Ho Myeong Kim

Bioethanol production from the nutrient stress-induced microalga Chlorella vulgaris by enzymatic hydrolysis and immobilized yeast fermentation.

The microalga Chlorella vulgaris is a potential feedstock for bioenergy due to its rapid growth, carbon dioxide fixation efficiency, and high accumulation of lipids and carbohydrates. In particular, the carbohydrates in microalgae make them a candidate for bioethanol feedstock. In this study, nutrient stress cultivation was employed to enhance the carbohydrate content of C. vulgaris. Nitrogen limitation increased the carbohydrate content to 22.4% from the normal content of 16.0% on dry weight basis. In addition, several pretreatment methods and enzymes were investigated to increase saccharification yields. Bead-beating pretreatment increased hydrolysis by 25% compared with the processes lacking pretreatment. In the enzymatic hydrolysis process, the pectinase enzyme group was superior for releasing fermentable sugars from carbohydrates in microalgae. In particular, pectinase from Aspergillus aculeatus displayed a 79% saccharification yield after 72h at 50°C. Using continuous immobilized yeast fermentation, microalgal hydrolysate was converted into ethanol at a yield of 89%.

Efficient approach for bioethanol production from red seaweed Gelidium amansii

Gelidium amansii (GA), a red seaweed species, is a popular source of food and chemicals due to its high galactose and glucose content. In this study, we investigated the potential of bioethanol production from autoclave-treated GA (ATGA). The proposed method involved autoclaving GA for 60min for hydrolysis to glucose. Separate hydrolysis and fermentation processing (SHF) achieved a maximum ethanol concentration of 3.33mg/mL, with a conversion yield of 74.7% after 6h (2% substrate loading, w/v). In contrast, simultaneous saccharification and fermentation (SSF) produced an ethanol concentration of 3.78mg/mL, with an ethanol conversion yield of 84.9% after 12h. We also recorded an ethanol concentration of 25.7mg/mL from SSF processing of 15% (w/v) dry matter from ATGA after 24h. These results indicate that autoclaving can improve the glucose and ethanol conversion yield of GA, and that SSF is superior to SHF for ethanol production.

Enhanced lignocellulosic biomass hydrolysis by oxidative lytic polysaccharide monooxygenases (LPMOs) GH61 from Gloeophyllum trabeum

Lignocellulose is a renewable resource that is extremely abundant, and the complete enzymatic hydrolysis of lignocellulose requires a cocktail containing a variety of enzyme groups that act synergistically. The hydrolysis efficiency can be improved by introducing glycoside hydrolase 61 (GH61), a new enzyme that belongs to the auxiliary activity family 9 (AA9). GH61was isolated from Gloeophyllum trabeum and cleaves the glycosidic bonds on the cellulose surface via oxidation of various carbons. In this study, we investigated the properties of GH61. GtGH61 alone did not exhibit any notable activity, but the synergistic activity of GtGH61 with xylanase (GtXyl10G) or cellulase (GtCel5B) showed efficient bioconversion rates of 56 and 174% in pretreated kenaf (Hibiscus cannabinus L.) and oak (Quercus spp.), respectively. Furthermore, the GtGH61 activity was strongly accelerated in the presence of cobalt Co(2+). Enzyme cocktails (GtXyl10G, GtCel5B, and GtGH61) increased the amount of sugar released by 7 and 6% for pretreated oak and kenaf, respectively, and the addition of Co(2+) stimulated bioconversion by 12 and 11% in pretreated oak and kenaf, respectively.

Efficient function and characterization of GH10 xylanase (Xyl10g) from Gloeophyllum trabeum in lignocellulose degradation.

The xylanase gene from Gloeophyllum trabeum was cloned and expressed in Pichia pastoris GS115. Xyl10g has a molecular weight of approximately 50kDa, and exhibits maximum specific activity at 70°C and a broad range of pH 4.0-7.0. Purified recombinant Xyl10g efficiently degraded popping-pretreated corn stover and newspaper waste at 50°C and pH 4.0 after 24h, and showed synergistic effects with Cel5B (endoglucanase) and BglB (β-glucosidase) to increase reduced sugar levels by about 1.71- to 1.88-fold and 2.26- to 2.48-fold, respectively. Although Xyl10g has low specific activity for beechwood xylan, as compared to XynA, Xyl10g more efficiently degraded corn stover than did XynA. According to immunogold labeling analysis, Xyl10g can attack highly substituted, unsubstituted, and low-substituted xylans, whereas XynA cannot efficiently attack highly substituted xylans, which is important for lignocellulose degradation. These results suggest that GH10 Xyl10g can be used for lignocellulose degradation.

Cellulosic bioethanol production from Jerusalem artichoke (Helianthus tuberosus L.) using hydrogen peroxide-acetic acid (HPAC) pretreatment.

Jerusalem artichoke (JA) is recognized as a suitable candidate biomass crop for bioethanol production because it has a rapid growth rate and high biomass productivity. In this study, hydrogen peroxide-acetic acid (HPAC) pretreatment was used to enhance the enzymatic hydrolysis and to effectively remove the lignin of JA. With optimized enzyme doses, synergy was observed from the combination of three different enzymes (RUT-C30, pectinase, and xylanase) which provided a conversion rate was approximately 30% higher than the rate with from treatment with RUT-C30 alone. Fermentation of the JA hydrolyzates by Saccharomyces cerevisiae produced a fermentation yield of approximately 84%. Therefore, Jerusalem artichoke has potential as a bioenergy crop for bioethanol production.

