Ok Kyung Lee

Chemo-enzymatic saccharification and bioethanol fermentation of lipid-extracted residual biomass of the microalga, Dunaliella tertiolecta

Chemo-enzymatic saccharification and bioethanol fermentation of the residual biomass of Dunaliella tertiolecta after lipid extraction for biodiesel production were investigated. HCl-catalyzed saccharification of the residual biomass at 121 °C for 15 min produced reducing sugars with a yield of 29.5% (w/w) based on the residual biomass dry weight. Various enzymes were evaluated for their ability to saccharify the residual biomass. Enzymatic saccharification using AMG 300 L produced 21.0 mg/mL of reducing sugar with a yield of 42.0% (w/w) based on the residual biomass at pH 5.5 and 55 °C. Bioethanol was produced from the enzymatic saccharification products without additional pretreatment by Saccharomyces cerevisiae with yields of 0.14 g ethanol/g residual biomass and 0.44 g ethanol/g glucose produced from the residual biomass. The waste residual biomass generated during microalgal biodiesel production could be used for the production of bioethanol to improve the economic feasibility of microalgal biorefinery.

Highly efficient extraction and lipase-catalyzed transesterification of triglycerides from Chlorella sp. KR-1 for production of biodiesel

We developed a method for the highly efficient lipid extraction and lipase-catalyzed transesterification of triglyceride from Chlorella sp. KR-1 using dimethyl carbonate (DMC). Almost all of the total lipids, approximately 38.9% (w/w) of microalgae dry weight, were extracted from the dried microalgae biomass using a DMC and methanol mixture (7:3 (v/v)). The extracted triglycerides were transesterified into fatty acid methyl esters (FAMEs) using Novozyme 435 as the biocatalyst in DMC. Herein, DMC was used as the reaction medium and acyl acceptor. The reaction conditions were optimized and the FAMEs yield was 293.82 mg FAMEs/g biomass in 6 h of reaction time at 60 °C in the presence of 0.2% (v/v) water. Novozyme 435 was reused more than ten times while maintaining relative FAMEs conversion that was greater than 90% of the initial FAMEs conversion.

Dimethyl carbonate-mediated lipid extraction and lipase-catalyzed in situ transesterification for simultaneous preparation of fatty acid methyl esters and glycerol carbonate from Chlorella sp. KR-1 biomass

Fatty acid methyl esters (FAMEs) and glycerol carbonate were simultaneously prepared from Chlorella sp. KR-1 containing 40.9% (w/w) lipid using a reactive extraction method with dimethyl carbonate (DMC). DMC was used as lipid extraction agent, acyl acceptor for transesterification of the extracted triglycerides, substrate for glycerol carbonate synthesis from glycerol, and reaction medium for the solvent-free reaction system. For 1g of biomass, 367.31 mg of FAMEs and 16.73 mg of glycerol carbonate were obtained under the optimized conditions: DMC to biomass ratio of 10:1 (v/w), water content of 0.5% (v/v), and Novozyme 435 to biomass ratio of 20% (w/w) at 70°C for 24h. The amount of residual glycerol was only in the range of 1-2.5mg. Compared to conventional method, the cost of FAME production with the proposed technique could be reduced by combining lipid extraction with transesterification and omitting the extraction solvent recovery process.

Bioethanol production from carbohydrate-enriched residual biomass obtained after lipid extraction of Chlorella sp. KR-1

The residual biomass of Chlorella sp. KR-1 obtained after lipid extraction was used for saccharification and bioethanol production. The carbohydrate was saccharified using simple enzymatic and chemical methods using Pectinex at pH 5.5 and 45°C and 0.3N HCl at 121°C for 15min with 76.9% and 98.2% yield, respectively, without any pretreatment. The residual biomass contained 49.7% carbohydrate consisting of 82.4% fermentable sugar and 17.6% non-fermentable sugar, which is valuable for bioethanol fermentation. Approximately 98.2% of the total carbohydrate was converted into monosaccharide (fermentable+non-fermentable sugar) using dilute acid saccharification. The fermentable sugar was subsequently fermented to bioethanol through separate hydrolysis and fermentation with a fermentation yield of 79.3%. Overall, 0.4g ethanol/g fermentable sugar and 0.16g ethanol/g residual biomass were produced.

Lipase-catalyzed in-situ biosynthesis of glycerol-free biodiesel from heterotrophic microalgae, Aurantiochytrium sp. KRS101 biomass

Heterotrophic microalgae, Aurantiochytrium sp. KRS101 had a large amount of lipid (56.8% total lipids). The cells in the culture medium were easily ruptured due to thin cell wall of Aurantiochytrium sp., which facilitated in-situ fatty acid methyl esters (FAMEs) production directly from biomass. The harvested biomass had a high content of free fatty acids (FFAs), which was advantageous for glycerol-free FAMEs production. FAMEs were directly produced from Aurantiochytrium sp. KRS101 biomass (48.4% saponifiable lipids) using Novozyme 435-catalyzed in-situ esterification in dimethyl carbonate (DMC). DMC was used as a lipid extraction reagent, acyl acceptor and reaction medium. A 433.09mg FAMEs/g biomass was obtained with 89.5% conversion under the optimal condition: DMC to biomass ratio of 5:1 (v/w) and enzyme to biomass ratio of 30% (w/w) at 50°C for 12h. Glycerol could not be detected in the produced FAMEs.

