Comparative Research on Facultative Anaerobic Cellulose Decomposing Bacteria Screened from Soil and Rumen Content and Diet of Dairy Cow

Introduction

Cellulose as the most abundant, wasteless resource in the biosphere has caught the attention of researchers(Ulrich and Wirth, 1999).Cellulose is a polysaccharide constituted of D-glucose linked by β-1, 4-glycosidic bonds and a helpful raw material for producing important chemicals (Ragauskas et al., 2006; Hahn-Häegerdal et al., 2006).Enzymatic hydrolysis is difficult because of insolubility and heterogeneity of cellulose.However, microorganisms could hydrolyse it with the help of a multi-enzyme system (Schwarz,2001).The cellulases consist of endoglucanases and cellobiohydrolases enzymes, which function together in degrading microbes (Dashtban et al., 2009).Consequently, there is an urgent need for the studies about cellulolytic microorganisms as well as their degradation abilities in cellulose were carried out.Some celluloses are digested by protozoa, but it is indicated that degrading activity of protozoa is lower than that of bacteria (Cheng and Costerton, 1980),which are able to degrade cellulose by the action of extracellular cellulase.For years, cellulase-producing bacteria have been isolated from multitude of sources such as the feces of ruminants, soils and organic matter,agricultural residues and forestry (Lynd et al., 1991).

为了充分的挖掘电子邮件写作的自身特点，我们还需要对非语言因素进行研究[3]。由于电子邮件文本在内容上具有格式、结构明显的特点，因此在非语言因素方面，我们将从结构特征与格式特征两个角度来进行研究。

In the soil ecosystem, the cellulolytic bacteria play important roles in the decomposition and transformation of organic matter, and they also provide a significant carbon source to the microbial community in the soil.In the rumen, cellulolytic microorganisms are important to the rumen ecosystem for their role in providing volatile fatty acids (VFA) by fermenting plant polysaccharides, and VFA is the primary energy source for the ruminant animal (Mcdonald et al.,2012).The reports of the cellulose decomposition bacteria isolated from the diary cow diets are rarely.

Majority of isolated cellulolytic bacteria belong to the aerobic bacteria (Lynd et al., 2002), because culturing the anaerobic bacteria needs specialized techniques and methods.Wenzel et al.(2002) isolated the facultative anaerobic cellulolytic bacteria from the gut of the termite Zootermopsis angusticollis.Cheng and Costerton (1980) indicated that extensive important populations of bacteria have been found not only in rumen fluid, but also in feed particles.Therefore, the fact that the cellulose-decomposing bacteria isolated from rumen contents might be associated with the cellulose-decomposing bacteria adhered on ration fed by animals was speculated in our laboratory.

The comparison researches about the facultative anaerobic cellulose-decomposing bacteria from different sources and their abilities of degrading cellulose are rare.Hence, the objective of this study was to isolate cellulose-decomposing bacteria from the rumen content of dairy cow, the soil around the barn and the ration of cow, respectively, and then evaluated what the relationship was among them by 16SrDNA gene sequences molecular biology methods,meanwhile, compared with their cellulase activities.The test would better qualify facultative anaerobic cellulolytic bacteria and provide basic data for using the bacteria in the future.

Animal care

Materials and Methods

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基于上述理论模型，本节取裂隙倾角α=0°、30°、60°和90°，浆液扩散方位角θ=0°、60°、120°和180°，并将表1中的计算参数代入公式(19)、公式(20)，深入探讨岩体倾斜裂隙在不同产状下的注浆压力与注浆时间和浆液扩散距离的关系。

以上机制若能建立，一旦出现事故就能及时准确的定位事故环节，查清事故原因。追踪机制可以和标识制度联动建立，以商品为中心设立转基因食品专用条形码或二维码查询追踪系统，既适应我国食品经营分散的特点，又能节省人力物力财力。此外，由于该追踪机制的涉及面特别广，所以，必须由政府各部门主导，监督各个关键环节的运行。

The experimental procedure was approved by the Institutional Animal Care and Use Committee in Northeast Agricultural University.

