Lizhen Hu’s research while affiliated with Xi'an Jiaotong University and other places

Background The dramatic increase in greenhouse gas (GHG) emissions, which causes serious global environmental issues and severe climate changes, has become a global problem of concern in recent decades. Currently, native and/or non-native C1-utilizing microbes have been modified to be able to effectively convert C1-gases (biogas, natural gas, and CO2) into isobutanol via biological routes. Even though the current experimental results are satisfactory in lab-scale research, the techno-economic feasibility of C1 gas-derived isobutanol production at the industrial scale still needs to be analyzed and evaluated, which will be essential for the future industrialization of C1-gas bioconversion. Therefore, techno-economic analyses were conducted in this study with comparisons of capital cost (CAPEX), operating cost (OPEX), and minimum isobutanol selling price (MISP) derived from biogas (scenario #1), natural gas (scenario #2), and CO2 (scenario #3) with systematic economic assessment. Results By calculating capital investments and necessary expenses, the highest CAPEX ($317 MM) and OPEX ($67 MM) were projected in scenario #1 and scenario #2, respectively. Because of the lower CAPEX and OPEX from scenario #3, the results revealed that bioconversion of CO2 into isobutanol temporally exhibited the best economic performance with an MISP of $1.38/kg isobutanol. Furthermore, a single sensitivity analysis with nine different parameters was carried out for the production of CO2-derived isobutanol. The annual plant capacity, gas utilization rate, and substrate cost are the three most important economic-driving forces on the MISP of CO2-derived isobutanol. Finally, a multiple-point sensitivity analysis considering all five parameters simultaneously was performed using ideal targets, which presented the lowest MISP of$0.99/kg in a long-term case study. Conclusions This study provides a comprehensive assessment of the bioconversion of C1-gases into isobutanol in terms of the bioprocess design, mass/energy calculation, capital investment, operating expense, sensitivity analysis, and minimum selling price. Compared with isobutanol derived from biogas and natural gas, the CO2-based isobutanol showed better economic feasibility. A market competitive isobutanol derived from CO2 is predicable with lower CO2 cost, better isobutanol titer, and higher annual capacity. This study will help researchers and decision-makers explore innovative and effective approaches to neutralizing GHGs and focus on key economic-driving forces to improve techno-economic performance.

C1 gaseous substrates (CH4, CO2, and CO) derived from natural gas, biogas, and syngas, are of interest due to their threats to the environment or inefficient utilization. Benefiting from advanced genetic editing tools and bioconversion strategies, metabolically engineered C1-gas-utilizing microorganisms (CGUM), such as methanotrophs, cyanobacteria, and acetogens, are capable of utilizing C1 gaseous feedstocks as the sole substrates for cell growth and synthesis of chemicals and biofuels. In this paper, we critically review metabolic pathways related to the assimilation of C1 gaseous substrates for alcohol biosynthesis in several model CGUM. Metabolic engineering approaches utilized to enhance the carbon conversion efficiency, microbial growth and biosynthesis of desired alcohols are summarized, including the regulation of C1 gaseous substrates activation and electron and energy supply, the accumulation of key intermediates, and the manipulation of target gene expression to optimize carbon flux to bioalcohols. In addition, challenges in the efficient microbial conversion of C1 gaseous substrates are explored and discussed. The strategies of bioalcohol biosynthesis presented here could guide the development of a variety of efficient biological routes for CH4, CO2, and CO utilization in the future.

Background The dramatic increase in emissions of greenhouse gases (GHGs) has led to an irreversible effect on the ecosystem, which in turn caused significant harm to human beings and other species. Exploring innovative and effective approaches to neutralizing GHGs is urgently needed. Considering the advancement of synthetic biology and the bioconversion process, C1-utilizing cell factories (CUCFs) have been modified to be able to effectively convert C1-gases includes biogas, natural gas, and carbon dioxide (CO 2 ) into chemicals or fuels via biological routes, which greatly facilitates the inedible carbon sources used in biomanufacturing, increases the potential value of GHGs and meanwhile reduces the GHG emissions. Process design and resultsEven though the current experimental results are satisfactory in lab-scale research, the evaluation of economic feasibility as well as applications of CUCFs in industrial-scale still need to be analyzed. This study designed three scenarios of CUCFs-based conversion of biogas, natural gas, and CO 2 into isobutanol, the detailed techno-economic analyses of these scenarios were conducted with the comparisons of capital cost, operating cost, and minimum isobutanol selling price (MISP). Results revealed that direct bio-conversion of CO 2 by CUCFs into isobutanol exhibited the best economic performance with a MISP of $1.38/kg isobutanol. The single sensitivity analysis showed that the gas utilization rate, flow rate, and CO 2 cost are the three most significant economic-driving forces on MISP of CO 2 -derived biological isobutanol. Multiple-point sensitivity analysis presented that the MISP for the long-term case can be as low as 0.99$/kg with using ideal targets. Conclusions Our findings provide a comprehensive assessment of bio-conversion of C1-gases via CUCFs to isobutanol in terms of the bioprocess design, mass/energy calculation, capital investment, operating expense, sensitivity analysis, and environmental impact. It is expected that this study may lead to the paradigm shift in isobutanol synthesis with C1-gases as substrates.

