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A novel nutritional induction strategy flexibly switching biosynthesis of food-like products from methane by a methanotrophic bacterium
Abstract
From the view of a circular economy, the bioconversion of methane into cell protein and carbohydrates could provide alternative food resources while cutting greenhouse gases, considering renewable gases from anaerobic...
... The conversion GHGs into valuable products may offer a promising solution for mitigating climate change and addressing current shortages in feedstock. Recently, both CO2 and CH4 have been demonstrated as a substrate for the biosynthesis of carbohydrates and proteins, which are significant sources of human diets or functional feed additives in the biomanfacturing (Cai et al., 2021;Gao et al., 2024). However, achieving efficient conversion of CO2 and CH4 in biocatalysis presents challenges due to the high energy barrier required for their activation. ...
Proteins are indispensable for maintaining a healthy diet and performing crucial functions in a multitude of physiological processes. The growth of the global population and the emergence of environmental concerns have significantly increased the demand for protein-rich foods such as meat and dairy products, exerting considerable pressure on global food supplies. Single-cell proteins (SCP) have emerged as a promising alternative source, characterized by their high protein content and essential amino acids, lipids, carbohydrates, nucleic acids, inorganic salts, vitamins, and trace elements. SCP offers several advantages over the traditional animal and plant proteins. These include shorter production cycles, the use of diverse raw material sources, high energy efficiency, and minimal environmental impact. This review is primarily concerned with the microbial species employed in SCP production, utilization of non-food renewable materials as a source of feedstock, and application of rational and non-rational metabolic engineering strategies to increase SCP biomass and protein content. Moreover, the current applications, production shortages, and safety concerns associated with SCP are discussed.
The rapid increase of the potent greenhouse gas methane in the atmosphere creates great urgency to develop and deploy technologies for methane mitigation. One approach to removing methane is to use bacteria for which methane is their carbon and energy source (methanotrophs). Such bacteria naturally convert methane to CO2 and biomass, a value-added product and a cobenefit of methane removal. Typically, methanotrophs grow best at around 5,000 to 10,000 ppm methane, but methane in the atmosphere is 1.9 ppm. Air above emission sites such as landfills, anaerobic digestor effluents, rice paddy effluents, and oil and gas wells contains elevated methane in the 500 ppm range. If such sites are targeted for methane removal, technology harnessing aerobic methanotroph metabolism has the potential to become economically and environmentally viable. The first step in developing such methane removal technology is to identify methanotrophs with enhanced ability to grow and consume methane at 500 ppm and lower. We report here that some existing methanotrophic strains grow well at 500 ppm methane, and one of them, Methylotuvimicrobium buryatense 5GB1C, consumes such low methane at enhanced rates compared to previously published values. Analyses of bioreactor-based performance and RNAseq-based transcriptomics suggest that this ability to utilize low methane is based at least in part on extremely low non-growth-associated maintenance energy and on high methane specific affinity. This bacterium is a candidate to develop technology for methane removal at emission sites. If appropriately scaled, such technology has the potential to slow global warming by 2050.
Predicted famines due to population increase created an interest in the development of protein alternatives during the 1950s. Currently, a renewed interest in protein alternatives has developed as a potential strategy to decrease the environmental impact of protein production and meet the global demand for protein as the population increases. Fusarium venenatum A3/5/3, the organism used for mycoprotein production has been commercially available since the 1980s, however new fungal protein companies are currently interested in scaling up production. To aid guide efforts in this domain, we created an economic model with over 340 inputs that examines the continuous production of mycoprotein utilizing airlift bioreactors. Utilizing a sensitivity analysis, we identified critical processing inputs and then developed a user-friendly Excel model that allows for the exploration of customized production scenarios for interested stakeholders. Our findings indicate that mycoprotein can be cost competitive with beef on a price per protein basis. The findings also indicate that mycoprotein may not be an economically competitive alternative for other types of commodity meats (chicken) or for inexpensive meat-derived products (pet food) that utilize offal or meat byproducts not traditionally consumed in the modern western diet.
Methanotrophs oxidize most of the methane (CH 4) produced in natural and anthropogenic ecosystems. Often living close to soil surfaces, these microorganisms must frequently adjust to temperature change. While many environmental studies have addressed temperature effects on CH 4 oxidation and methanotrophic communities, there is little knowledge about the physiological adjustments that underlie these effects. We have studied thermal acclimation in Methylobacter, a widespread, abundant, and environmentally important methanotrophic genus. Comparisons of growth and CH 4 oxidation kinetics at different temperatures in three members of the genus demonstrate that temperature has a strong influence on how much CH 4 is consumed to support growth at different CH 4 concentrations. However, the temperature effect varies considerably between species, suggesting that how a methanotrophic community is composed influences the temperature effect on CH 4 uptake. To understand thermal acclimation mechanisms widely we carried out a transcriptomics experiment with Methylobacter tundripaludum SV96 T. We observed, at different temperatures, how varying abundances of transcripts for glycogen and protein biosynthesis relate to cellular glycogen and ribosome concentrations. Our data also demonstrated transcriptional adjustment of CH 4 oxidation, oxidative phosphorylation, membrane fatty acid saturation, cell wall composition, and exopolysaccharides between temperatures. In addition, we observed differences in M. tundripaludum SV96 T cell sizes at different temperatures. We conclude that thermal acclimation in Methylobacter results from transcriptional adjustment of central metabolism, protein biosynthesis, cell walls and storage. Acclimation leads to large shifts in CH 4 consumption and growth efficiency, but with major differences between species. Thus, our study demonstrates that physiological adjustments to temperature change can substantially influence environmental CH 4 uptake rates and that consideration of methanotroph physiology might be vital for accurate predictions of warming effects on CH 4 emissions. The ISME Journal; https://doi.
Integration of methanogens with semiconductors is an effective approach to sustainable solar-driven methanogenesis. However, the H2 production rate by semiconductors largely exceeds that of methanogen metabolism, resulting in abundant H2 as side product. Here, we report that binary metallic active sites (namely, NiCu alloys) are incorporated into the interface between CdS semiconductors and Methanosarcina barkeri. The self-assembled Methanosarcina barkeri-NiCu@CdS exhibits nearly 100% CH4 selectivity with a quantum yield of 12.41 ± 0.16% under light illumination, which not only exceeds the reported biotic-abiotic hybrid systems but also is superior to most photocatalytic systems. Further investigation reveal that the Ni-Cu-Cu hollow sites in NiCu alloys can directly supply hydrogen atoms and electrons through photocatalysis to the Methanosarcina barkeri for methanogenesis via both extracellular and intracellular hydrogen cycles, effectively turning down the H2 production. This work provides important insights into the biotic-abiotic hybrid interface, and offers an avenue for engineering the methanogenesis process.
