Isolation and characterization of bioconversion microbes and aerobic conversion of crude glycerol to value-added bioproducts

Abstract

Renewable forms of energy are of significant interest in today’s economies. Biofuel is a renewable fuel produced by biological means. Biofuel is very useful because it does not have a finite supply, and it produces fewer pollutants than fossil fuels. Recently, there is a serious concern about future of fossil-based fuels due to increasing price of crude oil around the world, depletion of its sources, several important environmental issues (greenhouse effect and global warming) and increasing energy demands. Thus, this situation has imposed researchers for further exploring the renewable energy sources to produce environmentally friendly fuel that not cause any pollution to the atmosphere. Additionally, nowadays, supplies like plants, organic wastes, and agricultural products are often used to produce biofuel. Until now, bioethanol and biodiesel are the most widely produced and used as biofuel. Biodiesel-derived crude glycerol and lignocellulosic (from cell walls of woody plants) biomass are two attractive low-cost renewable resources for biofuel production. Efficient and cost-effective production of biofuel from these sources through biotechnological methods depends on the development of a suitable bioconversion process. The main aims of this study were to i) isolate and characterize the novel as well as highly efficient cellulase or glycerol dehydrogenase (GDH) producing bacterial strains, and ii) optimize the enzyme production parameters during fermentation. These strains could be considered for the biorefinery industry for hydrolysis of lignocellulosic biomass or bioconversion of glycerol so as to accelerate biofuel commercialization. In this research project, three potential glycerol metabolizing strains of Serratia sp. were isolated from paper mill waste of Resolute Forest Products, Thunder Bay, Ontario, Canada. The glycerol bioconversion abilities of the promising strain S. proteamaculans SRWQ1 were analyzed ii using crude glycerol (byproduct from biodiesel production) as the sole carbon source. During shake flask fermentation under aerobic conditions at an optimal incubation temperature of 25°C, the strain SRWQ1 used 98% of the crude glycerol and produced 18.43 ± 1.55 g/L 2,3-butanediol (BDO) and 8.38 ± 0.76 g/L acetoin, with yields of 0.4 and 0.06 g/g, respectively. When the culture medium (minimal salt medium) was supplemented with 50 g/L of glycerol as the sole carbon source, and 5 g/L yeast extract and 5 g/L peptone as the nitrogen sources, the maximum glycerol dehydrogenase (DGH) activity was 408.69 ± 0.069 U/mg protein. The incubation temperature, pH, glycerol concentration and nitrogen sources are the most important factors ruling the GDH activity. Slightly acidic initial pH (pH 6.0) led to enhanced GDH activity and biomass production. This is the first report that S. proteamaculans species can efficiently convert glycerol to produce green products 2,3-BDO and acetoin. Twenty cellulase-producing bacterial strains were isolated from Kingfisher Lake, Ontario, Canada, and screened for cellulase activity using the carboxymethyl cellulose (CMC) agar plate assay. Isolates showing large halos of depolymerization were further assayed to quantify enzyme production ability and identified by using 16S rDNA sequence analysis. The molecular weight of crude cellulase samples was determined ∼50 kDa with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The Bacillus strain IM7 showed the highest carboxymethyl cellulase (CMCase) activity (17.7 ± 0.17 IU/mL) after 48 h of incubation at a yeast extract concentration of 15 g/L. A temperature of at 30°C and pH 5.0 were the optimal conditions for cellulase production. The highest activity (24.59 ± 0.09 IU/mL) was recorded when the culture medium was supplemented with 2% mannose as a co-substrate. The increased glucose content by using mannose in the hydrolysate process resulted in the dramatic increase in enzyme activity.