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.

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.

Regulation of Reactive Oxygen Species Promotes Growth and Carotenoid Production Under Autotrophic Conditions in Rhodobacter sphaeroides.

Industrial demand for capture and utilization using microorganisms to reduce CO2, a major cause of global warming, is significantly increasing. Rhodobacter sphaeroides is a suitable strain for the process of converting CO2 into high-value materials because it can accept CO2 and has various metabolic pathways. However, it has been mainly studied for heterotrophic growth that uses sugars and organic acids as carbon sources, not autotrophic growth. Here, we report that the regulation of reactive oxygen species is critical for growth when using CO2 as a sole carbon source in R. sphaeroides. In general, the growth rate is much slower under autotrophic conditions compared to heterotrophic conditions. To improve this, we performed random mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine (NTG). As a result, we selected the YR-1 strain with a maximum specific growth rate (µ) 1.44 day-1 in the early growth phase, which has a 110% faster growth rate compared to the wild-type. Based on the transcriptome analysis, it was confirmed that the growth was more sensitive to reactive oxygen species under autotrophic conditions. In the YR-1 mutant, the endogenous contents of H2O2 levels and oxidative damage were reduced by 33.3 and 42.7% in the cells, respectively. Furthermore, we measured that concentrations of carotenoids, which are important antioxidants. The total carotenoid is produced 9.63 g/L in the YR-1 mutant, suggesting that the production is 1.7-fold higher than wild-type. Taken together, our observations indicate that controlling ROS promotes cell growth and carotenoid production under autotrophic conditions.

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.

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 hydrogen production by pH adjustment in pressurized gas fermentation.

Gas fermentation utilizes syngas converted from biomass or waste as feedstock. A bubble column reactor for pressurizing was designed to increase the mass transfer rate between gas and liquid, and reduce energy consumption by medium agitation. Thermococcus onnurineus, a hydrogenic CO-oxidizer, was cultured initially under ambient pressure with the initial inlet gas composition; 60% CO and 40% N2. The maximum H2 productivity was 363 mmol/l/h, without pH adjustment. When additional pressure was applied, the pH rapidly declined; this may be attributed to the increased CO2 solubility under pressure. By controlling pH, H2 productivity increased up to 450 mmol/l/h; which is comparable to the previously reported H2 productivity in a continuous stirred tank reactor. The results may suggest energy saving potentials of bubble column reactors in gas fermentation. This finding may be applied to other gas fermentation processes, as syngas itself contains CO2 and many microbial processes also release CO2.

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 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.

Pressurized cultivation strategies for improved microbial hydrogen production by Thermococcus onnurineus NA1.

While the hydrogen economy is receiving growing attention, research on microbial hydrogen production is also increasing. Microbial water-gas shift reaction is advantageous as it produces hydrogen from by product gas including carbon monoxide (CO). However, CO solubility in water is the bottleneck of this process by low mass transfer. Thermococcus onnurineus NA1 strain can endure a high-pressure environment and can enhance hydrogen production in a pressurized reactor by increasing CO solubility. As CO causes cell toxicity, two important factors, pressure and input gas flow rate, should be considered for process control during cultivation. Hence, we employed different operational strategies for enhancing hydrogen production and obtained 577 mmol/L/h of hydrogen productivity. This is the highest hydrogen productivity reported to date from microbial water-gas shift reaction.

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.

Bubble coalescence suppression driven carbon monoxide (CO)-water mass transfer increase by electrolyte addition in a hollow fiber membrane bioreactor (HFMBR) for microbial CO conversion to ethanol.

This study investigated the effects of electrolytes (CaCl2, K2HPO4, MgSO4, NaCl, and NH4Cl) on CO mass transfer and ethanol production in a HFMBR. The hollow fiber membranes (HFM) were found to generate tiny gas bubbles; the bubble coalescence was significantly suppressed in electrolyte solution. The volumetric gas-liquid mass transfer coefficients (kLa) increased up to 414% compared to the control. Saturated CO (C∗) decreased as electrolyte concentrations increased. Overall, the maximum mass transfer rate (Rmax) in electrolyte solution ranged from 106% to 339% of the value obtained in water. The electrolyte toxicity on cell growth was tested using Clostridium autoethanogenum. Most electrolytes, except for MgSO4, inhibited cell growth. The HFMBR operation using a medium containing 1% MgSO4 achieved 119% ethanol production compared to that without electrolytes. Finally, a kinetic simulation using the parameters got from the 1% MgSO4 medium predicted a higher ethanol production compared to the control.

Enhancement of volatile fatty acids production from rice straw via anaerobic digestion with chemical pretreatment.

