Waste to Energy: Steam explosion-based torrefaction process to produce solid biofuel for power generation utilizing various waste biomasses.

Currently, humankind is facing a serious environmental and climate crisis, which has accelerated the research on producing bioenergy from waste biomass as a carbon-neutral feedstock. In this study, the aim was to develop an upcycling strategy for waste biomass to solid-type biofuel conversion for power generation. Various types of waste biomass (i.e., waste wood after lumbering, sawdust-type mushroom waste wood, kudzu vine, and empty fruit bunches from palm) were used as sustainable feedstocks for steam explosion-based torrefaction. The reaction conditions were optimized for each waste biomass by controlling the severity index (Ro); the higher heating value increased proportional to the Ro increase. Additionally, component analysis revealed that steam explosion torrefaction mainly degraded hemicellulose, and most of the torrefied waste biomass met the Bio-Solid Refuse Fuel quality standard. The results provide not only a viable waste-to-energy strategy but also insights to address global climate change.

Synergetic effect of lytic polysaccharide monooxygenase from Thermobifida fusca on saccharification of agrowastes.

Saccharification is one of the most noteworthy processes in biomass-based biorefineries. In particular, the lytic polysaccharide monooxygenase has recently emerged as an oxidative cleavage-recalcitrant polysaccharide; however, there is insufficient information regarding its application to actual biomass. Accordingly, this study focused optimizing the recombinant expression level of a bacterial lytic polysaccharide monooxygenase from Thermobifida fusca (TfLPMO), which was characterized as a cellulolytic enzyme. Finally, the synergistic effect of the lytic polysaccharide monooxygenase and a commercial cellulase cocktail on the saccharification of agrowaste was investigated. TfLPMO functioned on various cellulosic and hemicellulosic substrates, and the combination of TfLPMO with cellulase exhibited a synergistic effect on the saccharification of agrowastes, resulting in a 19.2% and 14.1% increase in reducing sugars from rice straw and corncob, respectively. The results discussed herein can lead to an in-depth understanding of enzymatic saccharification and suggest viable options for valorizing agrowastes as renewable feedstocks in biorefineries.

Development of bioprocess for corncob-derived levulinic acid production.

Levulinic acid is a significant platform chemical obtained from biomass and can potentially be used to produce value-added biofuels, biopolymers, and biopharmaceuticals. This study aims at statistically optimizing levulinic acid production from agrowastes. Based on the total carbohydrate content (71.93 %), corncob was selected as the target feedstock. A Box-Behnken design with four factors, such as feedstock concentration, reaction time, reaction temperature, and catalyst concentration, was used to optimize the hydrothermal conversion of corncob to levulinic acid at 180 °C for 30 min using 1 M H2SO4 as the acid catalyst and 120 g/L corncob. The maximum yield of 19.9 % was obtained. Additionally, 8.1 g/L formic acid was co-produced. The results of this study can contribute toward valorization of levulinic acid. Moreover, our results can be useful in developing strategies to utilize agrowastes as a renewable feedstock for recent biorefineries to cope with the climate crisis.

Rice straw-derived lipid production by HMF/furfural-tolerant oleaginous yeast generated by adaptive laboratory evolution.

Research on producing medium- and long-chain hydrocarbons as drop-in biofuels has recently accelerated. In addition, lipids are emerging as precursors for biofuel production, and thus, microbial lipid production utilizing agrowastes is becoming a feasible platform technology. Nonetheless, microorganisms are often inhibited by furan aldehydes in biomass-derived hydrolysates. Accordingly, this study aimed to develop oleaginous yeast strains that can tolerate furan aldehydes for producing lipids as biofuel precursors. Rhodosporidium toruloides was selected as the target for adaptive laboratory evolution. The evolved strain, which was obtained from 16 rounds of subcultures, showed a 2.5-fold higher specific growth rate than the wild-type strain in the presence of furan aldehydes and slightly higher lipid production in rice straw hydrolysate. The results discussed in this study provide insights into the production of lipid production by oleaginous yeast utilizing agrowastes as feedstock to obtain drop-in biofuels and contribute to feasible strategies to address climate crises.

Valorization of CO2 to ß-farnesene in Rhodobacter sphaeroides.