Data collection and analysis

Generally, bacteria with high level of 16SrDNA sequence similar to the genera at NCBI GenBank were thought to belong to the same species of genera.In the test, the sequences similar with databases sequences reached to more than 94% for most isolates.It was observed that only L1 strain pertained to Acinetobacter, all other isolates were Bacillus genus(Table 2).For the isolates from rumen fluid, L3 belonged to Bacillus sonorensis, L6 pertained to Bacillus subtilis, while L8 belonged to Bacillus amyloliquefaciens; for the isolates from rumen residue,many of them belonged to Bacillus amyloliquefaciens,except N2, N5 and N7 belonged to Bacillus subtilis;for the isolates from rations, all of them belonged to Bacillus subtilis apart from S2 was Bacillus amyloliquefacien; for the isolates from soil, most of them pertained to Bacillusn amyloliquefacien, besides T5 belonged to Bacillus subtilis.

About 1 kg soil was randomly collected from five sites, which was near the dairy barn, but not polluted by the cow waste, and then sieved to remove vegetation waste.One g of mixed soil sample was dissolved in 100 mL sterile water, then cultured 2 h at 37℃ on an orbital shaker at 180 r · min-1 (Ulrich and Wirth, 1999).One g of diet was also suspended in 100 mL distilled water and cultured shakily 2 h at 37℃ at 180 r · min-1.

Bacteria isolation and screening

3) Wλ=1，WMAC=0.2，wp=1，以1∶6阶模态频率和1∶6阶振型相关系数为目标(图10-图12)。

The bacteria suspension of rumen fluid and residues,soil, and dairy diet were consecutively diluted into a serial solutions from 10-2 to 10-6, then 100 μL of each of the dilutions was spread on the nutrient agar plate medium.Bacterial colonies capable of utilizing cellulose as sole carbon source were selected on sodium CMC agar media (composing of KH2PO4 0.5 g, MgSO4 0.25 g, gelatin 2 g, sodium car boxy methyl cellulose 20 g, and agar 12 g in 1 L of distilled water).

Morphological characteristic of bacterial colonies

Phenotypic characteristics of the isolated bacterial colonies were determined after cultured 48 h on the sodium CMC plate medium based on the microbial application laboratory manual in general microbiology 8th ed (Benson, 2005).Macroscopic and microscopic analyses were included, morphological feature about bacteria included color, surface, shape, border, eleva-tion, Gram's features and spore.

Cellulose degradation capability of bacteria

Bacteria were cultured for 48 h at 39℃ on Congo-Red agar medium.The diameter size of clear zones around strains indicated the capability of degrading cellulose of bacteria.In this paper, bacteria with more than 10 cm diameter of clear zone were described as stronger capability for degradation.

Cellulase activity of bacteria

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β-glucosidases assay

It is widely accepted that complete enzymatic degradation of cellulose need synergistic action of system of three major types of enzymes, which consist of endoglucanases (EG), exoglucanases (ExG) and β-glucosidases (βG) (Dashtban et al., 2010).The measurements of the individual enzymes and the total cellulase activities were described as the followings:

Filter paper assay (FPase activity): the total cellulase activities

Filter paper assay (FPAase) is the key method for analysis of the total cellulase activity based on Mandels et al (1976).Filter paper activity (FPase) was defined as the micromole of glucose from generated filter per minute.

Carboxymethyl cellulase assay (CMCase)

Endoglucanase (EG) activity was measured using CMC as substrate according to Ghose (1987).One unit(IU) of EG was defined as the amount of enzyme that liberated 1 µmol of glucose per minute under assay conditions (Mandels et al., 1976).

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The activity of β-glucosidases was measured at pH 4.8 using 1 mmol · L-1 p-nitrophenyl-β-D-glucopyranoside as substrate described previously by Kubicek (1982).One unit (IU) of β-glucosidase activity was defined as the amount of enzyme that liberated 1 µmol of reducing sugar as glucose equivalents per minute (Chandra et al., 2009).

