A quantitative metabolic analysis reveals Acetobacterium woodii as a flexible and robust host for formate-based bioproduction.

Cheap and renewable feedstocks such as the one-carbon substrate formate are emerging for sustainable production in a growing chemical industry. We investigated the acetogen Acetobacterium woodii as a potential host for bioproduction from formate alone and together with autotrophic and heterotrophic co-substrates by quantitatively analyzing physiology, transcriptome, and proteome in chemostat cultivations in combination with computational analyses. Continuous cultivations with a specific growth rate of 0.05 h-1 on formate showed high specific substrate uptake rates (47 mmol g-1 h-1). Co-utilization of formate with H2, CO, CO2 or fructose was achieved without catabolite repression and with acetate as the sole metabolic product. A transcriptomic comparison of all growth conditions revealed a distinct adaptation of A. woodii to growth on formate as 570 genes were changed in their transcript level. Transcriptome and proteome showed higher expression of the Wood-Ljungdahl pathway during growth on formate and gaseous substrates, underlining its function during utilization of one-carbon substrates. Flux balance analysis showed varying flux levels for the WLP (0.7-16.4 mmol g-1 h-1) and major differences in redox and energy metabolism. Growth on formate, H2/CO2, and formate + H2/CO2 resulted in low energy availability (0.20-0.22 ATP/acetate) which was increased during co-utilization with CO or fructose (0.31 ATP/acetate for formate + H2/CO/CO2, 0.75 ATP/acetate for formate + fructose). Unitrophic and mixotrophic conversion of all substrates was further characterized by high energetic efficiencies. In silico analysis of bioproduction of ethanol and lactate from formate and autotrophic and heterotrophic co-substrates showed promising energetic efficiencies (70-92%). Collectively, our findings reveal A. woodii as a promising host for flexible and simultaneous bioconversion of multiple substrates, underline the potential of substrate co-utilization to improve the energy availability of acetogens and encourage metabolic engineering of acetogenic bacteria for the efficient synthesis of bulk chemicals and fuels from sustainable one carbon substrates.

Increasing ATP turnover boosts productivity of 2,3-butanediol synthesis in Escherichia coli.

BACKGROUND: The alcohol 2,3-butanediol (2,3-BDO) is an important chemical and an Escherichia coli producer strain was recently engineered for bio-based production of 2,3-BDO. However, further improvements are required for realistic applications. RESULTS: Here we report that enforced ATP wasting, implemented by overexpressing the genes of the ATP-hydrolyzing F1-part of the ATPase, leads to significant increases of yield and especially of productivity of 2,3-BDO synthesis in an E. coli producer strain under various cultivation conditions. We studied aerobic and microaerobic conditions as well as growth-coupled and growth-decoupled production scenarios. In all these cases, the specific substrate uptake and 2,3-BDO synthesis rate (up to sixfold and tenfold higher, respectively) were markedly improved in the ATPase strain compared to a control strain. However, aerobic conditions generally enable higher productivities only with reduced 2,3-BDO yields while high product yields under microaerobic conditions are accompanied with low productivities. Based on these findings we finally designed and validated a three-stage process for optimal conversion of glucose to 2,3-BDO, which enables a high productivity in combination with relatively high yield. The ATPase strain showed again superior performance and finished the process twice as fast as the control strain and with higher 2,3-BDO yield. CONCLUSIONS: Our results demonstrate the high potential of enforced ATP wasting as a generic metabolic engineering strategy and we expect more applications to come in the future.

Potential applications of halophilic microorganisms for biological treatment of industrial process brines contaminated with aromatics.

Saline wastewater contaminated with aromatic compounds can be frequently found in various industrial sectors. Those compounds need to be degraded before reuse of wastewater in other process steps or release to the environment. Halophiles have been reported to efficiently degrade aromatics, but their application to treat industrial wastewater is rare. Halophilic processes for industrial wastewater treatment need to satisfy certain requirements: a continuous process mode, low operational expenditures, suitable reactor systems and a monitoring and control strategy. The aim of this review is to provide an overview of halophilic microorganisms, principles of aromatic biodegradation, and sources of saline wastewater containing aromatics and other contaminants. Finally, process examples for halophilic wastewater treatment and potential process monitoring strategies are discussed. To further illustrate the significant potential of halophiles for saline wastewater treatment and to facilitate development of ready-to-implement processes, future research should focus on scale-up and innovative process monitoring and control strategies.

A robust flow cytometry-based biomass monitoring tool enables rapid at-line characterization of S. cerevisiae physiology during continuous bioprocessing of spent sulfite liquor.