The valorization of CO2 into valuable products is a sustainable strategy to help overcome the climate crisis. In particular, biological conversion is attractive as it can produce long-chain hydrocarbons such as terpenoids. This study reports the high yield of ß-farnesene production from CO2 by expressing heterologous ß-farnesene synthase (FS) into Rhodobacter sphaeroides. To increase the expression of FS, a strong active promoter and a ribosome binding site (RBS) were engineered. Moreover, ß-farnesene production was improved further through the supply of exogenous antioxidants and additional nutrients. Finally, ß-farnesene was produced from CO2 at a titer of 44.53 mg/L and yield of 234.08 mg/g, values that were correspondingly 23 times and 46 times higher than those from the initial production of ß-farnesene. Altogether, the results here suggest that the autotrophic production of ß-farnesene can provide a starting point for achieving a circular carbon economy.

Lytic polysaccharide monooxygenase (LPMO)-derived saccharification of lignocellulosic biomass.

Given that traditional biorefineries have been based on microbial fermentation to produce useful fuels, materials, and chemicals as metabolites, saccharification is an important step to obtain fermentable sugars from biomass. It is well-known that glycosidic hydrolases (GHs) are responsible for the saccharification of recalcitrant polysaccharides through hydrolysis, but the discovery of lytic polysaccharide monooxygenase (LPMO), which is a kind of oxidative enzyme involved in cleaving polysaccharides and boosting GH performance, has profoundly changed the understanding of enzyme-based saccharification. This review briefly introduces the classification, structural information, and catalytic mechanism of LPMOs. In addition to recombinant expression strategies, synergistic effects with GH are comprehensively discussed. Challenges and perspectives for LPMO-based saccharification on a large scale are also briefly mentioned. Ultimately, this review can provide insights for constructing an economically viable lignocellulose-based biorefinery system and a closed-carbon loop to cope with climate change.

Development of a glutaric acid production system equipped with stepwise feeding of monosodium glutamate by whole-cell bioconversion.

In the bioproduction of glutaric acid, an emerging bioplastic monomer, α-ketoglutaric acid (α-KG) is required as an amine acceptor for 4-aminobutyrate aminotransferase (GabT)-driven conversion of 5-aminovalerate (5-AVA) to glutarate semialdehyde. Herein, instead of using expensive α-KG, an indirect α-KG supply system was developed using a relatively cheap alternative, monosodium glutamate (MSG), for l-glutamate oxidase (Gox)-based whole-cell conversion. Using 200 mM 5-AVA and 30 mM MSG initially with Gox, 67.1 mM of glutaric acid was produced. By applying the stepwise feeding strategy of MSG, the glutaric acid production capability was increased to 159.1 mM glutaric acid with a conversion yield of 79.6%. In addition, a buffer-free one-pot reaction from l-lysine was also applied in a 5 L bioreactor to evaluate its industrial applicability, resulting in a conversion yield of 54.2%. The system developed herein might have great potential for the large-scale, economically feasible production of glutaric acid by whole-cell conversion.

Newly explored formate dehydrogenases from Clostridium species catalyze carbon dioxide to formate.

With concerns over global warming and climate change, many efforts have been devoted to mitigate atmospheric CO2 level. As a CO2 utilization strategy, formate dehydrogenase (FDH) from Clostridium species were explored to discover O2-tolerant and efficient FDHs that can catalyze CO2 to formate (i.e. CO2 reductase). With FDH from Clostridium ljungdahlii (ClFDH) that plays as a CO2 reductase previously reported as the reference, FDH from C.autoethanogenum (CaFDH), C. coskatii (CcFDH), and C. ragsdalei (CrFDH) were newly discovered via genome-mining. The FDHs were expressed in Escherichia coli and the recombinant FDHs successfully catalyzed CO2 reduction with a specific activity of 15 U g-1-CaFDH, 17 U g-1-CcFDH, and 8.7 U g-1-CrFDH. Interestingly, all FDHs newly discovered retain their catalytic activity under aerobic condition, although Clostridium species are strict anaerobe. The results discussed herein can contribute to biocatalytic CO2 utilization.

Effect of manganese peroxidase on the decomposition of cellulosic components: Direct cellulolytic activity and synergistic effect with cellulase.

