Materials and methods

Sample preparation: Samples of rice straw were chopped (2-5 cm) and were soaked in NaOH solution (4.5, 7 and 12 % w/v) in a closed plastic bag for 48 h. Treated straw was washed under tap water until the yellow colour resulting from the NaOH treatment was eliminated. The same amount of chopped samples was also washed under tap water (untreated, URSW). Then treated and untreated rice straw samples were dried using air forced oven dry (95[degrees]C, 24 h), weighed and milled (1mm screen). Rice straw was used as the carbon source for growth of the rumen anaerobic fungi. Therefore samples were: URSW: untreated rice straw; T1RSW: 45 g NaOH/kg DM treated rice straw; T2RSW: 70 g NaOH/kg DM treated rice straw, T3RSW: 120 g NaOH /kg DM treated rice straw.

Culture condition of rumen anaerobic fungi: Rumen fungi were isolated from the rice straw incubated in the rumen of fistulated steer. Method of Joblin (1981) was used to grow fungi under anaerobic conditions at 39[degrees]C for 3 days. These isolates were used (1:9) as a source of rumen fungi inoculum. Serum bottles of containing fungi culture medium, 1 g of rice straw as untreated or treated with 45, 70 and or 120 g/kg DM NaOH (URSW, T1RSW and T2RSW, respectively) and 1 ml antibiotic solution were used to culture the isolated fungi at 39 [degrees]C for 1, 3, 9 and 12 days after three times subculturing.

Measurement of DM and NDF disappearance: Samples of rice straw used as the substrate of culture media were collected from each bottle after washing twice with distilled water followed by filtration using grade 1 sintered glass crucibles. They were then freeze dried to constant weight for DM determination. The dry matter disappearance of each sample was calculated as the difference between initial and the residual weight of the dried substrate. Content of NDF of samples were determined from the freeze-dried samples using the method of Van Soest et al. (1991), and losses of each sample were calculated as the difference between initial and the residual weight of the dried substrate.

Measurement of chitin: About 150 mg of freeze-dried samples from in vitro cultures hydrolysed with 6ml of 6N HCL for 4 hour in 105[degrees] C. After cooling, the hydrolysate was centrifuged at 3200 rpm for 30 min at 4[degrees] C. Then supernatant was filtered using a 0.45 mm filter and freeze dried. Then their chitin content was determined from the glucosamine hydrochloride equivalent resulting from hydrolysis as described by Chen and Johnson (1983).

Statistical analysis: Data of DM and NDF disappearance and chitin content of medium were analysed as repeated measurement using the General Linear Model (GLM) procedure of SAS (1990). Duncan's multiple range tests was used to compare the means at P < 0.05.

Result and Discussion

Disappearance of DM and NDF: Disappearance of DM and NDF of untreated and treated rice straw with NaOH using rumen fungi culture are shown in Table 1. Disappearance rate of DM and NDF of T3RSW was significantly higher than the other samples (P <0.05). Anaerobic fungi are able to degrade between 25% and 60% of the dry weight of plant tissues (Grenet et al., 1993). Also, Gordon and Phillips (1989) reported, the loss of about 40% of straw dry matter due to digestion by Neocallimastix sp. after 10 day incubation.

The results showed higher disappearance of DM was always observed in the cultures of treated straw with NaOH. There was a high decrease in dry matter of substrates over the 3 days of the growth of anaerobic fungi in which the highest disappearance occurred in the rice straw treated with 120 g/kg DM. Results of the present study prove results of Rezaeian et al. (2004) which concluded after incubation NaOH treated straw with rumen fungi, dry matter losses were 38%, and 20% loss in the initial DM following sodium hydroxide treatment reported by Fahey et al. (1991). The higher amounts in the dry matter losses of NaOH treated rice straw is also in agreement with previously reported in sacco degradability measurements of treated barely straw (Chaudry et al., 2000) and in vitro digestibility of both leaf and stem fractions (Vadivello, 2001).

The results of this experiment showed higher disappearance of NDF was always observed in the cultures of treated rice straw with NaOH. It was indicated that 32.5% of the hemicellulose present in the untreated wheat straw was solubilized upon NaOH treatment (Lesong and Klopefestine, 1981). Bas et al. (1989) reported DM and NDF digestion of the straw treated with an alkali was greater than untreated straw. It is reported that the alkali treatment cleaves these linkages (Theander and Aman, 1984), resulting in the hemicellulose becoming more accessible to the action of rumen micro-organisms. Borneman et al. (1989) concluded that anaerobic fungi play a significant role in degrading xylans of plant cell walls. The ability of anaerobic fungi to degrade and utilize lignin anaerobically would be an important attribute for rumen micro organisms. Orpin (1983) reported that between 16% and 20% of its lignin content was lost within 4 days of incubation with rumen fungi. Chesson (1993) suggests that lignin-carbohydrate complexes are released from the cell wall as a result of the digestion of the surrounding carbohydrates and not from the direct degradation of the lignin. In most studies the partial solubilisation of hemicellulose has been demonstrated following alkali treatment (Wadhwa and Makkar, 1995).