16SrDNA gene sequencing analysis

Genomic DNA was extracted from the strains that showed stronger capability of cellulose decomposition following the methods of Johnson (1994).16SrDNA genes were amplified using universal primer of bacteria 27f (5-3'): AGAGTTTGATCCTGGCTCAG AACGAACGCT and 1 492f (5-3'): TACGGCTACC TTGTTACGACTTCACCCC (Weisburg et al., 1991)by polymerase chain reaction (PCR).Amplification was conducted in a total reaction volume of 50 µL,containing 2 µL primers, 3 µL dNTPs, 25 µL Polymerase Taq and 20 µL nuclease free water.

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16SrDNA gene sequences were compared with database at GeneBank using Blast-N search program in the website of the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov).In light of similar results, the generic affiliation and homology of the bacterial were con firmed.The accession numbers of strains were also applied for by Bankit.

Samples for separating bacteria

All the data were analyzed by one way ANOVA using MIXED procedure of SAS 9.2.

The model used for cellulase activity was: Yij=μ+Ti+eij, where, Yij was an observation of the dependent variable ij; μ was the population mean for the variable;Ti was effect of the bacterial strains ( fixed effect); and eij was random error that associated with the observed ij.

In all the cases, Duncan test was carried out to determine the significant difference within the variables with significance declared p＜0.05.The average mean of results of the three replicates was represented with standard deviations.

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Results

Morphological characteristics of cellulose decomposing bacteria

For 29 strains of bacteria, both the gram-negative(GN) bacteria with coccus cell morphology and grampositive (GP) bacteria with bacilli cell morphology were found.By observation, many of GN bacteria were opaque yellowish-white colony with wet, convex and rounded characteristic.In addition, majority of GP bacteria were white colony with dry, convex and rounded profiles.Of the 29 strains, only four strains were GN bacteria with no spore, three of the four strains were isolated from rumen fluid, and the rest were from soil.Other 25 strains were GP bacteria with spore.The growth status of the strains in aerobic condition was not different compared with anaerobic condition, therefore, these strains were considered as facultative anaerobic bacteria.The diameters of clear zone of cellulose decomposing microorganisms were between 7-15 mm, and L5 was the largest, reaching to 15 mm.Table 1 showed the diameters of clear zone no less than 10 mm.

Table 1　BLAST-N results of 16SrDNA gene of isolates which diameter of clear circles ≥10 mm and their diameters of clear circles dyed by Congo red

L, Bacterial strains isolated from rumen fluid; N, Bacterial strains isolated from rumen residue; S, Bacterial strains isolated from rations; T, Bacterial strains isolated from soil.

Bacterial strain　Acession number　Species　Query cover (%)　Maxmum identity (%)　Diameter (mm)L5　KX426073　Bacillus subtilis subsp.subtilis str.　100　99　15 L7　KX426074　Bacillus amyloliquefaciens DSM7　99　99　11 N5　KX426075　Bacillus subtilis subsp. subtilis str.　99　99　12 N9　KX426076　Bacillus subtilis subsp.subtilis str.　100　99　11 S6　KX426077　Bacillus subtilis subsp.subtilis str.　100　99　11 T1　KX426078　Bacillus subtilis subsp.subtilis str DSM7　100　99　10

Molecular identification of cellulolytic bacteria

Three fistulaed dairy cows (three and a half years old)in dry period were provided a total mixed ration (TMR)with concentrate: forage at a 45 : 55 ratio to fulfill their nutrient requirement for maintenance.Compositions of the ration (kg · kg-1) were: 0.50 L.chinensis, 0.33 ground corn, 0.05 soybean cake, 0.05 rapeseed dregs,0.04 cottonseed meal, and 0.03 vitamins and minerals.The cows were fed twice daily at 6:00 a.m.and 6:00 p.m.,and accessed to fresh water ad libitum.Whole rumen contents withdrawn from each of the three cows prior to the first meal in the morning were mixed equally,and then were poured into a pre-warmed thermos which filled with carbon dioxide.Subsequently,1 mL rumen fluid and 1 g residue were put into two sterilized tubes, into which were added 9 mL sterile water and fully shaken for preparation of bacterial suspension.