Assessment of viable biomass is challenging in bioprocesses involving complex media with distinct biomass and media particle populations. Biomass monitoring in these circumstances usually requires elaborate offline methods or sophisticated inline sensors. Reliable monitoring tools in an at-line capacity represent a promising alternative but are still scarce to date. In this study, a flow cytometry-based method for biomass monitoring in spent sulfite liquor medium as feedstock for second generation bioethanol production with yeast was developed. The method is capable of (i) yeast cell quantification against medium background, (ii) determination of yeast viability, and (iii) assessment of yeast physiology though morphological analysis of the budding division process. Thus, enhanced insight into physiology and morphology is provided which is not accessible through common online and offline biomass monitoring methods. To demonstrate the capabilities of this method, firstly, a continuous ethanol fermentation process of Saccharomyces cerevisiae with filtered and unfiltered spent sulfite liquor media was analyzed. Subsequently, at-line process monitoring of viability in a retentostat cultivation was conducted. The obtained information was used for a simple control based on addition of essential nutrients in relation to viability. Thereby, inter-dependencies between nutrient supply, physiology, and specific ethanol productivity that are essential for process design could be illuminated. Graphical abstract.

Metabolic engineering of Escherichia coli W for isobutanol production on chemically defined medium and cheese whey as alternative raw material.

The aim of this study was to establish isobutanol production on chemically defined medium in Escherichia coli. By individually expressing each gene of the pathway, we constructed a plasmid library for isobutanol production. Strain screening on chemically defined medium showed successful production in the robust E. coli W strain, and expression vector IB 4 was selected as the most promising construct due to its high isobutanol yields and efficient substrate uptake. The investigation of different aeration strategies in combination with strain improvement and the implementation of a pulsed fed-batch were key for the development of an efficient production process. E. coli W ΔldhA ΔadhE Δpta ΔfrdA enabled aerobic isobutanol production at 38% of the theoretical maximum. Use of cheese whey as raw material resulted in longer process stability, which allowed production of 20 g l-1 isobutanol. Demonstrating isobutanol production on both chemically defined medium and a residual waste stream, this study provides valuable information for further development of industrially relevant isobutanol production processes.

Towards continuous industrial bioprocessing with solventogenic and acetogenic clostridia: challenges, progress and perspectives.

The sustainable production of solvents from above ground carbon is highly desired. Several clostridia naturally produce solvents and use a variety of renewable and waste-derived substrates such as lignocellulosic biomass and gas mixtures containing H2/CO2 or CO. To enable economically viable production of solvents and biofuels such as ethanol and butanol, the high productivity of continuous bioprocesses is needed. While the first industrial-scale gas fermentation facility operates continuously, the acetone-butanol-ethanol (ABE) fermentation is traditionally operated in batch mode. This review highlights the benefits of continuous bioprocessing for solvent production and underlines the progress made towards its establishment. Based on metabolic capabilities of solvent producing clostridia, we discuss recent advances in systems-level understanding and genome engineering. On the process side, we focus on innovative fermentation methods and integrated product recovery to overcome the limitations of the classical one-stage chemostat and give an overview of the current industrial bioproduction of solvents.

Microbial Upgrading of Acetate into Value-Added Products-Examining Microbial Diversity, Bioenergetic Constraints and Metabolic Engineering Approaches.

Ecological concerns have recently led to the increasing trend to upgrade carbon contained in waste streams into valuable chemicals. One of these components is acetate. Its microbial upgrading is possible in various species, with Escherichia coli being the best-studied. Several chemicals derived from acetate have already been successfully produced in E. coli on a laboratory scale, including acetone, itaconic acid, mevalonate, and tyrosine. As acetate is a carbon source with a low energy content compared to glucose or glycerol, energy- and redox-balancing plays an important role in acetate-based growth and production. In addition to the energetic challenges, acetate has an inhibitory effect on microorganisms, reducing growth rates, and limiting product concentrations. Moreover, extensive metabolic engineering is necessary to obtain a broad range of acetate-based products. In this review, we illustrate some of the necessary energetic considerations to establish robust production processes by presenting calculations of maximum theoretical product and carbon yields. Moreover, different strategies to deal with energetic and metabolic challenges are presented. Finally, we summarize ways to alleviate acetate toxicity and give an overview of process engineering measures that enable sustainable acetate-based production of value-added chemicals.

Engineered E. coli W enables efficient 2,3-butanediol production from glucose and sugar beet molasses using defined minimal medium as economic basis.