Disasters resulting from climate change and extreme weather events adversely impact crop and livestock production. While the direct impacts of these events on productivity are generally well known, the indirect supply-chain repercussions (spillovers) are still unclear. Here, applying an integrated modelling framework that considers economic and physical factors, we estimate spillovers in terms of social impacts (for example, loss of job and income) and health impacts (for example, nutrient availability and diet quality) resulting from disruptions in food supply chains, which cascade across regions and sectors. Our results demonstrate that post-disaster impacts are wide-ranging and diverse owing to the interconnected nature of supply chains. We find that fruit, vegetable and livestock sectors are the most affected, with effects flowing on to other non-food production sectors such as transport services. The ability to cope with disasters is determined by socio-demographic characteristics, with communities in rural areas being most affected. The complex nature of food supply chains makes it a crucial exercise to estimate the impacts of disruptions caused by climate disasters. By applying an integrated modelling framework to Australia and considering heatwaves, cyclones and other climate events, this study presents novel ways of quantifying regional and sectoral spillover effects—including job and income losses, food and nutrient availability, and diet quality.
Fucoxanthin is a new dietary ingredient applied in healthy foods with specific benefits of body weight loss and liver fat reduction. The marine diatom Phaeodactylum tricornutum is a highly suitable species for fucoxanthin production. In the present study, aiming to promote fucoxanthin biosynthesis in mixotrophic P. tricornutum, NaNO3, tryptone, and urea were evaluated as nitrogen sources with 0.10 mol L⁻¹ of glycerol as the organic carbon source for mixotrophic growth in shake flasks. Compared to NaNO3, the mixture of tryptone and urea (referred to as T+U, 1:1, mol N:mol N) as organic nitrogen sources could induce a higher biomass and fucoxanthin production. Through nitrogen utilization analysis, leucine, arginine, lysine, and phenylalanine in the T+U medium were identified as the amino acids that primarily support cell growth. Among those amino acids, arginine causes the highest rate of nitrogen utilization and cell growth promotion. After 12 days of cultivation, the highest biomass concentration (3.18 g L⁻¹), fucoxanthin content (12.17 mg g⁻¹), and productivity (2.68 mg L⁻¹ day⁻¹) were achieved using 25 mmol N L⁻¹ of arginine and 5 mmol N L⁻¹ of urea as nitrogen sources, indicating that arginine and urea performed synergistically on enhancing biomass and pigment production. This study provides new insights into the promotion of fucoxanthin biosynthesis by nitrogen utilization analysis and verifies the synergetic effect of arginine and urea on facilitating the development of a promising strategy for efficient enhancement of fucoxanthin production through mixotrophic cultivation of P. tricornutum.
Global catastrophes such as a supervolcanic eruption, asteroid impact, or nuclear winter could cause global agricultural collapse due to reduced sunlight reaching the Earth’s surface. The human civilization’s food production system is unprepared to respond to such events, but methane single cell protein (SCP) could be a key part of the solution. Current preparedness centers around food stockpiling, an excessively expensive solution given that an abrupt sunlight reduction scenario (ASRS) could hamper conventional agriculture for 5–10 years. Instead, it is more cost-effective to consider resilient food production techniques requiring little to no sunlight. This study analyses the potential of SCP produced from methane (natural gas and biogas) as a resilient food source for global catastrophic food shocks from ASRS. The following are quantified: global production potential of methane SCP, capital costs, material and energy requirements, ramp-up rates, and retail prices. In addition, potential bottlenecks for fast deployment are considered. While providing a more valuable, protein-rich product than its alternatives, the production capacity could be slower to ramp up. Based on 24/7 construction of facilities, 7%–11% of the global protein requirements could be fulfilled at the end of the first year. Despite significant remaining uncertainties, methane SCP shows significant potential to prevent global protein starvation during an ASRS at an affordable price—US$3–5/kg dry.
Methanotrophs are naturally occurring microorganisms capable of oxidizing methane, having an impact on global net methane emissions. Additionally, they have also gained interest for their biotechnological applications in single-cell protein production, biofuels, and bioplastics.
Methanotrophs capable of converting C1-based substrates play an important role in the global carbon cycle. As one of the essential macronutrient components in the medium, the uptake of nitrogen sources severely regulates the cell’s metabolism. Although the feasibility of utilizing nitrogen gas (N2) by methanotrophs has been predicted, the mechanism remains unclear. Herein, the regulation of nitrogen fixation by an essential nitrogen-fixing regulator (NifA) was explored based on transcriptomic analyses of Methylomicrobium buryatense 5GB1. A deletion mutant of the nitrogen global regulator NifA was constructed, and the growth of M. buryatense 5GB1ΔnifA exhibited significant growth inhibition compared with wild-type strain after the depletion of nitrate source in the medium. Our transcriptome analyses elucidated that 22.0% of the genome was affected in expression by NifA in M. buryatense 5GB1. Besides genes associated with nitrogen assimilation such as nitrogenase structural genes, genes related to cofactor biosynthesis, electron transport, and post-transcriptional modification were significantly upregulated in the presence of NifA to enhance N2 fixation; other genes related to carbon metabolism, energy metabolism, membrane transport, and cell motility were strongly modulated by NifA to facilitate cell metabolisms. This study not only lays a comprehensive understanding of the physiological characteristics and nitrogen metabolism of methanotrophs, but also provides a potentially efficient strategy to achieve carbon and nitrogen co-utilization.
Key points
• N2 fixation ability of M. buryatense 5GB1 was demonstrated for the first time in experiments by regulating the supply of N2.
• NifA positively regulates nif-related genes to facilitate the uptake of N2 in M. buryatense 5GB1.
• NifA regulates a broad range of cellular functions beyond nif genes in M. buryatense 5GB1.
More food needs to be produced for the growing human population, but the possibilities of expanding the area of arable land are limited. Cellular Agriculture is an emerging field of biotechnology, aimed at finding alternatives to agricultural production of various commodities. As a part of Cellular Agriculture, the use of microbes and microalgae as food and feed with high protein content, so-called single cell protein (SCP), is gaining renewed scientific and commercial interest. In this review, we give an introduction to SCP production by heterotrophic microbial species, phototrophs, methanotrophs and autotrophic hydrogen oxidizers, as well as highlight some challenges and the latest developments in the growing SCP industry.
