| Lignocellulosic biomass（LCB）in the form of agricultural,forestry,and agro-industrial wastes is globally generated in large volumes every year.The chemical components of lignocellulosic biomass render them a substrate valuable for biofuel production.LCB consists mainly of sugar polymers（cellulose and hemicellulose）and lignin,which are closely linked by covalent and hydrogen bonds.The sugar polymers of LCB can be converted into simple fermentable sugars by chemical or enzymatic hydrolysis.However,the rigid association of lignin,cellulose,and hemicellulose component of LCB forms a complex,hierarchical,and recalcitrant structure,which inhibits the solubilization of LCB resources for biofuel production.Owing to these restrictions,the junk production of LCB waste has recently become a significant worldwide environmental problem resulting from inefficient disposal techniques and increased persistence.In addition,burning LCB waste,such as paddy straws,is a widespread practice that causes considerable air pollution and endangers the environment and human existence.Besides environmental pollution from LCB waste,the increasing industrialization has resulted in the production of billion tons of dyeing wastewater from several industries,including textiles,pharmaceuticals,tanneries,and food processing units.The massive use of synthetic dyes in the various industries can be detrimental to the environment due to the recalcitrant aromatic structure of synthetic dyes similar to the polymeric phenol lignin in LCB structure and their persistent color.Discharging dyeing effluents into the environment without prior treatment could drastically affect soil microbes as well as inhibit photosynthetic activities.Furthermore,synthetic dyes have been described as possessing carcinogenic and toxic properties that could be harmful to public health.Environmental pollution emanating from LCB wastes and dyeing wastewater is of great concern and should be carefully handled to mitigate their catastrophic effects.An effective strategy to curtail these problems is to learn from analogous systems in nature,such as termites,where woody lignocellulose is digested by wood-feeding termites,and humus recalcitrant aromatic compounds are decomposed by soil-feeding termites.It is well known that termite success in degrading lignocellulose or soil humus material is largely dependent on their gut microbial symbionts.However,some essential information on gut bacterial profiles and their unique contributions to food digestion in wood-feeding and soil-feeding termites is still inadequate.We hypothesize that the feeding type of termites may shape their gut bacterial composition and influence their functions to degrade lignocellulose or other organic chemicals,which could potentially offer alternative solutions to degrade some recalcitrant environmental chemicals.Inferring from our stated hypothesis,we employed culture-independent（metagenomics analysis）and culture-dependent（isolation process）techniques to analyze the composition and functional profiles of gut bacterial symbionts in wood-feeding termite,Microcerotermes sp.and soil-feeding termite,P.nitobei which are abundantly accessible in the southern part of China.Consequently,the objective of this dissertation aims to identify and analyze the phylogenetic properties of gut bacterial symbionts between Microcerotermes sp.,wood-feeding termite,and P.nitobei,soil-feeding termite;determine their functional profiles for processing lignocellulose and other recalcitrant aromatic compounds;explore the potential application of a gut bacterial isolate from the wood-feeding termite,Microcerotermes sp.in biorefinery and bioremediation processing.The main results of the investigations are reported below:1.The findings,based on the metagenomic analysis of the 16S r RNA gene sequences of gut bacterial symbionts in the wood-feeding termite,Microcerotermes sp.,and the soil-feeding termite,P.nitobei,demonstrated that there were a total of 26 major bacterial phyla,where 18 phyla were commonly represented in both termites,but in different abundances.The bacterial symbionts in Microcerotermes sp.were dominated by Spirochaetes（55%）,followed by Fibrobacters,whereas Firmicutes（95%）were prevalent among the gut bacteria symbionts in P.nitobei,followed by Actinobacteria（2%）.Furthermore,Shannon and PD tree diversity indices and observed OTUs and Chao 1 richness indices were all identified to be higher in the wood-feeding termites than the soil-feeding termites deduced from the Alpha diversity analysis.In addition,Beta diversity based on principal coordinate analysis（PCo A）exhibited a significant distance dissimilarity between the gut bacterial symbionts.The results showed that the gut bacterial composition differed significantly between the wood-and soil-feeding termites in the present investigation.Furthermore,bacterial functions were evaluated with Tax4Fun analysis,showing the predominance of carbohydrate metabolism,followed by amino acid metabolism and energy metabolism in both Microcerotermes sp.and P.nitobei termites.The results implicated that bacterial symbiont inhabiting the gut of both termites may be involved in the degradation of lignocellulose and other recalcitrant compounds.As most of these bacterial symbionts were uncultured,their physiology and potential applications are unknown.To this end,culture- dependent studies through isolation techniques were adopted to characterize lignocellulose-degrading bacteria in Microcerotermes sp.and P.nitobei.2.For culturable gut bacterial symbionts,a total of 40 and 67 bacterial isolates were indeed identified from the gut system of soil-and wood-feeding termites（P.nitobei,Microcerotermes