Herein, it was unearthed that manganese peroxidase (MnP) from Phanerochaete chrysosporium, a lignin-degrading enzyme, is capable of not only directly decomposing cellulosic components but also boosting cellulase activity. MnP decomposes various cellulosic substrates (carboxymethyl cellulose, cellobiose [CMC], and Avicel®) and produces reducing sugars rather than oxidized sugars such as lactone and ketoaldolase. MnP with MnII in acetate buffer evolves the MnIII-acetate complex functioning as a strong oxidant, and the non-specificity of MnIII-acetate enables cellulose-decomposition. The catalytic mechanism was proposed by analyzing catalytic products derived from MnP-treated cellopentaose. Notably, MnP also boosts cellulase activity on CMC and Avicel®, even considering the cellulolytic activity of MnP itself. To the best of the authors' knowledge, this is the first report demonstrating a previously unknown fungal MnP activity in cellulose-decomposition in addition to a known delignification activity. Consequently, the results provide a promising insight for further investigation of the versatility of lignin-degrading biocatalysts.

Paradigm shift in algal biomass refinery and its challenges.

Microalgae have been studied and tested for over 70 years. However, biodiesel, the prime target of the algal industry, has suffered from low competitiveness and current steps toward banning the internal combustion engine all over the world. Meanwhile, interest in reducing CO2 emissions has grown as the world has witnessed disasters caused by global warming. In this situation, in order to maximize the benefits of the microalgal industry and surmount current limitations, new breakthroughs are being sought. First, drop-in fuel, mandatory for the aviation and maritime industries, has been discussed as a new product. Second, methods to secure stable and feasible outdoor cultivation focusing on CO2 sequestration were investigated. Lastly, the need for an integrated refinery process to simultaneously produce multiple products has been discussed. While the merits of microalgae industry remain valid, further investigations into these new frontiers would put algal industry at the core of future bio-based economy.

Improving the catalytic performance of xylanase from Bacillus circulans through structure-based rational design.

Endo-1,4-ß-xylanase is one of the most important enzymes employed in biorefineries for obtaining fermentable sugars from hemicellulosic components. Herein, we aimed to improve the catalytic performance of Bacillus circulans xylanase (Bcx) using a structure-guided rational design. A systematic analysis of flexible motions revealed that the R49 component of Bcx (i) constrains the global conformational changes essential for substrate binding and (ii) is involved in modulating flexible motion. Site-saturated mutagenesis of the R49 residue led to the engineering of the active mutants with the trade-off between flexibility and rigidity. The most active mutant R49N improved the catalytic performance, including its catalytic efficiency (7.51-fold), conformational stability (0.7 °C improvement), and production of xylose oligomers (2.18-fold higher xylobiose and 1.72-fold higher xylotriose). The results discussed herein can be applied to enhance the catalytic performance of industrially important enzymes by controlling flexibility.

Chemoenzymatic valorization of agricultural wastes into 4-hydroxyvaleric acid via levulinic acid.

Given that (i) levulinic acid (LA) is one of the most significant platform chemicals derived from biomass and (ii) 4-hydroxyvaleric acid (4-HV) is a potential LA derivative, the aim of this study is to achieve chemoenzymatic valorization of LA, which was obtained from agricultural wastes, to 4-HV. The thermochemical process utilized agricultural wastes (i.e., rice straw and corncob) as feedstocks and successfully produced LA, ranging from 25.1 to 65.4 mM. Additionally, formate was co-produced and used as a hydrogen source for the enzymatic hydrogenation of LA. Finally, engineered 3-hydroxybutyrate dehydrogenase from Alcaligenes faecalis (eHBDH) was applicable for catalyzing the conversion of agricultural wastes-driven LA, resulting in a maximum concentration of 11.32 mM 4-HV with a conversion rate of 48.2%. To the best of our knowledge, this is the first report describing the production of 4-HV from actual biomass, and the results might provide insights into the valorization of agricultural wastes.

Recent progress and challenges in microbial polyhydroxybutyrate (PHB) production from CO2 as a sustainable feedstock: A state-of-the-art review.