Single-cell proteins are attracting growing attention as viable alternatives for fishmeal (FM) in aquatic feed. Methanotroph (Methylococcus capsulatus, Bath) bacteria meal FeedKind® (FK) is a type of single cell protein with high protein content (75.14%) and desirable amino acids profile, produced by Methylococcus capsulatus (Bath) living on methane consumption. The present study evaluated the potential of replacing FM with FK in the diet of black sea bream (Acanthopagrus schlegelii). Five iso-energetic and iso-nitrogenous diets were designed with FK replacing 0, 4.13, 8.27, 16.53, and 24.80% FM protein in the basal diet (40% FM content), respectively. All the diets were fed to three replicates of fish (initial weight 6.56 ± 0.02 g) for 70 days. After the feeding trial, replacing dietary 8.27% FM protein with FK significantly improved the weight gain and specific growth rate of fish (P < 0.05), while other groups showed no significant difference in the growth performance (P > 0.05). The fish fed diets with 8.27 and 16.53% replacement levels exhibited significantly increased feeding rates. The 8.27% FK diet significantly increased the whole-body and muscle crude protein contents, apparent digestibility of crude lipid, foregut, and midgut amylase activities. The microvillus density in the midgut of fish fed the 24.80% FK diet significantly increased. The diet with 8.27% FK increased the serum triglyceride content of the fish, while the 24.80% FK diet reduced the serum triglyceride, total cholesterol, and low-density lipoprotein cholesterol contents of the fish. In conclusion, the results indicated that replacing dietary FM protein with up to 24.80% FK had no adverse effects on the growth of black sea bream, whilst replacing 8.27% FM protein with FK enhanced its growth performance and feed utilization.
Methane emitted and flared from industrial sources across the United States is a major contributor to global climate change. Methanotrophic bacteria can transform this methane into useful protein-rich biomass, already approved for inclusion into animal feed. In the rapidly growing aquaculture industry, methanotrophic additives have a favourable amino acid profile and can offset ocean-caught fishmeal, reducing demands on over-harvested fisheries. Here we analyse the economic potential of producing methanotrophic microbial protein from stranded methane produced at wastewater treatment plants, landfills, and oil and gas facilities. Our results show that current technology can enable production, in the United States alone, equivalent to 14% of the global fishmeal market at prices at or below the current cost of fishmeal (roughly US$1,600 per metric ton). A sensitivity analysis highlights technically and economically feasible cost reductions (such as reduced cooling or labour requirements), which could allow stranded methane from the United States alone to satisfy global fishmeal demand.
From carbon dioxide to starch: no plants required
Many plants turn glucose from photosynthesis into polymers that form insoluble starch granules ideal for long-term energy storage in roots and seeds. Cai et al . developed a hybrid system in which carbon dioxide is reduced to methanol by an inorganic catalyst and then converted by enzymes first to three and six carbon sugar units and then to polymeric starch. This artificial starch anabolic pathway relies on engineered recombinant enzymes from many different source organisms and can be tuned to produce amylose or amylopectin at excellent rates and efficiencies relative to other synthetic carbon fixation systems—and, depending on the metric used, even to field crops. —MAF
In this study, a novel exopolysaccharide (EPS) was extracted from Leuconostoc mesenteroides Shen Nong’s (SN)-8 which can be obtained from Dajiang. After the purification step, EPS-8-2 was obtained with molecular weights of 1.46 × 105 Da. The structural characterization of EPS indicated that the EPS belonged to the class polysaccharide, mainly composed of glucan and also contained certain mannose residues that were found to be connected by α-1,6 glycosidic bonds. Moreover, the results demonstrated that EPS displayed a significant capacity to scavenge free radical to some extent, and this anti-oxidant potential was found to be concentration dependent. The results further revealed that EPS displayed a significant inhibitory potential on the growth of HepG2 cells by promoting apoptosis and induced cell cycle arrest in G1 and G2 phases. Overall, these results suggested that EPS can be explored as a possible anti-cancer agent.
With the global supply of forage fish at a plateau, fed aquaculture must continue to reduce dependence on fishmeal and oil in feeds to ensure sustainable sector growth. The use of novel aquaculture feed ingredients is growing, but their contributions to scalable and sustainable aquafeed solutions are unclear. Here, we show that global adoption of novel aquafeeds could substantially reduce aquaculture’s forage fish demand by 2030, maintaining feed efficiencies and omega-3 fatty acid profiles. We combine production data, scenario modelling and a decade of experimental data on forage fish replacement using microalgae, macroalgae, bacteria, yeast and insects to illustrate how reducing future fish oil demand, particularly in high-value species such as salmonids, will be key for the sustainability of fed aquaculture. However, considerable uncertainties remain surrounding novel feed efficacy across different life-cycle stages and taxa, and various social, environmental, economic and regulatory challenges will dictate their widespread use. Yet, we demonstrate how even limited adoption of novel feeds could aid sustainable aquaculture growth, which will become increasingly important for food security. Novel aquaculture feeds are rapidly developing, but their contributions to sustainable industry growth are unknown. Cottrell et al. model feed efficiency and fatty acid profiles, showing that replacing forage fish with novel feed ingredients could strengthen aquaculture’s role in global food security.
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.
Significance
Cellulosic biofuels have not yet reached cost parity with conventional petroleum fuels. One strategy to address this challenge is to generate valuable coproducts alongside biofuels. Engineering bioenergy crops to generate value-added bioproducts in planta can reduce input requirements relative to microbial chassis and skip costly deconstruction and conversion steps. Although rapid progress has been made in plant metabolic engineering, there has been no systematic analysis devoted to quantifying the impact of such engineered bioenergy crops on biorefinery economics. Here, we provide new insights into how bioproduct accumulation in planta affects biofuel selling prices. We present the range of bioproduct selling prices and accumulation rates needed to compensate for additional extraction steps and reach a target $2.50/gal minimum biofuel selling price.
The global demand for high-quality, protein-rich foods will continue to increase as the global population grows, along with income levels. Aquaculture is poised to help fulfill some of this demand, and is thus the fastest growing animal protein industry. A key challenge for it, though, is sourcing a sustainable, renewable protein ingredient. Single cell protein (SCP) products, protein meals based on microbial or algal biomass, have the potential to fulfill this need. Here, we review potential sources of SCP strains and their respective production processes, highlight recent advances on identification of new SCP strains and feedstocks, and, finally, review new feeding trial data on important aquaculture species, specifically Atlantic salmon, rainbow trout, and whiteleg shrimp.
To satisfy the growing demand for limonene, novel pathways for microbial production of limonene have been sought. A techno-economic analysis is carried out for one such process producing limonene from sugar at an industrial plant scale to assess potential economic viability. A conceptual design of the process is developed, in which a gas stripping-solvent scrubbing method is chosen for recovering limonene from bioreactors based on consideration of payback time and process operability. Minimum limonene selling prices are estimated over a range of fermentation productivity based on the calculation of net present value using discounted cash flow method. Under 45% of the maximum theoretical yield, the selling price reaches $19.9/kg, which could be competitive with established production processes when fermentation productivity is above 0.7 kg/(m3·h). Reduction of cost could be realised through improvement of microbial strains, utilisation of cheaper feedstocks, reduction in capital investment and strategic business planning.
