Improvement of succinate production from methane by combining rational engineering and laboratory evolution in Methylomonas sp. DH-1.

Recently, methane has been considered a next-generation carbon feedstock due to its abundance and it is main component of shale gas and biogas. Methylomonas sp. DH-1 has been evaluated as a promising industrial bio-catalyst candidate. Succinate is considered one of the top building block chemicals in the agricultural, food, and pharmaceutical industries. In this study, succinate production by Methylomonas sp. DH-1 was improved by combining adaptive laboratory evolution (ALE) technology with genetic engineering in the chromosome of Methylomonas sp. DH-1, such as deletion of bypass pathway genes (succinate dehydrogenase and succinate semialdehyde dehydrogenase) or overexpression of genes related with succinate production (citrate synthase, pyruvate carboxylase and phosphoenolpyruvate carboxylase). Through ALE, the maximum consumption rate of substrate gases (methane and oxygen) and the duration maintaining high substrate gas consumption rates was enhanced compared to those of the parental strain. Based on the improved methane consumption, cell growth (OD600) increased more than twice, and the succinate titer increased by ~ 48% from 218 to 323 mg/L. To prevent unwanted succinate consumption, the succinate semialdehyde dehydrogenase gene was deleted from the genome. The first enzyme of TCA cycle (citrate synthase) was overexpressed. Pyruvate carboxylase and phosphoenolpyruvate carboxylase, which produce oxaloacetate, a substrate for citrate synthase, were also overproduced by a newly identified strong promoter. The new strong promoter was screened from RNA sequencing data. When these modifications were combined in one strain, the maximum titer (702 mg/L) was successfully improved by more than three times. This study demonstrates that successful enhancement of succinic acid production can be achieved in methanotrophs through additional genetic engineering following adaptive laboratory evolution.

Enhancing microbial CO2 electrocatalysis for multicarbon reduction in a wet amine-based catholyte.

Microbial CO2 electroreduction (mCO2ER) offers a promising approach for producing high-value multicarbon reductants from CO2 by combining CO2 fixing microorganisms with conducting materials (i. e., cathodes). However, the solubility and availability of CO2 in an aqueous electrolyte pose significant limitations in this system. This study demonstrates the efficient production of long-chain multicarbon reductants, specifically carotenoids (~C40), within a wet amine-based catholyte medium during mCO2ER. Optimizing the concentration of the biocompatible CO2 absorbent, monoethanolamine (MEA), led to enhanced CO2 fixation in the electroautotroph bacteria. Molecular biological analyses revealed that MEA in the catholyte medium redirected the carbon flux towards carotenoid biosynthesis during mCO2ER. The faradaic efficiency of mCO2ER with MEA for carotenoid production was 4.5-fold higher than that of the control condition. These results suggest the mass transport bottleneck in bioelectrochemical systems could be effectively addressed by MEA-assissted mCO2ER, enabling highly efficient production of valuable products from CO2.

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.

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.

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.

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.

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.

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.

Biological Carbon Recovery from Sugar Refinery Washing Water into Microalgal DHA: Medium Optimization and Stress Induction.

Sugar refinery washing water (SRWW) contains abundant levels of carbon sources and lower levels of contaminants than other types of wastewater, which makes it ideal for heterotrophic cultivation of microalgae. Here, carbon sources in SRWW were utilized for conversion into the form of value-added docosahexaenoic acid (DHA) using Aurantiochytrium sp. KRS101. Since SRWW is not a defined medium, serial optimizations were performed to maximize the biomass, lipid, and DHA yields by adjusting the nutrient (carbon, nitrogen, and phosphorus) concentrations as well as the application of salt stress. Optimum growth performance was achieved with 30% dilution of SRWW containing a total organic carbon of 95,488 mg L-1. Increasing the nutrient level in the medium by supplementation of 9 g L-1 KH2PO4 and 20 g L-1 yeast extract further improved the biomass yield by an additional 14%, albeit at the expense of a decrease in the lipid content. Maximum biomass, lipid, and DHA yields (22.9, 6.33, and 2.03 g L-1, respectively) were achieved when 35 g L-1 sea salt was applied on a stationary phase for osmotic stress. These results demonstrate the potential of carbon-rich sugar refinery washing water for DHA production using Aurantiochytrium sp. KRS101 and proper cultivation strategy.

Optimization of heterotrophic cultivation of Chlorella sp. HS2 using screening, statistical assessment, and validation.