The recalcitrance of petroleum-based plastics causes severe environmental problems and has accelerated research into production of biodegradable polymers from inexpensive and sustainable feedstocks. Various microorganisms are capable of producing Polyhydroxybutyrate (PHB), a representative biodegradable polymer, under nutrient-limited conditions, among which CO2-utilizing microorganisms are of primary interest. Herein, we discuss recent progress on bacterial strains including proteobacteria, purple non-sulfur bacteria, and cyanobacteria in terms of CO2-containing carbon sources, PHB-production capability, and genetic modification. In addition, this review introduces recent technical approaches used to improve PHB production from CO2 such as two-stage bioprocesses and bioelectrochemical systems. Challenges and future perspectives for the development of economically feasible PHB production are also discussed. Finally, this review might provide insights into the construction of a closed-carbon-loop to cope with climate change.

Improving the organic solvent resistance of lipase a from Bacillus subtilis in water-ethanol solvent through rational surface engineering.

Given that lipase is an enzyme applicable in various industrial fields and water-miscible organic solvents are important reaction media for developing industrial-scale biocatalysis, a structure-based strategy was explored to stabilize lipase A from Bacillus subtilis in a water-ethanol cosolvent. Site-directed mutagenesis of ethanol-interacting sites resulted in 4 mutants, i.e., Ser16Gly, Ala38Gly, Ala38Thr, and Leu108Asn, which were stable in 50% ethanol and had up to 1.8-fold higher stability than the wild-type. In addition, Leu108Asn was more thermostable at 45 °C than the wild type. The results discussed in this study not only provide insights into strategies for enzyme engineering to improve organic solvent resistance but also suggest perspectives on pioneering routes for constructing enzyme-based biorefineries to produce value-added fuels and chemicals.

Recent developments and key barriers to microbial CO2 electrobiorefinery.

The electrochemical conversion of CO2 can include renewable surplus electricity storage and CO2 utilisation. This review focuses on the microbial CO2 electrobiorefinery based on microbial electrosynthesis (MES) which merges electrochemical and microbial conversion to produce biofuels and higher-value chemicals. In this review, recent developments are discussed about bioelectrochemical conversion of CO2 into biofuels and chemicals in MES via microbial CO2-fixation and electricity utilisation reactions. In addition, this review examines technical approaches to overcome the current limitations of MES including the following: engineering of the biocathode, application of electron mediators, and reactor optimisation, among others. An in-depth discussion of strategies for the CO2 electrobiorefinery is presented, including the integration of the biocathode with inorganic catalysts, screening of novel electroactive microorganisms, and metabolic engineering to improve target productivity from CO2.

Elevated conversion of CO2 to versatile formate by a newly discovered formate dehydrogenase from Rhodobacter aestuarii.

Due to climate change, recent research interests have increased towards CO2 utilization as a strategy to mitigate the atmospheric CO2 level. Herein, we aimed to explore formate dehydrogenases (FDHs) from chemoautotroph to discover an efficient and O2-tolerant biocatalyst for catalyzing the CO2 reduction to a versatile formate. Through genome-mining and phylogenetic analysis, the FDH from Rhodobacter aestuarii (RaFDH) was newly discovered as a promising O2-tolernat CO2 reductase and was successfully expressed in Escherichia coli. In this study, the optimum conditions and turnover rates of RaFDH were examined for CO2 reduction and formate oxidation. In particular, the RaFDH-driven CO2 reduction far surpassed the formate oxidation with a turnover rate of 48.3 and 15.6 min-1, respectively. The outstanding superiority of RaFDH towards CO2 reduction can be applicable for constructing a feasible electroenzymatic system that produce a versatile formate from CO2 as a cheap, abundant, and renewable resource.

A perspective on the biotechnological applications of the versatile tyrosinase.

Tyrosinase (E.C. 1.14.18. 1) is a type of Cu-containing oxidoreductase which has bifunctional activity for various phenolic substrates: ortho-hydroxylation of monophenols to diphenols (a cresolase activity) and oxidation of diphenols to quinones (a catecholase activity). Based on the broad substrate spectrum, tyrosinase has been used in bioremediation of phenolic pollutants, constructing biosensors for identifying phenolic compounds, and L-DOPA synthesis. Furthermore, not only tyrosinase has been used to produce useful polyphenol derivatives, but also it is recently revealed that the promiscuous activity of tyrosinase is closely related with delignification in the biorefinery. Accordingly, tyrosinase might be a potential biocatalyst for industrial applications (e.g., electroenzymatic L-DOPA production, but its long-term stability and reusability should be further explored. In this review, we emphasize the versatility of tyrosinase, which includes conventional applications, and suggest new perspectives as an industrial biocatalyst (e.g., electroenzymatic L-DOPA production). Especially, this review focuses on and comprehensively discusses recent innovative studies.