Glycogen, a randomly-branched glucose polymer, provides energy storage in organisms. It forms small β particles which in animals bind to form composite α particles, which give better glucose release. Simulations imply β particle size is controlled only by activities and sizes of glycogen biosynthetic enzymes and sizes of polymer chains. Thus storing more glucose requires forming more β particles, which are expected to sometimes form α particles. No α particles have been reported in bacteria, but the extraction techniques might have caused degradation. Using milder glycogen extraction techniques on E. coli, TEM and size-exclusion chromatography showed α particles, consistent with this hypothesis for α-particle formation. Molecular density and size distributions show similarities with animal glycogen, despite very different metabolic processes. These general polymer constraints are such that any organism which needs to store and then release glucose will have similar α and β particle structures: a type of convergent evolution.
Feed protein supplements are one of the most expensive and limiting feed ingredients. This review offers a comprehensive analysis of how the expected expansion of animal production, driven by the rising world population and living standards for more animal-sourced foods, is creating a global shortage of feed protein supply. Because ruminants, chickens, and pigs contribute to 96% of the global supply of animal protein and aquaculture is growing fast, means of meeting the feed protein requirements of these species are elaborated. Geographic variation and interdependence among China, Europe, and North America in the demand and supply of feed protein are compared. The potential and current state of exploration into alternative feed proteins, including microalgae, insects, single-cell proteins, and coproducts, are highlighted. Strategic innovations are proposed to upgrade feed protein processing and assessment, improve protein digestion by exogenous enzymes, and genetically select feed-efficient livestock breeds. An overall successful and sustainable solution in meeting global feed protein demands will lead to a substantial net gain of human-edible animal protein with a minimal environmental footprint. Expected final online publication date for the Annual Review of Animal Biosciences Volume 7 is February 15, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Anaerobic digestion (AD) of waste substrates, and renewable biomass and crop residues offers a means to generate energy-rich biogas. However, at present, AD-derived biogas is primarily flared or used for combined heat and power (CHP), in part due to inefficient gas-to-liquid conversion technologies. Methanotrophic bacteria are capable of utilizing methane as a sole carbon and energy source, offering promising potential for biological gas-to-liquid conversion of AD-derived biogas. Here, we report cultivation of three phylogenetically diverse methanotrophic bacteria on biogas streams derived from AD of a series of energy crop residues. Strains maintained comparable central metabolic activity and displayed minimal growth inhibition when cultivated under batch configuration on AD biogas streams relative to pure methane, although metabolite analysis suggested biogas streams increase cellular oxidative stress. In contrast to batch cultivation, growth arrest was observed under continuous cultivation configuration, concurrent with increased biosynthesis and excretion of lactate. We examined the potential for enhanced lactate production via the employ of a pyruvate dehydrogenase mutant strain, ultimately achieving 0.027 g lactate/g DCW/h, the highest reported lactate specific productivity from biogas to date.
Background
Recent advances in metabolic engineering enable the production of chemicals from sugars through microbial bio-conversion. Terpenes have attracted substantial attention due to their relatively high prices and wide applications in different industries. To this end, we synthesize and assess processes for microbial production of terpenes.
Results
To explain a counterintuitive experimental phenomenon where terpenes such as limonene (normal boiling point 176 °C) are often found to be 100% present in the vapor phase after bio-conversion (operating at only ~ 30 °C), we first analyze the vapor–liquid equilibrium for systems containing terpenes. Then, we propose alternative production configurations, which are further studied, using limonene as an example, in several case studies. Next, we perform economic assessment of the alternative processes and identify the major cost components. Finally, we extend the assessment to account for different process parameters, terpene products, ways to address terpene toxicity (microbial engineering vs. solvent use), and cellulosic biomass as a feedstock. We identify the key cost drivers to be (1) feed glucose concentration (wt%), (2) product yield (% of maximum theoretical yield) and (3) VVM (Volume of air per Volume of broth liquid per Minute, i.e., aeration rate in min⁻¹). The production of limonene, based on current experimental data, is found to be economically infeasible (production cost ~ 465 /kg), but higher glucose concentration and yield can lower the cost. Among 12 terpenes studied, limonene appears to be the most reasonable short-term target because of its large market size (~ 160 million $/year in the US) and the relatively easier to achieve break-even yield (~ 30%, assuming a 14 wt% feed glucose concentration and 0.1 min⁻¹ VVM).
Conclusions
The methods proposed in this work are applicable to a range of terpenes as well as other extracellular insoluble chemicals with density lower than that of water, such as fatty acids. The results provide guidance for future research in metabolic engineering toward terpenes production in terms of setting targets for key design parameters.
Electronic supplementary material
The online version of this article (10.1186/s13068-018-1285-7) contains supplementary material, which is available to authorized users.
As a potential feedstock for biofuel production, a high-cell-density continuous culture for the lipid production by Cryptococcus albidus was investigated in this study. The influences of dilution rates in the single-stage continuous cultures were explored first. To reach a high-cell-density culture, a single-stage continuous culture coupled with a membrane cell recycling system was carried out at a constant dilution rate of 0.36/h with varied bleeding ratios. The maximum lipid productivity of 0.69 g/L/h was achieved with the highest bleeding ratio of 0.4. To reach a better lipid yield and content, a two-stage continuous cultivation was performed by adjusting the C/N ratio in two different stages. Finally, a lipid yield of 0.32 g/g and lipid content of 56.4% were obtained. This two-stage continuous cultivation, which provided a higher lipid production performance, shows a great potential for an industrial-scale biotechnological production of microbial lipids and biofuel production.