The heterotrophic cultivation of microalgae has a number of notable advantages, which include allowing high culture density levels as well as enabling the production of biomass in consistent and predictable quantities. In this study, the full potential of Chlorella sp. HS2 is explored through optimization of the parameters for its heterotrophic cultivation. First, carbon and nitrogen sources were screened in PhotobioBox. Initial screening using the Plackett-Burman design (PBD) was then adopted and the concentrations of the major nutrients (glucose, sodium nitrate, and dipotassium phosphate) were optimized via response surface methodology (RSM) with a central composite design (CCD). Upon validation of the model via flask-scale cultivation, the optimized BG11 medium was found to result in a three-fold improvement in biomass amounts, from 5.85 to 18.13 g/L, in comparison to a non-optimized BG11 medium containing 72 g/L glucose. Scaling up the cultivation to a 5-L fermenter resulted in a greatly improved biomass concentration of 35.3 g/L owing to more efficient oxygenation of the culture. In addition, phosphorus feeding fermentation was employed in an effort to address early depletion of phosphate, and a maximum biomass concentration of 42.95 g/L was achieved, with biomass productivity of 5.37 g/L/D.

Author Correction: Statistical optimization of light intensity and CO2 concentration for lipid production derived from attached cultivation of green microalga Ettlia sp.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Increased biomass and lipid production of Ettlia sp. YC001 by optimized C and N sources in heterotrophic culture.

The culture conditions and media composition for the heterotrophic culture of an axenic strain of Ettlia sp. YC001 were firstly optimized using the Plackett-Burman design (PBD) and response surface methodology (RSM). The strain successfully showed higher productivity in the basal media without any light illumination at 32.2 to 33.3 °C. The PBD results showed that the most effective components for biomass productivity of Ettlia sp. were fructose and yeast extract for sources of C and N, respectively. The RSM results showed an optimal level of 72.2 g/L for fructose and 21.5 g/L for yeast extract, resulting in 46.1 g/L biomass with a lipid content of 13.8% over a course of 9 days. Using a 5 L scaled-up fermentation system for 6 days, the production of biomass and lipids was 7.21 g/L/day and 1.18 g/L/day, respectively. Consequently, heterotrophic cultivation of Ettlia sp. YC001 provided much higher production of biomass and lipids than those of autotrophic cultivation. As further research, the use of substitute substrates instead of fructose and yeast extract should be developed to reduce production costs.

Statistical optimization of light intensity and CO2 concentration for lipid production derived from attached cultivation of green microalga Ettlia sp.

Attached cultivation systems have been receiving extensive attention as a breakthrough in microalgae cultivation technology. However, there is a lack of studies that emphasize precise optimization of important parameters in attached cultivation of microalgae. In this study, the effects of two major environmental parameters in photoautotrophic cultivation, light intensity and CO2 concentration, on the biomass and lipid surface productivity of Ettlia sp. YC001 were optimized by employing Response Surface Methodology (RSM) and validated experimentally. The optimum initial conditions for attached cultivation were use of seed from the late exponential phase (LE) and an inoculum surface density of 2.5 g/m2. By optimization, maximum biomass surface productivity of 28.0 ± 1.5 g/m2/day was achieved at 730 µE/m2/s with 8% CO2. The maximum lipid surface productivity was 4.2 ± 0.3 g/m2/day at 500 µE/m2/s with 7% CO2. Change of the fatty acid composition with respect to changes in environment parameters led to improvement of biodiesel quality at higher light intensity and higher CO2 concentration. Attached cultivation of Ettlia sp. YC001 has successfully produced biomass and lipids at a high production rate with relatively low light energy demand and high CO2 utilization.

Evaluation of various harvesting methods for high-density microalgae, Aurantiochytrium sp. KRS101.

Five technologies, coagulation, electro-flotation (EF), electro-coagulation-flotation (ECF), centrifugation, and membrane filtration, were systematically assessed for their adequacy of harvesting Aurantiochytrium sp. KRS101, a heterotrophic microalgal species that has much higher biomass concentration than photoautotrophic species. Coagulation, EF, and ECF were found to have limited efficiency. Centrifugation was overly powerful to susceptible cells like Aurantiochytrium sp. KRS101, inducing cell rupture and consequently biomass loss of over 13%. Membrane filtration, in particular equipped with an anti-fouling turbulence generator, turned out to be best suited: nearly 100% of harvesting efficiency and low water content in harvested biomass were achieved. With rotation rate increased, high permeate fluxes could be attained even with extremely concentrated biomass: e.g., 219.0 and 135.0 L/m(2)/h at 150.0 and 203.0 g/L, respectively. Dynamic filtration appears to be indeed a suitable means especially to obtain highly concentrated biomass that have no need of dewatering and can be directly processed.