Molecular characterization of a novel oligoalginate lyase consisting of AlgL- and heparinase II/III-like domains from Stenotrophomonas maltophilia KJ-2 and its application to alginate saccharification

Molecular identification and development of a novel recombinant alginate lyase as the biocatalyst for alginate saccharification are prerequisite for bioethanol fermentation from brown seaweed biomass. We identified and characterized a novel oligoalginate lyase for complete degradation of alginate from Stenotrophomonas maltophilia KJ-2 that grow on alginate as the sole carbon source. KJ-2 oligoalginate lyase consisted of AlgL- and heparinase II/III-like domains. The recombinant KJ-2 oligoalginate lyase exhibited substrate preference toward polymannuronate and alginate as well as oligoalginate. The recombinant KJ-2 oligoalginate lyase completely degraded alginate into unsaturated uronate monomer most efficiently at pH 7.5 and 37 °C. Interestingly, AlgL-like recombinant proteins showed more like endolytic activity. The recombinant KJ-2 oligoalginate lyase was a novel oligoalginate lyase consisting of AlgL- and heparinase-like domains and could be used as a candidate for biocatalyst selection to saccharify alginate for bioethanol production from brown seaweed.

Biological conversion of methane to chemicals and fuels: technical challenges and issues

Methane is a promising next-generation carbon feedstock for industrial biotechnology due to its low price and huge availability. Biological conversion of methane to valuable products can mitigate methane-induced global warming as greenhouse gas. There have been challenges for the conversion of methane into various chemicals and fuels using engineered non-native hosts with synthetic methanotrophy or methanotrophs with the reconstruction of synthetic pathways for target products. Herein, we analyze the technical challenges and issues of potent methane bioconversion technology. Pros and cons of metabolic engineering of methanotrophs for methane bioconversion, and perspectives on the bioconversion of methane to chemicals and liquid fuels are discussed.

Systematic metabolic engineering of Methylomicrobium alcaliphilum 20Z for 2,3-butanediol production from methane

Methane is considered a next-generation feedstock, and methanotrophic cell-based biorefinery is attractive for production of a variety of high-value compounds from methane. In this work, we have metabolically engineered Methylomicrobium alcaliphilum 20Z for 2,3-butanediol (2,3-BDO) production from methane. The engineered strain 20Z/pBudK.p, harboring the 2,3-BDO synthesis gene cluster (budABC) from Klebsiella pneumoniae, accumulated 2,3-BDO in methane-fed shake flask cultures with a titer of 35.66u202fmg/L. Expression of the most efficient gene cluster was optimized using selection of promoters, translation initiation rates (TIR), and the combination of 2,3-BDO synthesis genes from different sources. A higher 2,3-BDO titer of 57.7u202fmg/L was measured in the 20Z/pNBM-Re strain with budA of K. pneumoniae and budB of Bacillus subtilis under the control of the Tac promoter. The genome-scale metabolic network reconstruction of M. alcaliphilum 20Z enabled in silico gene knockout predictions using an evolutionary programming method to couple growth and 2,3-BDO production. The ldh, ack, and mdh genes in M. alcaliphilum 20Z were identified as potential knockout targets. Pursuing these targets, a triple-mutant strain ∆ldh ∆ack ∆mdh was constructed, resulting in a further increase of the 2,3-BDO titer to 68.8u202fmg/L. The productivity of this optimized strain was then tested in a fed-batch stirred tank bioreactor, where final product concentrations of up to 86.2u202fmg/L with a yield of 0.0318u202fg-(2,3-BDO) /g-CH4 were obtained under O2-limited conditions. This study first demonstrates the strategy of in silico simulation-guided metabolic engineering and represents a proof-of-concept for the production of value-added compounds using systematic approaches from engineered methanotrophs.

Identification of 4-Deoxy-L-Etychro-Hexoseulose Uronic Acid Reductases in an Alginolytic Bacterium Vibrio splendidus and their Uses for L-Lactate Production in an Escherichia coli Cell-Free System

Abstract4-Deoxy-L-erythro-hexoseulose uronic acid (DEH) reductase is a key enzyme in alginate utilizing metabolism, but the number of characterized DEH reductase is quite limited. In this study, novel two DEH reductases, VsRed-1 and VsRed-2, were identified in marine bacterium Vibrio splendidus, and the recombinant enzymes were expressed in an Escherichia coli system and purified by Ni-NTA chromatography. The optimal pH and temperature of the recombinant VsRed-1 and VsRed-2 were pHxa07.5, 30xa0°C, and pHxa07.0, 35xa0°C, respectively. The specific activities of VsRed-1 (776xa0U/mg for NADH) and VsRed-2 (176xa0U/mg for NADPH) were the highest among the DEH reductases reported so far. We also demonstrated that DEH could be converted to L-lactate with a yield of 76.7 and 81.9% in E. coli cell-free system containing VsRed-1 and VsRed-2 enzymes, respectively, indicating that two DEH reductases can be employed for production of biofuels and bio-chemicals from brown macroalgae biomass.