The alkali solution hydrolyse the ester linkages between lignin and the cell wall polysaccharides (Chesson, 1981), reduce the physical enmeshment of cellulose, and solubilise the inhibitory phenolic compounds; and facilitates microbial colonization and improve of the ruminal degradation plant cell walls (Euna et al., 2006). In addition, alkali treatment increased fungi populations in cell wall (Chen et al., 2007). Gould (1984) proposed that the dilute solutions of alkali; react with lignocellulosics to yield partially delignified products that are highly susceptible to enzymatic and microbial attach. The enhanced degradability has been ascribed to a solubilisation of total phenolics (Chesson, 1981), arabinoxylans and cellulose (Lindberg et al., 1984) and arising from the cleavage of alkali-labile lignin-carbohydrate linkages (Alexander et al., 1987).

Chitin measurement: The highest chitin content was for culture containing 120 g/ kg DM sodium hydroxide treated rice straw (P <0.05). A significantly higher chitin content was observed in treated rice straw compared with untreated rice straw cultures during the 12 days growth of anaerobic fungi (Figure 1) (P <0.05) that prove the result of Rezaeian et al. (2004). Chitin content of cultures increased linearly during the first 3 days of fungal growth reaching 4.02 and 5.96 g/kg DM of initial substrate for the untreated rice straws and treated with 120 g/kg DM, respectively.

[FIGURE 1 OMITTED]

Conclusions

The result showed sodium hydroxide caused to increase the fungal digestion of DM and NDF of rice straw and chitin content of media that indicator of increase of fungi growth, and the effect of 120 g/kg DM sodium hydroxide was more than 45 and 70 g/kg DM NaOH. Therefore it is suggested that sodium hydroxide used for improving DM and NDF digestion of rice straw by rumen fungi.

References

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T. Mohammadabadi * and M. Chaji

Department of Animal Science, Ramin (Khuzestan) Agriculture and Natural Resource University, Molasani, Ahwaz, Iran E-mail: mohammadabadi2002@yahoo.com
Table 1: Dry matter disappearance of rice straw treated sodium
hydroxide using anaerobic rumen fungi culture.

 IT DM disappearance (g / 100g DM)
(day) URSW T1RSW T2RSW T3RSW s.e.m

 1 3.5 (d) 4.1 (c) 4.8 (b) 6.3 (a) 0.04
 3 21.6 (d) 23.9 (c) 25.7 (b) 31.2 (a) 0.02
 9 .5 (d) 30 34.2 (c) 37.2 (b) 46.2 (a) 0.05
 12 33.2 (d) 38.3 (c) 40.3 (b) 49.3 (a) 0.03

IT: Incubation time; URSW: Untreated rice straw; T1RSW: 45 g
NaOH /kg DM treated rice straw; T2RSW: 70 g NaOH /kg DM treated
rice straw, T3RSW: 120 g NaOH /kg DM treated rice straw;
s.e.m: Standard error of mean; Means with different
letters within each row differed significantly (P < 0.05).

Table 2: Neutral detergent fibre disappearance of sodium hydroxide
treated rice straw using anaerobic rumen fungi culture.

 IT NDF disappearance (mg /g DM)
(day) URSW T1RSW T2RSW T3RSW s.e.m

 1 3.2 (d) 4.7 (c) 6.1 (b) 9.6 (a) 0.2
 3 69.6 (d) 82.1 (c) 97.3 (b) 122.2 (a) 0.3
 9 .1 (d) 125 133.2 (c) 142.3 (b) 164.5 (a) 0.7
 12 128.2 (d) 137.1 (c) 146.2 (b) 168.6 (a) 0.8

IT: Incubation time; URSW: Untreated rice straw; T1RSW: 45 g
NaOH /kg DM treated rice straw; T2RSW: 70 g NaOH /kg DM treated
rice straw, T3RSW: 120 g NaOH /kg DM treated rice straw; s.e.m:
Standard error of mean; Means with different letters within each
row differed significantly (P < 0.05).
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Author:Mohammadabadi, T.; Chaji, M.
Publication:International Journal of Biotechnology & Biochemistry
Date:Feb 1, 2011
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