One-carbon (C1) substrates such as methane and methanol have been considered as the next-generation carbon source in industrial biotechnology with the characteristics of low cost, availability, and bioconvertibility. Recently, methanotrophic bacteria naturally capable of converting C1 substrates have drawn attractive attention for their promising applications in C1-based biomanufacturing for the production of chemicals or fuels. Although genetic tools have been explored for metabolically engineered methanotroph construction, there is still a lack of efficient methods for heterologous gene expression in methanotrophs. Here, a rapid and efficient electroporation method with a high transformation efficiency was developed for a robust methanotroph of Methylomicrobium buryatense 5GB1. Based on the homologous recombination and high transformation efficiency, gene deletion and heterologous gene expression can be simultaneously achieved by direct electroporation of PCR-generated linear DNA fragments. In this study, the influence of several key parameters (competent cell preparation, electroporation condition, recovery time, and antibiotic concentration) on the transformation efficiency was investigated for optimum conditions. The maximum electroporation efficiency of 719 ± 22.5 CFU/μg DNA was reached, which presents a 10-fold improvement. By employing this method, an engineered M. buryatense 5GB1 was constructed to biosynthesize isobutyraldehyde by replacing an endogenous fadE gene in the genome with a heterologous kivd gene. This study provides a potential and efficient strategy and method to facilitate the cell factory construction of methanotrophs.

Commercial cellulosic biofuel operation requires a reliable, low-cost, and stable feedstock logistic system. One great challenge is its long-term storage at least for one harvest cycle (1 year for agricultural crop residues) with a minimum loss of bulky, geographically dispersed, inflammable, and easily degradable lignocellulosic biomass. This study conducted an investigation of year-round storage of the agricultural crop residue feedstock under the scenario of performing dry acid pretreatment at the distributed regional collection depots, instead of the central biorefinery plant. The dry acid pretreatment method provides a practical basis for the storage operation by its ability for high preservation of polysaccharide solids, highly compacted accumulation density, being free from wastewater generation, low capital investment, and low energy consumption. Three major agricultural crop residues (corn stover, wheat straw, and rice straw) were pretreated by dry acid pretreatment and then stored in their major planting regions under varying natural conditions of temperature, rain and snow fall, humidity, wind, and sunlight. The pretreated corn stover, wheat straw, and rice straw contained approximately 50% (w/w) of moisture, and their high water absorption capacity maintained the crop residues in solid and fine-particle forms without free wastewater generation and flammability. Meanwhile, the pretreated crop residues were of low pH value and contained various inhibitory compounds for microbial growth. The results show that the crop residue feedstocks were well preserved with negligible solid and fermentable sugar loss after year-round storage in different regions. The physical properties, chemical compositions, enzymatic hydrolysis yields, and ethanol fermentability were maintained essentially constant with a few positive exceptions such as the increased hydrolysis yield and reduced inhibitor content. A case study shows that the feedstock transportation cost of the long-term stored feedstocks under the scenario of dry acid pretreatment at collection depots was significantly reduced compared to that of the direct transportation of virgin crop residual feedstocks. This study provided an efficient and practical logistic system for large-scale biorefinery plants.

The methane (CH4)/oxygen (O2) gas supply ratios significantly affect the cell growth and metabolic pathways of aerobic obligate methanotrophs. However, few studies have explored the CH4/O2 ratios of the inlet gas, especially for the CH4 concentrations within the explosion range (5∼15% of CH4 in air). This study thoroughly investigated the molecular mechanisms associated with the impact of different CH4/O2 ratios on cell growth of a model type I methanotroph Methylomicrobium buryatense 5GB1 cultured at five different CH4/O2 supply molar ratios from 0.28 to 5.24, corresponding to CH4 content in gas mixture from 5% to 50%, using RNA-Seq transcriptomics approach. In the batch cultivation, the highest growth rate of 0.287 h–1 was achieved when the CH4/O2 supply molar ratio was 0.93 (15% CH4 in air), and it is crucial to keep the availability of carbon and oxygen levels balanced for optimal growth. At this ratio, genes related to methane metabolism, phosphate uptake system, and nitrogen fixation were significantly upregulated. The results indicated that the optimal CH4/O2 ratio prompted cell growth by increasing genes involved in metabolic pathways of carbon, nitrogen and phosphate utilization in M. buryatense 5GB1. Our findings provided an effective gas supply strategy for methanotrophs, which could enhance the production of key intermediates and enzymes to improve the performance of bioconversion processes using CH4 as the only carbon and energy source. This research also helps identify genes associated with the optimal CH4/O2 ratio for balancing energy metabolism and carbon flux, which could be candidate targets for future metabolic engineering practice.