Rice straw is one of the most abundant renewable biomass sources and was selected as the feedstock for the production of volatile fatty acids (VFAs) from which microbial biodiesel can be produced. Two kinds of chemical pretreatments involving nitric acid and sodium hydroxide were investigated at 150 °C with 20 min of reaction time. The nitric acid pretreatment generated the most hemicellulose hydrolyzate, while significant reduction of the lignin occurred with sodium hydroxide pretreatment. Anaerobic digestion of 20 g/L rice straw yielded 6.00 and 7.09 g VFAs/L with 0.5% HNO3 and 2% NaOH, respectively. The VFAs yield with 2% NaOH was 0.35 g/g.

A comprehensive study on volatile fatty acids production from rice straw coupled with microbial community analysis.

Rice straw is one of the most abundant renewable energy sources available. Through anaerobic acidogenesis, the substance of rice straw can be converted to volatile fatty acids (VFAs). VFAs itself is of value and is a precursor to biofuels. Hence, it can be converted to mixed alcohols by addition of hydrogen, and biodiesel can be produced as a carbon source for oleaginous microorganism. To maximize VFAs production during anaerobic digestion (AD), response surface analysis (RSM) was carried out with respect to temperature, substrate concentration, and pH variables. Optimization results showed maximal VFAs concentration of 12.37 g/L at 39.23 °C, 52.85 g/L of rice straw, and pH 10. In quantification of microbial community by quantitative polymerase chain reaction, the bacterial profile showed that the growth of methanogens was effectively inhibited by methanogenic inhibitors. Furthermore, 454 pyrosequencing showed that members of the Ruminococcaceae family, capable of hydrolyzing lignocellulosic biomass, were the most dominant species in many RSM trials. This study provided a useful insight on the biological improvement of AD performance through the combinational linkage between process parameters and microbial information.

Optimization of volatile fatty acids and hydrogen production from Saccharina japonica: acidogenesis and molecular analysis of the resulting microbial communities.

Response surface methodology (RSM) was used to optimize the production of volatile fatty acids (VFAs) and hydrogen from mixed anaerobic cultures of Saccharina japonica with respect to two independent variables: methanogenic inhibitor concentration and temperature. The effects of four methanogenic inhibitors on acidogenic processes were tested, and qualitative microbial analyses were carried out. Escherichia, Acinetobacter, and Clostridium were the most predominant genera in samples treated with chloroform (CHCl3), iodoform (CHI3), 2-bromoethanesulfonate (BES), or ß-cyclodextrin (ß-CD), respectively. RSM showed that the production of VFAs reached a peak of 12.5 g/L at 38.6 °C in the presence of 7.4 g/L ß-CD; these were the conditions under which hydrogen production was also nearly maximal. The quantitative polymerase chain reaction (qPCR) showed that shifts in the bacterial community population correlated with the concentrations of ß-CD indicating that this compound effectively inhibited methanogens.

Efficient lactulose production from cheese whey using sodium carbonate.

An economical method of lactulose production from cheese whey was developed using sodium carbonate (Na2CO3). Three parameters such as temperature, reaction time, and Na2CO3 concentration were identified as experimental factors, and yield was selected as a response parameter. The experimental factors were optimised employing Response Surface Methodology (RSM). Maximum yield of 29.6% was obtained at reaction time of 20.41 min, Na2CO3 of 0.51% at 90 °C. To overcome this limited lactulose yield, due to the conversion of lactulose to galactose, fed batch system was applied using dried cheese whey as lactose source. By this system, limit was broken, and 15.8 g/L of lactulose is produced in hour.

Volatile fatty acids derived from waste organics provide an economical carbon source for microbial lipids/biodiesel production.

Volatile fatty acids (VFAs) derived from organic waste, were used as a low cost carbon source for high bioreactor productivity and titer. A multi-stage continuous high cell density culture (MSC-HCDC) process was employed for economic assessment of microbial lipids for biodiesel production. In a simulation study we used a lipid yield of 0.3 g/g-VFAs, cell mass yield of 0.5 g/g-glucose or wood hydrolyzates, and employed process variables including lipid contents from 10-90% of cell mass, bioreactor productivity of 0.5-48 g/L/h, and plant capacity of 20000-1000000 metric ton (MT)/year. A production cost of USD 1.048/kg-lipid was predicted with raw material costs of USD 0.2/kg for wood hydrolyzates and USD 0.15/kg for VFAs; 9 g/L/h bioreactor productivity; 100, 000 MT/year production capacity; and 75% lipids content. The variables having the highest impact on microbial lipid production costs were the cost of VFAs and lipid yield, followed by lipid content, fermenter cost, and lipid productivity. The cost of raw materials accounted for 66.25% of total operating costs. This study shows that biodiesel from microbial lipids has the potential to become competitive with diesels from other sources.