Characterization of ligninolytic microbial consortia and analysis of recalcitrant structural properties of pretreated lignocellulosic biomass

Abstract

The aims of this dissertation was to evaluate chemical and biological treatment methods to remove and degrade lignin from agriculture waste biomass for increasing the yield of biogas and biohydrogen. In chemical treatment approach, three alkali reagents at various dosages: NaOH (1-5%), KOH (1-5%), and Ca(OH)2 (0.5%) and three different heating processes, water bath, autoclave and short time microwave were tested for 10 different agriculture substrate. The Scanning Electron Microscopy (SEM) images showed visible degradation on the alkalies treated surface of biomass as compared to the untreated biomass. Additionally, disapperance and emergence of new peaks were observed in treated substrates using Fourier Transform Infrared spectroscopy (FT-IR). Microwave heating with 2% NaOH treated substrates showed more total biogas yield as compared to other treatment conditions. The Ca(OH)2 (0.5%) soaking of corn cob for 7, 15, and 30 days incubation was tested. The highest cumulative biogas was 360.5 NmL/gVS, 3-times higher than the cumulative biogas produced from the untreated corn cob 115.1 NmL/gVS. For biological treatment of waste material, 27 ligninolytic bacteria were isolated from soil, wood compost, and waste sludge. Seven of the most active strains were selected. The optimum yields of lignin peroxidase and laccase were achieved at pH 3-5. The co-cultures demonstrated 2.5 times more rice straw lignin degradation than using single culture. Likewise, the greatest enhancements of cumulative methane yield (70-76%) occurred from co-cultures treated rice straw as compared to individual culture. To produce biohydrogen and biomethane separatly in batch fermentation, 20 ligninolytic Bacillus sp. strains were isolated from granular sludge of full scale anaerobic digester. Among them, four ligninolytic Bacillus sp. strains were selected based on their lignin and Azure B degradation. Brevibacillus agri AN-3 exhibited the highest decrease in COD (88.4%) of lignin and (78.1%) of Azure B. Brevibacillus agri AN-3 showed hydrogen (H2) yield of 1.34 and 2.9 mol-H2/mol from xylose and cellulose respectively. In two-phase wheat straw batch fermentation, Brevibacillus agri AN-3 produced 72.5 and 125.5 NmL/gVS cumulative H2 and methane (CH4) respectively. It was perceived that using ligninolytic Bacillus sp. strains, 48.6% more methane yield could be obtained xx from the wheat straw than using the untreated wheat straw in batch fermentation. A consolidating bioprocessing of recombinantecombinantecombinant ecombinant ecombinant NeurosporaNeurosporaNeurospora NeurosporaNeurospora crassa F5 strain was used for saccharification of wheat straw (WS) to increase the biogas production. The WS was pretreated with 2% NaOH followed by 2,4, and 6 days hydrolysis with N. cN. c N. crassa F5 strain at 28±1℃ and 200 rpm using 0.5 g/L glucose in Vogel media. Scanning Electron Microscopy (SEM) analysis showed a visible change on the surface structure of the pretreated WS as compared to the untreated WS. The 2,4 and 6 days N. crassa F5 saccharified WS was used for biomethane potential (BMP) analysis using automatic methane potential testing system (AMPTS). A maximum cumulative biogas of 700.8 mL/gVS was obtained from 2% NaOH pretreated WS followed by 2 days N. crassa F5 treatment. The recombinant ecombinant ecombinant ecombinant ecombinant N. cN. c N. crassa F5 treated WS produced daily biogas which was 6-fold higher per day and 339.3% more in cumulative volume than the untreated WS sample. Finally, a single culture was tested for the potential of biohydrogen from Organic Fraction of Municipal Solid Waste (OFMSW). One hundred and twenty bacterial strains were isolated from heat-treated granular sludge of a full scale anaerobic digester. The best hydrolytic strains were assessed for H2 production from glucose and soluble starch. Two Bacillus sp. strains, namely F2.5 and F2.8, exhibited high H2 yields and were used as pure culture to convert OFMSW into hydrogen. The strains produced up to 61 mL of H2 per grams of volatile solids and could be considered as good candidates towards the development of industrially relevant H2-producing inoculants. This was the first successful application of pure microbial cultures in bio-hydrogen production from OFMSW.