sp.）,respectively.The identified isolates from both termite species were actually classified into four different phyla:Actinobacteria,Proteobacteria,Firmicutes,and Bacteroidetes,where the phylum Actinobacteria dominated gut bacteria isolates identified from P.nitobei at 47.5%while Proteobacteria were dominant in wood-feeding termite,Microcerotermes sp.at 46.27%.At the genus level,the bacterial isolates cultivated from the gut of Microcerotermes sp.belonged to 17different genera,among which the bacterial genus Streptomyces（28%）was most prevalent,followed by Enterobacter（11%）.Meanwhile,9 genera were recorded for gut bacterial isolates from P.nitobei and were dominated by Streptomyces（37.5%）,followed by Acinetobacter（25%）.In general,the gut bacterial symbionts from both termites showed a congruency at the phyla level but were more diverged at a lower classification,inferring that different termite species evolved a unique repertoire of gut bacteria.Moreover,61%and 55%of the gut bacterial isolates from the wood-and soil-feeding termites demonstrated a significant and multifunctional role in the hydrolysis of three lignocellulolytic substrates,including carboxymethyl cellulose,beechwood xylan,and aniline blue dye,indicating their unique functions and assistance to the host for the degradation of lignocellulose or other xenobiotic compounds from soil.However,the percentages of gut bacterial isolates from the wood-feeding termite,functioned and evaluated at an effective cellulolytic（27%）,xylanolytic（19.4%）,and ligninolytic（10.4%）performance was really higher than those of bacterial isolates identified from the soil-feeding termite,where 12%,10%,and 5%were recorded respectively.3.Subsequently,a viable lignocellulolytic bacteria strain,Streptomyces sp.MS-S2 among the gut cultures of the wood-feeding termite（Microcerotermes sp.）was then evaluated for its capacity to hydrolyze several lignocellulose substrates to produce cellulase and xylanase enzymes.The highest cellulase and xylanase productions were recorded for ten days when the bacterial isolate MS-S2 was cultured in the medium amended with 15 g/L of wheat straw and 1.5%yeast extract at p H 8.0,30℃.Under the optimum conditions,xylanase and cellulase activities were estimated to be 6.560±0.160 and 0.866±0.067 U/m L,respectively.With the assistance of scanning electron microscopy analysis,the surface accessibility of cellulose fibrils in treated wheat straw was clearly to be observed,indicating the efficiency of the hydrolysis process.In addition,functional group modifications such as CH3C-H,C-O,OH,C-C,and C=O stretching from Fourier transform infrared spectroscopy analysis demonstrated the successful depolymerization of wheat straw.Furthermore,excreted enzymes produced under optimal conditions from isolate MS-S2were further evaluated for the liberation of reducing sugars from the wheat straw substrate,where ethanol production was also obtained for a maximum concentration of 10.8 g/L.4.The ability of the MS-S2 strain to depolymerize wheat straw without pretreatment indicated its capacity to secrete lignin-degrading enzymes relevant to bioremediation processes and thus was explored for the degradation of xenobiotic dyes.The decolorization efficiency of five different dyes,including malachite green（MG）,methyl violet,anihiline blue,reactive red,and reactive blue after 12 h of degradation by MS-S2 bacterial strain were recorded to be 98.2%,17.5%,8.7%4.6%and 3.28%respectively.Several physico-chemical parameters were then optimized to accelerate MG degradation,including p H,temperature,glucose,and yeast extract concentration.Streptomyces sp.strain MS-S2 completely decolorized MG（50 mg/L）within six h under optimized conditions when the cultivation medium was amended with 5 g/L glucose and0.08 g/L yeast extract at p H 8 and incubated at 28℃.After MG degradation,the enzyme activities of manganese peroxidase（4.605 U/L）and laccase（45.185 U/L）were estimated.To comprehend the degradation process of MG by the MS-S2 strain,UV-vis spectroscopy,FTIR,and GC-MS analyses were carried out to ascertain the possible degradation mechanism.The metabolites identified,such as 2,6-Bis（tert-butyl）phenol and[4-（dimethylamino）phenyl]phenyl,suggest that MG was broken down into less toxic compounds for further degradation.MS-S2exhibited outstanding characteristics that could make it suitable for the bioremediation of MG dye in the industrial setting.The results obtained from this investigation corroborate our hypothesis,which revealed that the gut bacterial compositions in wood-feeding termites,Microcerotermes sp.,and soil-feeding termites,Pericapritermes nitobei,differed significantly.These symbiotic bacteria from termite gut systems may possess unique properties in degrading lignocellulose and other recalcitrant compounds.The findings further enhance our understanding of mechanisms adopted by the different feeding types of termites to thrive on woody lignocellulose and recalcitrant humus organic compounds,which are worthy of emulation for biorefinery and bioremediation processing.Furthermore,the enrichment techniques employed in this dissertation depict the gut system of the wood-feeding termite,Microcerotermes sp.,and soil-feeding termite,P.nitobei,as unique bioresources consisting of distinct bacterial species valued for the processing of lignocellulosic materials and the degradation of synthetic dyes which can be integrated into modern-biorefinery for processing LCB waste and bioremediation application for the treatment of dyeing wastewaters to help resolve environmental issues arising from LCB waste and dyeing wastewaters. |
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