Perspectives for biocatalytic lignin utilization: cleaving 4-O-5 and Cα-Cß bonds in dimeric lignin model compounds catalyzed by a promiscuous activity of tyrosinase.

BACKGROUND: In the biorefinery utilizing lignocellulosic biomasses, lignin decomposition to value-added phenolic derivatives is a key issue, and recently biocatalytic delignification is emerging owing to its superior selectivity, low energy consumption, and unparalleled sustainability. However, besides heme-containing peroxidases and laccases, information about lignolytic biocatalysts is still limited till date. RESULTS: Herein, we report a promiscuous activity of tyrosinase which is closely associated with delignification requiring high redox potentials (>1.4 V vs. normal hydrogen electrode [NHE]). The promiscuous activity of tyrosinase not only oxidizes veratryl alcohol, a commonly used nonphenolic substrate for assaying ligninolytic activity, to veratraldehyde but also cleaves the 4-O-5 and Cα-Cß bonds in 4-phenoxyphenol and guaiacyl glycerol-ß-guaiacyl ether (GGE) that are dimeric lignin model compounds. Cyclic voltammograms additionally verified that the promiscuous activity oxidizes lignin-related high redox potential substrates. CONCLUSION: These results might be applicable for extending the versatility of tyrosinase toward biocatalytic delignification as well as suggesting a new perspective for sustainable lignin utilization. Furthermore, the results provide insight for exploring the previously unknown promiscuous activities of biocatalysts much more diverse than ever thought before, thereby innovatively expanding the applicable area of biocatalysis.

Expression and characterization of Pantoea CO dehydrogenase to utilize CO-containing industrial waste gas for expanding the versatility of CO dehydrogenase.

Although aerobic CO dehydrogenases (CODHs) might be applicable in various fields, their practical applications have been hampered by low activity and no heterologous expression. We, for the first time, could functionally express recombinant PsCODH in E. coli and obtained a highly concentrated recombinant enzyme using an easy and convenient method. Its electron acceptor spectra, optimum conditions (pH 6.5 and 30 °C), and kinetic parameters (kcat of 12.97 s-1, Km of 0.065 mM, and specific activity of 0.86 Umg-1) were examined. Blast furnace gas (BFG) containing 20% CO, which is a waste gas from the steel-making process, was tested as a substrate for PsCODH. Even with BFG, the recombinant PsCODH retained 88.2% and 108.4% activity compared with those of pure CO and 20% CO, respectively. The results provide not only a promising strategy to utilize CO-containing industrial waste gases as cheap, abundant, and renewable resources but also significant information for further studies about cascade reactions producing value-added chemicals via CO2 as an intermediate produced by a CODH-based CO-utilization system, which would ultimately expand the versatility of CODH.

Electrochemical detoxification of phenolic compounds in lignocellulosic hydrolysate for Clostridium fermentation.

Lignocellulosic biomass is being preferred as a feedstock in the biorefinery, but lignocellulosic hydrolysate usually contains inhibitors against microbial fermentation. Among these inhibitors, phenolics are highly toxic to butyric acid-producing and butanol-producing Clostridium even at a low concentration. Herein, we developed an electrochemical polymerization method to detoxify phenolic compounds in lignocellulosic hydrolysate for efficient Clostridium fermentation. After the electrochemical detoxification for 10h, 78%, 77%, 82%, and 94% of p-coumaric acid, ferulic acid, vanillin, and syringaldehyde were removed, respectively. Furthermore, 71% of total phenolics in rice straw hydrolysate were removed without any sugar-loss. Whereas the cell growth and metabolite production of Clostridium tyrobutyricum and Clostridium beijerinckii were completely inhibited in un-detoxified hydrolysate, those in detoxifying rice straw hydrolysate were recovered to 70-100% of the control cultures. The electrochemical detoxification method described herein provides an efficient strategy for producing butanol and butyric acid through Clostridium fermentation with lignocellulosic hydrolysate.