Enrichment of cellulose-saccharifying bacteria communities from rat fed with high-fiber diet
Date Issued
2016
Date
2016
Author(s)
Chen, Chi-Chen
Abstract
Cellulose is one of the highly abundant organic compounds on earth, which may be one of the alternative energy resources. Cellulose can be chemically or biologically degraded into simple sugars, which can be used to generate bioenergy such as ethanol and hydrogen. Chemical hydrolysis of cellulose requires high energy input. Therefore, bio-degradation may be a feasible process. The objective of the study is to isolate and identify cellulose-saccharifying microorganisms from rat cecum fed with high-fiber diets. A Sprague Dawley (SD) rat was fed with high fiber diets for 6 months. Mandel-Reese (MR) media with sodium carboxymethyl cellulose (CMC) as the sole carbon source, was used to enrich cellulolytic microbes. Congo red overlay method was used to select the cellulose-degrading colonies on the isolation plates. The congo red-positive colonies were inoculated into MR media with CMC, and the reducing sugar were measured by 3,5-dinitrosalicylic acid (DNS) colorimetric method. Enrichment culture RCF (rat cecum colony F), the one with highest CMC-saccharifying rate, was selected for crystalline cellulose-saccharifying tests and further studies. The whole-genome sequencing result from the 30-day-incubation RCF culture showed a community of bacteria consisted with four Clostridium spp., Desulfotomaculum ruminis (D. ruminis), Escherichia coli (E. coli), Enterobacter aerogenes, Pseudoflavonifractor capillosus (P. capillosus) and Cellulomonas cellasea. In addition, time-coursed reducing sugar concentrations from α-cellulose indicated that microbial community RCF consumed more reducing sugars than it produced within 8 days of incubation. According to previous studies, Clostridium spp. were most likely to be cellulolytic and D. ruminis, E. coli and Enterobacter aerogenes were speculated to be non-cellulolytic in this microbial community. In order to enhance the sugar yields from crystalline cellulose, α-cellulose, three treatments based on the characteristics of community were conducted: antibiotic, heat and alcohol treatments. Nalidixic acid, an antibiotic against gram-negative bacteria, was used to inhibit D. ruminis, E. coli and Enterobacter aerogenes. Heat and alcohol were applied to favor spore formers, Clostridium spp. and D. ruminis. Species- distribution with time was analyzed using real-time PCR and 16S ribosome RNA gene amplicon sequencing. Amongst these three treatments, antibiotic enhanced the sugar yield successfully. However, real-time PCR and 16S rRNA gene amplicon sequencing results showed that both the microbial populations and fractions of Clostridum spp. were reduced substantially by antibiotic treatment. Three possible reasons for the sugar yield enhancement after antibiotic treatment were: 1. Antibiotic changed the Clostridium population structure into more cellulose-saccharifying community. 2. Antibiotic treatment provided the competitive advantage of minor microorganism having high cellulose-saccharifying ability. 3. The predicted non-cellulolytic E. coli could perform cellulosic saccharification.
Subjects
cellulose
cellulosic saccharification
cecum
enrichment
real-time PCR
Next-generation sequencing
Type
thesis
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