BLAST_N results of 16SrDNA gene of isolates are presented in Tables 1 and 2.The sequences of all the isolates had been reserved in GeneBank under the consecutive accession numbers of the bacterial strains which the clear zone diameter more than 10 mm were KX426073 to KX426078.And others were KX588160 to KX588172 except for the isolates which had lower Query cover compared with the database.

Determination of cellulase activity of bacteria

式中，∂为线性系数，可以根据光强检测仪器的分辨力结合线性系数，估算检测系统检测微悬臂偏移的分辨力.检测系统并不需要计算出实际的腔长，只需在当微悬臂的偏移造成F-P干涉仪的腔长改变而偏离参考点腔长时，将偏移量反馈给AFM系统中.

The cellulase production of different strains about the total cellulase, endoglucanases and β-glucosidases were presented in Table 3.Filter paper activity (FPase)and CMCase activity which represented the total cellulase and endoglucanases activity, respectively,were the highest in L5, moreover, FPase activity had no significantly different in L7, N5 and L5 and they produced 57% more glucose than T1, which implied that CMCase and FPase activities were the highest in the strains isolated from rumen fluid and the lowest isolated from soil.The differences of β-glucosidases activity among various strains were significant(p＜0.05), with the highest activity in T1 and the lowest in N5.Additional, β-glucosidases activity was the highest in strains isolated from soil, and the lowest isolated from rumen residues.CMCase activities were similar among L7, N5 and N9, but higher than those of S6 and T1.The data is shown in Table 3.

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Table 2　BLAST-N results of 16SrDNA gene isolate

Bacterial strain　Accession number　Species　Query cover (%)　Maxmum identity (%)L1　KX588172　Acinetobacter baumannii strain AB030　100　99 L2　KX588160 　Bacillus sp.EGD-AK1　100　99 L3　KX588161　Bacillus sonorensis 　99　99 L6　--------------　Bacillus subtilis subsp. subtilis str.　24　100 L8 　--------------　Bacillus amyloliquefaciens DSM7　100　99 N2　KX588162　Bacillus subtilis subsp.subtilis str.　99　99 N7　KX588163　Bacillus subtilis subsp.subtilis str.　100　99 N8　KX588164　Bacillus amyloliquefaciens DSM7　100　99 S1　KX588165　Bacillus subtilis subsp.subtilis str　99　99 S2　-------------　Bacillus amyloliquefacien DSM7　62　98 S3　KX588166　Bacillus subtilis subsp.subtilis str.　100　99 S4　KX588167　Bacillus subtilis subsp.subtilis str.　100　99 S6　KX426077　Bacillus subtilis subsp.subtilis str.　100　100 T3　KX588168　Bacillus amyloliquefacie DSM7　99　99 T4　KX588169　Bacillus amyloliquefacie DSM7　100　96 T5　KX588170　Bacillus subtilis subsp.　100　99 T6　KX588171　Bacillus amyloliquefacie DSM7　100　99

Nearest relatives on GenBank and percentage similarities are listed.L, Bacterial strains isolated from rumen fluid; N, Bacterial strains isolated from rumen residue; S, Bacterial strains isolated from rations; T, Bacterial strains isolated from soil.