BACKGROUND: Efficient microbial production of chemicals is often hindered by the cytotoxicity of the products or by the pathogenicity of the host strains. Hence 2,3-butanediol, an important drop-in chemical, is an interesting alternative target molecule for microbial synthesis since it is non-cytotoxic. Metabolic engineering of non-pathogenic and industrially relevant microorganisms, such as Escherichia coli, have already yielded in promising 2,3-butanediol titers showing the potential of microbial synthesis of 2,3-butanediol. However, current microbial 2,3-butanediol production processes often rely on yeast extract as expensive additive, rendering these processes infeasible for industrial production. RESULTS: The aim of this study was to develop an efficient 2,3-butanediol production process with E. coli operating on the premise of using cost-effective medium without complex supplements, considering second generation feedstocks. Different gene donors and promoter fine-tuning allowed for construction of a potent E. coli strain for the production of 2,3-butanediol as important drop-in chemical. Pulsed fed-batch cultivations of E. coli W using microaerobic conditions showed high diol productivity of 4.5 g l-1 h-1. Optimizing oxygen supply and elimination of acetoin and by-product formation improved the 2,3-butanediol titer to 68 g l-1, 76% of the theoretical maximum yield, however, at the expense of productivity. Sugar beet molasses was tested as a potential substrate for industrial production of chemicals. Pulsed fed-batch cultivations produced 56 g l-1 2,3-butanediol, underlining the great potential of E. coli W as production organism for high value-added chemicals. CONCLUSION: A potent 2,3-butanediol producing E. coli strain was generated by considering promoter fine-tuning to balance cell fitness and production capacity. For the first time, 2,3-butanediol production was achieved with promising titer, rate and yield and no acetoin formation from glucose in pulsed fed-batch cultivations using chemically defined medium without complex hydrolysates. Furthermore, versatility of E. coli W as production host was demonstrated by efficiently converting sucrose from sugar beet molasses into 2,3-butanediol.

Characterizing the effect of expression of an acetyl-CoA synthetase insensitive to acetylation on co-utilization of glucose and acetate in batch and continuous cultures of E. coli W.

BACKGROUND: Due to its high stress tolerance and low acetate secretion, Escherichia coli W is reported to be a good production host for several metabolites and recombinant proteins. However, simultaneous co-utilization of glucose and other substrates such as acetate remains a challenge. The activity of acetyl-CoA-synthetase, one of the key enzymes involved in acetate assimilation is tightly regulated on a transcriptional and post-translational level. The aim of this study was to engineer E. coli W for overexpression of an acetylation insensitive acetyl-CoA-synthetase and to characterize this strain in batch and continuous cultures using glucose, acetate and during co-utilization of both substrates. RESULTS: Escherichia coli W engineered to overexpress an acetylation-insensitive acetyl-CoA synthetase showed a 2.7-fold increase in acetate uptake in a batch process containing glucose and high concentrations of acetate compared to a control strain, indicating more efficient co-consumption of glucose and acetate. When acetate was used as the carbon source, batch duration could significantly be decreased in the overexpression strain, possibly due to alleviation of acetate toxicity. Chemostat cultivations with different dilution rates using glucose revealed only minor differences between the overexpression and control strain. Accelerostat cultivations using dilution rates between 0.20 and 0.70 h-1 indicated that E. coli W is naturally capable of efficiently co-utilizing glucose and acetate over a broad range of specific growth rates. Expression of acetyl-CoA synthetase resulted in acetate and glucose accumulation at lower dilution rates compared to the control strain. This observation can possibly be attributed to a higher ratio between acs and pta-ackA in the overexpression strain as revealed by gene expression analysis. This would result in enhanced energy dissipation caused by an imbalance in the Pta-AckA-Acs cycle. Furthermore, yjcH and actP, genes co-transcribed with acetyl-CoA synthetase showed significant down-regulation at elevated dilution rates. CONCLUSIONS: Escherichia coli W expressing an acetylation-insensitive acetyl-CoA synthetase was shown to be a promising candidate for mixed feed processes using glucose and acetate. Comparison between batch and continuous cultures revealed distinct differences in glucose-acetate co-utilization behavior, requiring additional investigations such as multi-omics analysis and further engineering towards even more efficient co-utilization strains of E. coli W.

Model based engineering of Pichia pastoris central metabolism enhances recombinant protein production.

The production of recombinant proteins is frequently enhanced at the levels of transcription, codon usage, protein folding and secretion. Overproduction of heterologous proteins, however, also directly affects the primary metabolism of the producing cells. By incorporation of the production of a heterologous protein into a genome scale metabolic model of the yeast Pichia pastoris, the effects of overproduction were simulated and gene targets for deletion or overexpression for enhanced productivity were predicted. Overexpression targets were localized in the pentose phosphate pathway and the TCA cycle, while knockout targets were found in several branch points of glycolysis. Five out of 9 tested targets led to an enhanced production of cytosolic human superoxide dismutase (hSOD). Expression of bacterial ß-glucuronidase could be enhanced as well by most of the same genetic modifications. Beneficial mutations were mainly related to reduction of the NADP/H pool and the deletion of fermentative pathways. Overexpression of the hSOD gene itself had a strong impact on intracellular fluxes, most of which changed in the same direction as predicted by the model. In vivo fluxes changed in the same direction as predicted to improve hSOD production. Genome scale metabolic modeling is shown to predict overexpression and deletion mutants which enhance recombinant protein production with high accuracy.