Background
Due to the success of shale gas development in the US, the production cost of natural gas has been reduced significantly, which in turn has made methane (CH4), the major component of natural gas, a potential alternative substrate for bioconversion processes compared with other high-price raw material sources or edible feedstocks. Therefore, exploring effective ways to use CH4 for the production of biofuels is attractive. Biological fixation of CH4 by methanotrophic bacteria capable of using CH4 as their sole carbon and energy source has obtained great attention for biofuel production from this resource. ResultsIn this study, a fast-growing and lipid-rich methanotroph, Methylomicrobium buryatense 5GB1 and its glycogen-knock-out mutant (AP18) were investigated for the production of lipids derived from intracellular membranes, which are key precursors for the production of green diesel. The effects of culture conditions on cell growth and lipid production were investigated in high cell density cultivation with continuous feeding of CH4 and O2. The highest dry cell weight observed was 21.4 g/L and the maximum lipid productivity observed was 45.4 mg/L/h obtained in batch cultures, which corresponds to a 2-fold enhancement in cell density and 3-fold improvement in lipid production, compared with previous reported data from cultures of 5GB1. A 90% enhancement of lipid content was achieved by limiting the biosynthesis of glycogen in strain AP18. Increased CH4/O2 uptake and CO2 evaluation rates were observed in AP18 cultures suggesting that more carbon substrate and energy are needed for AP18 growth while producing lipids. The lipid produced by M. buryatense was estimated to have a cetane number of 75, which is 50% higher than biofuel standards requested by US and EU. Conclusions
Cell growth and lipid production were significantly influenced by culture conditions for both 5GB1 and AP18. Enhanced lipid production in terms of titer, productivity, and content was achieved under high cell density culture conditions by blocking glycogen accumulation as a carbon sink in the strain AP18. Differences observed in CH4/O2 gas uptake and CO2 evolution rates as well as cell growth and glycogen accumulation between 5GB1 and AP18 suggest changes in the metabolic network between these strains. This bioconversion process provides a promising opportunity to transform CH4 into biofuel molecules and encourages further investigation to elucidate the remarkable CH4 biofixation mechanism used by these bacteria.
Recently, syngas has gained significant interest as renewable and sustainable feedstock, in particular for the biotechnological production of poly([R]-3-hydroxybutyrate) (PHB). PHB is a biodegradable, biocompatible polyester produced by some bacteria growing on the principal component of syngas, CO. However, working with syngas is challenging because of the CO toxicity and the explosion danger of H2 , another main component of syngas. In addition, the bioprocess control needs specific monitoring tools and analytical methods that differ from standard fermentations. Here, we present a syngas fermentation platform with a focus on safety installations and process analytical technology (PAT) that serves as a basis to assess the physiology of the PHB-producing bacterium Rhodospirillum rubrum. The platform includes (i) off-gas analysis with an online quadrupole mass spectrometer to measure CO consumption and production rates of H2 and CO2 , (ii) an at-line flow cytometer to determine the total cell count and the intracellular PHB content and (iii) different online sensors, notably a redox sensor that is important to confirm that the culture conditions are suitable for the CO metabolization of R. rubrum. Furthermore, we present as first applications of the platform a fed-batch and a chemostat process with R. rubrum for PHB production from syngas.
In light of the availability of low-cost methane (CH4) derived from natural gas and biogas along with increasing concerns of the greenhouse gas emissions, the production of alternative liquid biofuels directly from CH4 is a promising approach to capturing wasted energy. A novel biorefinery concept integrating biological conversion of CH4 to microbial lipids together with lipid extraction and generation of hydrocarbon fuels is demonstrated in this study for the first time. An aerobic methanotrophic bacterium, Methylomicrobium buryatense capable of using CH4 as the sole carbon source was selected on the basis of genetic tractability, cultivation robustness, and ability to accumulate phospholipids in membranes. A maximum fatty acid content of 10% of dry cell weight was obtained in batch cultures grown in a continuous gas sparging fermentation system. Although phospholipids are not typically considered as a good feedstock for upgrading to hydrocarbon fuels, we set out to demonstrate that using a combination of novel lipid extraction methodology with advanced catalyst design, we could prove the feasibility of this approach. Up to 95% of the total fatty acids from membrane-bound phospholipids were recovered by a two-stage pretreatment method followed by hexane extraction of the aqueous hydrolysate. The upgrading of extracted lipids was then demonstrated in a hydrodeoxygeation process using palladium on silica as a catalyst. Lipid conversion in excess of 99% was achieved, with a full selectivity to hydrocarbons. The final hydrocarbon mixture is dominated by 88% pentadecane (C15H32) based on decarbonylation/decarboxylation and hydrogenation of C16 fatty acids, indicating that a biological gas-to-liquid fuel (Bio-GTL) process is technically feasible.
This study aimed to assess the single-cell oil production integrated to a sugarcane-mill and molasses as main raw material. Rhodotorula glutinis lipid production with low-cost raw sugarcane molasses was accessed. The envisage process relies in an optimized sugarcane molasses culture media in a fed-batch strategy to achieve a high-cell density high-lipid cell content. A Process Flow Design (PFD) integrated to a typical Brazilian sugar mill was proposed according to fermentation experiments. Performed economic analysis provided data for discussion on strategic equipment that can facilitate the viability to the process. Typical results of established protocol were total biomass concentration 62.25 g l−1, lipid productivity 0.42 g l−1 h−1, sugar conversion yield to lipids 0.21 g of lipid g−1 of total reducing sugar. Microbial lipid production and defatted biomass plant production was proposed at a nominal capacity of respectively 16,720 ton and 21,600 ton per year, running 8300 h per year. The projected selling prices of these products were US 500.00/ton of defatted yeast. It was demonstrated that the industrial plant was potentially attractive when capital investment cost was decreased with the use of low cost epoxy-lined carbon steel stirred bioreactor. In this alternative, the internal rate of return (IRR) will be 24.61% leading the NPV (at 7% interest rate) of US$ 67,797,000/year (before taxes).
Methane, a carbon source for methanotrophic bacteria, is the principal component of natural gas and is produced during anaerobic digestion of organic matter (biogas). Methanotrophs are a viable source of single cell protein (feed supplement) and can produce various products, since they accumulate osmolytes (e.g. ectoine, sucrose), phospholipids (potential biofuels) and biopolymers (polyhydroxybutyrate, glycogen), among others. Other cell components, such as surface layers, metal chelating proteins (methanobactin), enzymes (methane monooxygenase) or heterologous proteins hold promise as future products. Here, scenarios are presented where ectoine, polyhydroxybutyrate or protein G are synthesised as the primary product, in conjunction with a variety of ancillary products that could enhance process viability. Single or dual-stage processes and volumetric requirements for bioreactors are discussed, in terms of an annual biomass output of 1000 tonnesyear(-1). Product yields are discussed in relation to methane and oxygen consumption and organic waste generation.
Background:
Methane-utilizing bacteria (methanotrophs) are capable of growth on methane and are attractive systems for bio-catalysis. However, the application of natural methanotrophic strains to large-scale production of value-added chemicals/biofuels requires a number of physiological and genetic alterations. An accurate metabolic model coupled with flux balance analysis can provide a solid interpretative framework for experimental data analyses and integration.
