Monitoring Of Cellulose Degradation With Microbial Community Of β-glucosidase Family During Composting

The thermophilic aerobic composting us ing cow dung and straw in in the self-made composting bioreactor lasted for a mounth. The temper atur e of the upper, middle and lower of the pile were measur ed, enzyme dynamics of CMCase and β-glucos idas e determined and the contents of cellulose, hemic ellulose and lignin in the compost pile wer e also meas ured, in addition the content of glucose and cellobiose were determined us ing HP LC. The β-glucos idase GH1、GH3 family gene librar y were constructed to analyze the m icrobial community compos ition and dominant micr obial spec ies w ith β- glucos idase GH1、GH3 family gene. The changes in gene copy number of the dominant microbial species in each per iod were determained us ing q-P CR. Af ter analyze the correlation of the var iations of m icrobial community succession and the changes in gene copy number of the dominant microbial spec ies with the changes of β- glucos idase enzyme dynamics, to further r eveal the potential r elationship of degradation of cellulose- based substance and the var iations of microbial spec ies and copy number which have β-glucosidas e function gene. The results of the research are following:The temper atur e of the pile up to 45 ℃ in the f irst 3 days of composting, the ther mophilic phas e started.The thermophilic phas e lasted for 8 days. The amounts of cellulose and hemicellulose wer e r educed dur ing the thermophilic phase while the amount of lignin was increased s lightly in the cooling phase. The amount of glucose was decreas ed in the f irst two days, reached to the f irst peak(1.85 mmol/kg) on the 4d, decreased in 4-7 d. The second peak reached on 23 d. In 0-4 d, the tr end of the content of cellobiose is decrease after increased f irst. The content of cellobiose was on a lower level at the end of thermophilic phase and at the beganing of the cooling phas e, stabilized range from 0mmol/kg-0.91mmol/kg, correosponded with the high level of β-glucosidase enzyme dynamics of these phases. The content of cellobiose increased obviously from 13 d, reaches the maximum value(1.29 mmol/kg) on 29 d. The activity of β- glucos idase reached the peak(1.482 μmol p-Nitr/g dw min) on day 4 and stabilized range from 0.429 μmol p-Nitr/g dw min to 0.533 μmol p- Nitr/g dw min. The activity trend of CMCase was similar to β-glucosidase, reached the peak of 47.67 μg glucose/g dw min at the 7 th day.Af ter the c loned sequence alignment ana lys is, Devosia sp. of GH1 bacter ial family found in heating phase, ther mophilic phas e and cooling phase; soybean B radyrhizobium and Microbacteri um have appear ed in var ious per iods; Rhizobi um have appear ed in var ious per iods, and mainly in the pre- and post-thermophilic phase; abnormal Thermotoga wer e mainly f ound in pre-thermophilic phase; In GH3 bacter ial family, Stenotrophomonas have appear ed in var ious per iods, most pr esent in the heating phase; crescent Pelosinus most pr esent in heating phase and pre-thermophilic phas e; Mycobacterium can be found in pre- thermophilic phase, post-thermophilic phas e and cooling phase. In GH3 fungal family, Hypocrea presented in heating phase, pre-thermophilic phase and cooling phas e, but more times in cooling phase; A spergillus in each per iod were encounter ed in frequency heating per iod appears more; Penicillium mainly in the heating and cooling phase.According to gene copy number analys is us ing q-P CR w ith β-glucos idase GH1 f amily universal pr imer can be found much more copy number in heating phas e and cooling phase. Analysis of eight pairs of β-glucosidase GH1 family specific primers can be found, GH1-5-2, GH1- 3-9, GH1-3-5 three pairs of pr imers in q-P CR wer e obtained s ignif icantly more copies of thermophilic phase than that of heating phase and cooling phas e, which means the gene copy numbers of Rhizobium and Thermotoga in thermophilic phase have pos itive correlation with the production of β- glucos idase and degr adation of cellulose. Copy numbers obtained us ing q-PCR with four pairs of GH3 bacter ial f amily specif ic pr imers ar e higher in the heating phase, lower in thermophilic phas e, which proves Stenotrophomonas, Pelosinus and Mycobacterium were produced and played a role in in cooling phase, but less effection in ther mophilic phas e. The copy numbers of GH3B-3 in cooling phase ar e mor e than the other thr ee phas es suggest that Mycobacteri um played the main r ole in the production of β- glucos idase and cellulose degradation. The copy numbers of GH3 fungal f amily obtained us ing q-P CR in each per iod ar e at a high level in ever y phase, which means fungus is mainly r elated to cellulose degradation. Meanwhile, the analys is of copy number of thr ee pairs of s pec if ic pr imers obtained can be found, the number of copies of three pairs of q-PCR pr imers showed the same trend in each per iod, which suggest Trichoderma, Aspergillus and Penicillium played a major role on production of β-glucos idase and degradation of cellulose, less effection in thermophilic phase.