Improving lignocellulose degradation using xylanase–cellulase fusion protein with a glycine–serine linker

The fungal hydrolytic system efficiently degrades lignocellulosics efficiently. We previously characterized two hydrolytic enzymes from Gloeophyllum trabeum, namely, endoglucanase (Cel5B) and xylanase (Xyl10g). To enhance lignocellulosic degradation, we designed a fusion protein (Xyl10g GS Cel5B) using a glycine-serine (GS) linker and expressed it in Pichia pastoris GS115, which produced a hydrolytic fusion enzyme for the degradation of lignocellulosics. Purified Xyl10g GS Cel5B protein has a molecular weight of approximately 97 kDa and shows a lower specific activity than Xyl10g or Cel5B. However, Xyl10g GS Cel5B can degrade popping-pretreated rice straw, corn stover, kenaf, and oak more efficiently than the mixture of Xyl10g and Cel5B, by about 1.41-, 1.37-, 1.32-, and 1.40-fold, respectively. Our results suggest that Xyl10g GS Cel5B is an efficient hydrolytic enzyme and a suitable candidate for degrading lignocellulosics to produce fermentable sugar.

Comparison of red microalgae (Porphyridium cruentum) culture conditions for bioethanol production

Microalgae biomass are useful resources in biofuel production. The objective of this study was to evaluate bioethanol production in response to Porphyridium cruemtum culture conditions. Enzymatic hydrolysis of seawater P. cruemtum (SPC) and freshwater P. cruemtum (FPC, 1% substrate loading, w/v) resulted in glucose conversion yields of 89.8 and 85.3%, respectively, without any pretreatment. However, FPC hydrolysate was more efficiently converted to ethanol about 7.1% than SPC hydrolysate. The comparison of separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) showed that SSF processing is a superior method for bioethanol production from both SPC and FPC. Though SSF processing (5% substrate loading, w/v) in a 500-mL twin-neck round bottom flask, we achieved ethanol conversion yields of 65.4 and 70.3% from SPC and FPC, respectively, after 9h. These findings indicate that P. cruemtum can grow in freshwater conditions and is an efficient candidate for bioethanol production.

Production of bio-sugar and bioethanol from coffee residue (CR) by acid-chlorite pretreatment

Nowadays, coffee residue (CR) after roasting is recognized as one of the most useful resources in the world for producing the biofuel and bio-materials. In this study, we evaluated the potential of bio-sugar and bioethanol production from acid-chlorite treated CR. Notably, CR treated three times with acid-chlorite after organic solvent extraction (OSE-3), showed the high monosaccharide content, and the efficient sugar conversion yield compared to the other pretreatment conditions. The OSE-3 (6% substrate loading, w/v) can produce bio-sugar (0.568g/g OSE-3). Also, simultaneous saccharification and fermentation (SSF) produced ethanol (0.266g/g OSE-3), and showed an ethanol conversion yield of 73.8% after a 72-h reaction period. These results suggest that acid-chlorite pretreatment can improve the bio-sugar and bioethanol production of CR by removing the phenolic and brown compounds.

Single step purification of concanavalin A (Con A) and bio-sugar production from jack bean using glucosylated magnetic nano matrix

Jack bean (JB, Canavalia ensiformis) is the source of bio-based products, such as proteins and bio-sugars that contribute to modern molecular biology and biomedical research. In this study, the use of jack bean was evaluated as a source for concanavalin A (Con A) and bio-sugar production. A novel method for purifying Con A from JBs was successfully developed using a glucosylated magnetic nano matrix (GMNM) as a physical support, which facilitated easy separation and purification of Con A. In addition, the enzymatic conversion rate of 2% (w/v) Con A extracted residue to bio-sugar was 98.4%. Therefore, this new approach for the production of Con A and bio-sugar is potentially useful for obtaining bio-based products from jack bean.

Engineering of the catalytic site of xylose isomerase to enhance bioconversion of a non-preferential substrate

Mutation in active site would either completely eliminate enzyme activity or may result in an active site with altered substrate-binding properties. The enzyme xylose isomerase (XI) is sterospecific for the α-pyranose and α-fructofuranose anomers and metal ions (M1 and M2) play a pivotal role in the catalytic action of this enzyme. Mutations were created at the M2 site of XI of Thermus thermophilus by replacing D254 and D256 with arginine. Mutants D254R and a double mutant (D254R/D256R) showed complete loss of activity while D256R showed an increase in the specificity on D-lyxose, L-arabinose and D-mannose which are non-preferential substrates for XI. Both wild type (WT) and D256R showed higher activity at pH 7.0 and 85°C with an increase in metal requirement. The catalytic efficiency Kcat/Km (S(-1) mM(-1)) of D256R for D-lyxose, L-arabinose and D-mannose were 0.17, 0.09 and 0.15 which are higher than WT XI of T.thermophilus. The altered catalytic activity for D256R could be explained by the possible role of arginine in catalytic reaction or the changes in a substrate orientation site. However, both the theories are only assumptions and have to be addressed with crystal study of D256R.