Results:
A stoichiometric flux balance model of Methylomicrobium buryatense strain 5G(B1) was constructed and used for evaluating metabolic engineering strategies for biofuels and chemical production with a methanotrophic bacterium as the catalytic platform. The initial metabolic reconstruction was based on whole-genome predictions. Each metabolic step was manually verified, gapfilled, and modified in accordance with genome-wide expression data. The final model incorporates a total of 841 reactions (in 167 metabolic pathways). Of these, up to 400 reactions were recruited to produce 118 intracellular metabolites. The flux balance simulations suggest that only the transfer of electrons from methanol oxidation to methane oxidation steps can support measured growth and methane/oxygen consumption parameters, while the scenario employing NADH as a possible source of electrons for particulate methane monooxygenase cannot. Direct coupling between methane oxidation and methanol oxidation accounts for most of the membrane-associated methane monooxygenase activity. However the best fit to experimental results is achieved only after assuming that the efficiency of direct coupling depends on growth conditions and additional NADH input (about 0.1-0.2 mol of incremental NADH per one mol of methane oxidized). The additional input is proposed to cover loss of electrons through inefficiency and to sustain methane oxidation at perturbations or support uphill electron transfer. Finally, the model was used for testing the carbon conversion efficiency of different pathways for C1-utilization, including different variants of the ribulose monophosphate pathway and the serine cycle.
Conclusion:
We demonstrate that the metabolic model can provide an effective tool for predicting metabolic parameters for different nutrients and genetic perturbations, and as such, should be valuable for metabolic engineering of the central metabolism of M. buryatense strains.
Background:
Methane is a feedstock of interest for the future, both from natural gas and from renewable biogas sources. Methanotrophic bacteria have the potential to enable commercial methane bioconversion to value-added products such as fuels and chemicals. A strain of interest for such applications is Methylomicrobium buryatense 5GB1, due to its robust growth characteristics. However, to take advantage of the potential of this methanotroph, it is important to generate comprehensive bioreactor-based datasets for different growth conditions to compare bioprocess parameters.
Results:
Datasets of growth parameters, gas utilization rates, and products (total biomass, extracted fatty acids, glycogen, excreted acids) were obtained for cultures of M. buryatense 5GB1 grown in continuous culture under methane limitation and O2 limitation conditions. Additionally, experiments were performed involving unrestricted batch growth conditions with both methane and methanol as substrate. All four growth conditions show significant differences. The most notable changes are the high glycogen content and high formate excretion for cells grown on methanol (batch), and high O2:CH4 utilization ratio for cells grown under methane limitation.
Conclusions:
The results presented here represent the most comprehensive published bioreactor datasets for a gamma-proteobacterial methanotroph. This information shows that metabolism by M. buryatense 5GB1 differs significantly for each of the four conditions tested. O2 limitation resulted in the lowest relative O2 demand and fed-batch growth on methane the highest. Future studies are needed to understand the metabolic basis of these differences. However, these results suggest that both batch and continuous culture conditions have specific advantages, depending on the product of interest.
The accumulation of a large amount of organic solid waste and the lack of sufficient protein supply worldwide are two major challenges caused by rapid population growth. Anaerobic digestion is the main force of organic waste treatment, and the high-value utilization of its products (biogas and digestate) has been widely concerned. These products can be used as nutrients and energy sources for microorganisms such as microalgae, yeast, methane-oxidizing bacteria(MOB), and hydrogen-oxidizing bacteria(HOB) to produce single cell protein(SCP), which contributes to the achievement of sustainable development goals. This new model of energy conversion can construct a bioeconomic cycle from waste to nutritional products, which treats waste without additional carbon emissions and can harvest high-value biomass. Techno-economic analysis shows that the SCP from biogas and digestate has higher profit than biogas electricity generation, and its production cost is lower than the SCP using special raw materials as the substrate. In this review, the case of SCP-rich microorganisms using anaerobic digestion products for growth was investigated. Some of the challenges faced by the process and the latest developments were analyzed, and their potential economic and environmental value was verified.
The engineered Methylococcus capsulatus Bath presents a promising approach for converting methane, a potent greenhouse gas, into valuable chemicals. High cell-density culture (HCDC) is necessary for high-titer growth-associated bioproducts, but it often requires time-consuming and labor-intensive optimization processes. In this study, we aimed to achieve efficient HCDC of M. capsulatus Bath by measuring the residual nutrient levels during bioreactor operations and analyzing the specific uptake of each medium component. By controlling the concentrations of nutrients, particularly calcium and phosphorus via intermittent feeding, we achieved a high cell density of 28.2 g DCW/L and a significantly elevated production of mevalonate at a concentration of 1.8 g/L from methane. Our findings demonstrate that the methanotroph HCDC approach presented herein offers a promising strategy for promoting sustainable development, with an exceptional g-scale production titer for value-added synthetic biochemicals.
Streptococcus thermophilus is a traditional starter for yogurt making. Here, an eps gene cluster encoded for exopolysaccharides (EPS) was identified in the genome of S. thermophilus CGMCC 7.179. After purifying by DEAE-Sepharose Fast-Flow column chromatography, three main components ST-ESP1, ST-EPS2 and ST-EPS3, consisted of mannose, glucuronic acid, galacturonic acid, glucose and N-acetylglucosamine, were obtained. ST-EPS2 showed stronger antioxidant activity than the others in vitro. Moreover, ST-EPS2 could significantly upregulate the transcription levels of NF-E2-related factor-2 (Nrf2) and promote the expression of antioxidant-related proteins involved in Nrf2, suggesting ST-EPS2 protected the intestinal epithelial NCM 460 cells by attenuating the intracellular reactive oxygen species (ROS) level rising against oxidative stress. The potential binding mode of the mannose, galacturonic acid and N-acetylglucosamine with Keap1 simulated through molecular docking confirmed the action of ST-EPS2 with the protein Nrf2. Those results demonstrated that the ST-EPS2 provided opportunities for a natural antioxidant agent exploration in the food industry.
2-Propanol is a widely used industrial solvents. Herein, we employed a unique feature of type I methanotrophic bacterium Methylotuvimicrobium alcaliphilum 20Z possessing only particulate methane monooxygenase (pMMO) for one-step direct production of pure 2-propanol from propane. By maintaining cell growth on glycerol, and after deletion of both Ca²⁺-dependent and La³⁺-dependent methanol dehydrogenases, propane was converted to 2-propanol by pMMO. Although most of the 2-propanol produced was further oxidized to acetone, deletion of active alcohol dehydrogenase, concomitant with synchronous overexpression of secondary alcohol dehydrogenase, significantly inhibited such undesirable oxidation. As a result, a remarkable enhancement (263 mg/L) of 2-propanol was achieved for 120 h by increasing cell growth with a supply of 50% (v/v) propane in headspace. This is the first demonstration to develop an engineered methanotrophic strain for the one-step direct production of pure 2-propanol from propane using one-phase cultivation without the supply of chemical inhibitors or additional reducing-power sources.