Table 3　Comparison of cellulase production among different strains on the total cellulase, endoglucanases and β-glucosidase

Strain No.　Enzyme activity (μ · umol-1 glucose)CMCase　FPase　　β-glucosidase L5　7.981±0.159a　6.274±0.756a　1.509±0.207d L7　4.911±0.067b　5.966±0.158a　3.543±0.125b N5　4.740±0.069b　5.705±0.035ab　0.696±0.044e N9　4.579±0.185b　4.721±0.250bc　2.686±0.145c S6　3.364±0.097c　4.509±0.183c　1.479±0.141d T1　3.214±0.100c　2.576±0.055d　6.175±0.268a

Different letters in the same column differ significantly (p＜0.05) (means±SD) (n=3).L, Bacterial strains isolated from rumen fluid; N, Bacterial strains isolated from rumen residue; S, Bacterial strains isolated from rations; T, Bacterial strains isolated from soil.

Discussion

Isolate and screening of cellulose decomposition microbes

图5(b)是温度对苯酚降解的影响。随着反应温度的升高，苯酚的降解速率有所加快，在45 ℃时，10 min即可达到降解平衡。但是温度对反应速率的影响并不明显，即使在较低温度下仍然可以获得较高的催化反应速率。因此，当利用Fe3O4-C空心微球处理含酚废水时，可以选择在室温下进行。

Sodium CMC medium is the traditional culture medium used for screening cellulose degradation bacteria, bacterial colonies capable of utilizing cellulose as sole source of carbon are isolated on cellulose agar media (Gupta et al., 2012).Based on morphological and molecular identification of 16SrDNA gene, the cellulose bacteria screened in this experiment mainly belonged to GP Bacillus, this result was in accordance with the reports that lots of producing cellulose bacteria are GP Bacillus (Deka et al., 2011; Mohammed et al., 2003).However,three strains that isolated from rumen fluid were GN bacteria, this might be related to the foraging situation of cows.When cows were fed a concentrate diet, the bacteria were predominantly of the gramnegative cell wall type, if cows were fed normal diets, the bacteria were gram-positive bacteria in rumen fluid (Cheng and Costerton, 1980).The phylogenetic analysis of the bacterial strains using its 16SrDNA sequence data showed that strains had the highest homology with Bacillus genus.Due to lacking overall genome relatedness, chemotaxonomic data, the specific epithet of the strains could not be assigned and identified as Bacillus sp., this still need lots of relevant researches to be proved.

Congo-Red is an indicator for cellulose degradation in an agar medium, which provides the basis for a rapid and sensitive screening for cellulolytic bacteria(Gupta et al., 2012).In this experiment, the range of clearing zone diameter was between 7-15 mm,which was smaller than that of previous reports, the clearing zone diameter reached to more than 28 mm,whereas the strains were isolated from four different invertebrates in previous study.This could be due to various sources of cellulase micro-organisms, but could also be the result of the problems associated with enzyme activity determinations.

Cellulolytic activity of bacterial isolates

Cellulase activity ranged from 2.578 to 6.274 IU · mL-1 for FPCase, and 3.214 to 7.981 IU · mL-1 for endoglucanase in our trial.However, Gupta et al.(2012)reported that the bacterial cellulase activities ranged from 0.012 to 0.196 IU · mL-1 for FPase and 0.162 to 0.400 IU · mL-1 for endoglucanase assay, which were much lower than ours, this could be related to the methods of enzyme activity determinations, kinds of strains, culture time, determination conditions and other factors.For CMCase and FPase activities, the strains isolated from rumen fluid were dominant,which was in accordance with the reports that domestic and international studies have confirmed,the reason might be that there existed fungi that could secrete cellulase in rumen fluid, the cellulolytic enzyme activity of rumen fungi was higher than that of rumen bacteria (Kamra, 2005).It was shown that CMCase and FPase activities were the lowest in soil cellulose-decomposing bacteria, of which the reasons needed to be researched.

Conclusions

All of the 29 strains cellulose-decomposing bacteria screened from rumen residues, rumen fluid, soil and ration of dairy cow were facultative anaerobic,and most of them were Bacillus genus, although the bacterial strains of four sources could not be completely separated by the genus.The activities of FPase and CMCase were significantly higher for L5 bacterial strains isolated from rumen fluid than other strains.However, β-glucosidase activity was the highest for T1 bacterial strain.

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