Current food production practices contribute significantly to climate change. To transition into a sustainable future, a combination of new food habits and a radical food production innovation must occur. Single-cell protein from microbial fermentation can profoundly impact sustainability. This review paper explores opportunities offered by gas fermentation to completely replace our reliance on fossil fuels for the production of food. Together with synthetic biology, designed microbial proteins from gas fermentation have the potential to reduce our dependence on fossil fuels and make food production more sustainable.
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.
Improper use of conventional plastics poses challenges for sustainable energy and environmental protection. Algal derivatives have been considered as a potential renewable biomass source for bioplastic production. Algae derivatives include a multitude of valuable substances, especially starch from microalgae, short-chain length polyhydroxyalkanoates (PHAs) from cyanobacteria, polysaccharides from marine and freshwater macroalgae. The algae derivatives have the potential to be used as key ingredients for bioplastic production, such as starch and PHAs or only as an additive such as sulfated polysaccharides. The presence of distinctive functional groups in algae, such as carboxyl, hydroxyl, and sulfate, can be manipulated or tailored to provide desirable bioplastic quality, especially for food, pharmaceutical, and medical packaging. Standardizing strains, growing conditions, harvesting and extracting algae in an environmentally friendly manner would be a promising strategy for pollution control and bioplastic production.
Microbial oils have become a research hotspot in alleviating energy challenges and environmental problems because of their potential to be green alternatives of traditional fossil fuels. Yarrowia lipolytica is a promising oleaginous yeast that can utilize various, especially low-cost carbon sources to synthesize considerable lipids more than 30% of dry cell weight, which is attracting researchers’ attention. Based on well understanding of its lipid synthesis and metabolism mechanism, various optimization approaches have been studied dispersedly to improve the lipid synthesis and realize the industrial-scale application of Y. lipolytica. Some approaches are focused on improving and optimizing culture conditions, such as temperature, pH value, and rotating speed, etc. Other approaches are dedicated to the optimization of nutrient elements, such as carbon source, nitrogen source type and/or concentration, and C/N ratio, etc. Some adjust the cultivation mode to facilitate nutrient assimilation and transformation, and others use genetic engineering to modify this yeast. This review focuses on the comprehensive and detailed analyses of feasible enhancement approaches for lipid synthesis. Some prospects will also be introduced as references for further study.
Climate change and food shortage are two of the defining challenges in the coming decades. Considering that conventional approaches for protein production may associate with negative environmental impacts and greenhouse gas emissions, alternative protein sources that rely on inexhaustible substrates/energy should be pursued. In this proof-of-concept study, we propose a two-stage bioinorganic electrosynthesis process that can first convert CO2 and excessive electricity into methane and then synthesize single-cell protein. With an external voltage of 3.5 V and a CO2 inflow rate of 50 mL·d−1, it was possible to produce methanotrophic biomass of 118.7 ± 9.2 mg·L−1 with an amino acids mass content of 54.6% ± 8.3%, resulting in nitrogen assimilation and CO2 conversion efficiency of 91.0% ± 1.3% and 71.0%. The applied voltages, CO2 inflow rates, and O2 supply were found to affect the process significantly. This process using renewable feedstocks was proved independent of conventional agriculture for protein production.
The objective of this study was to investigate the effects of repeated freeze-thaw cycles on microorganisms, amino acid composition profile, chemical composition, mineral concentrations, water mobility, and fat of beef and chicken meats. Pure cultures of specific fungi and bacteria were separately injected into the minced meat. Apart from Pseudomonas, the total count of microorganisms significantly increased (P < 0.05) during refreezing treatment with the increase of storage period in both beef and chicken meats. During freezing treatment, the total count of Staphylococcus aureus, spore forming bacteria, and lactic acid bacteria were meat-type dependent. In conclusion, freeze-thaw cycles increased the microbial counts and decreased the water holding capacity, amino acids, and mineral concentrations of beef and chicken meats.
Methylomicrobium buryatense 5 GB1 has been identified as a promising biocatalyst for industrial methane conversion to produce value-added products. However, despite recent advancements in understanding the metabolism of 5 GB1, existing knowledge on the differences between oxygen-limited and methane-limited phenotypes is still limited. In this work, both batch and continuous experiments were carried out to systematically examine the strain’s oxygen-limited and methane-limited phenotypes. Total carbon balances were performed to ensure the obtained measurements of CH4 and O2 consumption rates and CO2 production rate were accurate. Our results showed that the feed gas composition alone does not dictate the strain’s growth phenotype. In order to achieve a desired phenotype, both feed gas composition and cell growth rate have to be controlled. In addition, contrary to the common belief that oxygen-limited conditions lead to increased production of organic compounds, our results suggest that it is the methane-limited condition that has higher yield for organic compounds. Knowledge of these differences could provide key understanding into how M. buryatense 5 GB1 regulate its carbon flow among different pathways under different growth conditions, which will provide the key insights for both mutant design and process design (e.g., culture conditions) for desired outcomes such as increased production of organic acids. Finally, using data collected in this work and those published in literature, we further validated a published genome-scale model under optimal growth condition. In addition, our results suggest that the current model lacks key metabolic routes to explain the surprisingly robust growth exhibited by the strain under wide substrate availability conditions.
Background
Adequate human nutrition depends on the ingestion of many nutrients present in the diet. Proteins are indispensable macronutrients, including the essential amino acids. A protein source can vary its nutritional quality in terms of digestibility, amino acid profile, and its bioavailability. There are several disadvantages claimed to livestock and traditional animal protein sources. Thus, in a continuous increasing world population, a current challenge is the consumption of proteins with low-cost and easily supply, meeting environmental and social aspects.
Scope and approach
Plant proteins may be a critical step for reducing animal protein dependence by humans. This critical review contributes to facilitating the choice of a plant protein source based on the amino acid composition. Additionally, this overview can give insights into the development of new food products and add value to agro-industrial wastes and by-products. Furthermore, this paper shows the wide variety of sources of plant proteins, with balanced nutritional quality and high protein content as a potential protein supply for the human population and industrial applications.
Key findings and conclusion
A significant challenge is encountering the most suitable protein source since it depends on consumers' preferences, industrial availability, geographical location, and cultural elements. Nevertheless, it is possible to select a plant protein source comparing the essential amino acid composition of each source to the reference pattern.
Microbial exopolysaccharides (EPS) are an abundant and important group of compounds that can be secreted by bacteria, fungi and algae. The biotechnological production of these substances represents a faster alternative when compared to chemical and plant-derived production with the possibility of using industrial wastes as substrates, a feasible strategy after a comprehensive study of factors that may affect the synthesis by the chosen microorganism and desirable final product. Another possible difficulty could be the extraction and purification methods, a crucial part of the production of microbial polysaccharides, since different methods should be adopted. In this sense, this review aims to present the biotechnological production of microbial exopolysaccharides, exploring the production steps, optimization processes and current applications of these relevant bioproducts.
The effect of dissolved oxygen concentration on lipid accumulation in Trichosporon oleaginosus has been investigated. The experiment was performed in 15 L fermenters. The dissolved oxygen concentration varied by adjusting the agitation and aeration. High dissolved oxygen level at 50%–60% enhanced cell growth. Maintaining low dissolved oxygen concentration at 20%–30% during lipogenesis phase led to high final lipid content (51%) in Trichosporon oleaginosus. The consumptions of energy and cost of the process were evaluated. The energy consumption in the dissolved oxygen level optimized process was 41% less than that with dissolved oxygen level at 50%–60%. In addition, the cost was also reduced around one time in the dissolved oxygen level optimized process compared to the one with dissolved oxygen level at 50%–60%. The study provided a feasible way of enhancing lipid accumulation in Trichosporon oleaginosus and reducing the consumption of energy and cost of lipid production from Trichosporon oleaginosus.
This study constitutes the first-proof-of-concept of a methane biorefinery based on the multi-production of high profit margin substances (ectoine, hydroxyectoine, polyhydroxyalkanoates (PHAs) and exopolysaccharides (EPS)) using methane as the sole carbon and energy source. Two bubble column bioreactors were operated under different magnesium concentrations (0.2, 0.02 and 0.002 g L-1) to validate and optimize this innovative strategy for valorization of CH4 emissions. High Mg2+ concentrations promoted the accumulation of ectoine (79.7-94.2 mg g biomass-1), together with high hydroxyectoine yields (up to 13 mg g biomass-1) and EPS concentrations (up to 2.6 g L culture broth-1). Unfortunately, PHA synthesis was almost negligible (14.3 mg L-1) and only found at the lowest Mg2+ concentration tested. Halomonas, Marinobacter, Methylophaga and Methylomicrobium, previously described as ectoine producers, were dominant in both bioreactors, Methylomicrobium being the only described methanotroph. This study encourages further research on CH4 biorefineries capable of creating value out of GHG mitigation.
Methane, with a global warming potential twenty five times higher than that of CO 2 is the second most important greenhouse gas emitted nowadays. Its bioconversion into microbial molecules with a high retail value in the industry offers a potential cost-efficient and environmentally friendly solution for mitigating anthropogenic diluted CH 4-laden streams. Methane bio-refinery for the production of different compounds such as ectoine, feed proteins, biofuels, bioplastics and polysaccharides, apart from new bioproducts characteristic of methanotrophic bacteria, has been recently tested in discontinuous and continuous bioreactors with promising results. This review constitutes a critical discussion about the state-of-the-art of the potential and research niches of biotechnologies applied in a CH 4 biorefinery approach.
The present study aimed at: (1) determining the effect of sulfur addition on biomass growth and (2) assessing the effect of sulfur, phosphorus and nitrogen limitation on lipid accumulation by C. vulgaris SAG 211-11b. The sulfur cellular content was more than two-fold higher under nitrogen and phosphorus limitation (0.52% and 0.54% w w⁻¹, respectively) compared to sulfur requirements (0.20% w w⁻¹) under sulfur limiting conditions. The nitrogen needs are significantly lower (2.81-3.35% w w⁻¹) when compared to other microalgae and become 23% lower under nitrogen or phosphorus limitation. The microalga exhibited substrate inhibition above 30g L⁻¹ initial glucose concentration. Sulfur limitation had the most significant effect on lipid accumulation, resulting in maximum total lipid content of 53.43±3.93% g gDW⁻¹. In addition to enhancing lipid productivity, adopting the optimal nutrient limitation strategy can result in cost savings by avoiding unnecessary nutrient additions and eliminate the environmental burden due to wasted resources.
Microbial conversion of methane to high-value bio-based chemicals and materials offers a path to mitigate GHG emissions and valorize this abundant-yet -underutilized carbon source. In addition to fermentation optimization strategies, rational methanotrophic bacterial strain engineering offers a means to reach industrially relevant titers, carbon yields, and productivities of target products. The phosphoketolase pathway functions in heterofermentative bacteria where carbon flux through two sugar catabolic pathways to mixed acids (lactic acid and acetic acid) increases cellular ATP production. Importantly, this pathway also serves as an alternative route to produce acetyl-CoA that bypasses the CO2 lost through pyruvate decarboxylation in the Embden-Meyerhof-Parnas pathway. Thus, the phosphoketolase pathway can be leveraged for carbon efficient biocatalysis to acetyl-CoA-derived intermediates and products. Here, we show that the industrially promising methane biocatalyst, Methylomicrobium buryatense, encodes two phosphoketolase isoforms that are expressed in methanol- and methane-grown cells. Overexpression of the PktB isoform led to a 2-fold increase in intracellular acetyl-CoA concentration, and a 2.6-fold yield enhancement from methane to microbial biomass and lipids compared to wild-type, increasing the potential for methanotroph lipid-based fuel production. Off-gas analysis and metabolite profiling indicated that global metabolic rearrangements, including significant increases in post-translational protein acetylation and gene expression of the tetrahydromethanopterin-linked pathway, along with decreases in several excreted products, coincided with the superior biomass and lipid yield observed in the engineered strain. Further, these data suggest that phosphoketolase may play a key regulatory role in methanotrophic bacterial metabolism. Given that acetyl-CoA is a key intermediate in several biosynthetic pathways, phosphoketolase overexpression offers a viable strategy to enhance the economics of an array of biological methane conversion processes.
Oxygen uptake rate (OUR) and respiratory quotient (RQ) are key respiratory parameters for docosahexaenoic acid (DHA) production by Schizochytrium sp. HX-308 under dissolved oxygen limited conditions. To investigate the relationship of OUR and RQ with culture status, three independent cultures with different aeration rates were performed in a 50 L bioreactor. OUR was found to be positively correlated with the aeration rate, which reflected the oxygen supply level in each culture. The highest biomass, reaching 124.5 g/L, was achieved under the highest OUR. DHA content was found to be highly correlated with the RQ value, and the highest DHA content (44.85% in total fatty acids, w/w) was achieved in the highest RQ level, which implies that the polyketide synthase pathway was more active. OUR and RQ, which reflect the physiological state of microorganisms, are suggested as synergistic real-time bioprocess monitoring parameters for DHA fermentation.