Recombinant Aspergillus ß-galactosidases as a robust glycomic and biotechnological tool.

Galactosidases are widespread enzymes that are used for manifold applications, including production of prebiotics, biosynthesis of different transgalactosylated products, improving lactose tolerance and in various analytical approaches. The nature of these applications often require galactosidases to be present in a purified form with clearly defined properties, including precisely determined substrate specificities, low sensitivity to inhibitors, and high efficiency and stability under distinct conditions. In this study, we present the recombinant expression and purification of two previously uncharacterized ß-galactosidases from Aspergillus nidulans as well as one ß-galactosidase from Aspergillus niger. All enzymes were active toward p-nitrophenyl-ß-D-galactopyranoside as substrate and displayed similar temperature and pH optima. The purified recombinant galactosidases digested various complex substrates containing terminal galactose ß-1,4 linked to either N-acetylglucosamine or fucose, such as N-glycans derived from bovine fibrin and Caenorhabditis elegans. In our comparative study of the recombinant galactosidases with the commercially available galactosidase from Aspergillus oryzae, all enzymes also displayed various degrees of activity toward complex oligosaccharides containing ß-1,3-linked terminal galactose residues. All recombinant enzymes were found to be robust in the presence of various organic solvents, temperature variations, and freeze/thaw cycles and were also tested for their ability to synthesize galactooligosaccharides. Furthermore, the use of fermentors considerably increased the yield of recombinant galactosidases. Taken together, we demonstrate that purified recombinant galactosidases from A. niger and from A. nidulans are suitable for various glycobiological and biotechnological applications.

A fluorescent reporter system for anaerobic thermophiles.

Owing to their inherent capacity to make invisible biological processes visible and quantifiable, fluorescent reporter systems have numerous applications in biotechnology. For classical fluorescent protein systems (i.e., GFP and derivatives), chromophore maturation is O2-dependent, restricting their applications to aerobic organisms. In this work, we pioneered the use of the oxygen-independent system FAST (Fluorescence Activating and absorption Shifting tag) in the thermophilic anaerobe Thermoanaerobacter kivui. We developed a modular cloning system that was used to easily clone a library of FAST expression cassettes in an E. coli-Thermoanaerobacter shuttle plasmid. FAST-mediated fluorescence was then assessed in vivo in T. kivui, and we observed bright green and red fluorescence for cells grown at 55°C. Next, we took advantage of this functional reporter system to characterize a set of homologous and heterologous promoters by quantifying gene expression, expanding the T. kivui genetic toolbox. Low fluorescence at 66°C (Topt for T. kivui) was subsequently investigated at the single-cell level using flow cytometry and attributed to plasmid instability at higher temperatures. Adaptive laboratory evolution circumvented this issue and drastically enhanced fluorescence at 66°C. Whole plasmid sequencing revealed the evolved strain carried functional plasmids truncated at the Gram-positive origin of replication, that could however not be linked to the increased fluorescence displayed by the evolved strain. Collectively, our work demonstrates the applicability of the FAST fluorescent reporter systems to T. kivui, paving the way for further applications in thermophilic anaerobes.

Corrigendum: Reducing Organic Load From Industrial MDA Residual Process Brine With a Novel Halophilic Mixed Culture: Scale-Up and Long-Term Piloting of an Integrated Bioprocess.

[This corrects the article DOI: 10.3389/fbioe.2022.896576.].

Reducing Organic Load From Industrial Residual Process Brine With a Novel Halophilic Mixed Culture: Scale-Up and Long-Term Piloting of an Integrated Bioprocess.

Integrating bioprocess solutions for treatment and subsequent reuse of saline residual process brine into industrial processes could increase the sustainability of production chains. However, such bioprocesses require large-scales and a robust operation over a prolonged period. Consequently, the aim of this study was to analyze scale-up equivalence as well as continuous and stable process performance of a previously established lab scale process for the degradation of organic contaminants (formate and aromatic compounds) in an industrial context. To that end, a pilot-scale bubble column bioreactor system equipped with a membrane-based cell retention system for process intensification was integrated at an industrial production site. The process was successfully scaled-up and continuously operated for more than 210 days. Overall, the process proved to be robust towards changing compositions of the residual process brine stream and degradation rates for organic contaminants were close to 100%. Interestingly, due to the unsterile process conditions, the original Haloferax mediterranei culture was replaced by a novel halophilic bacterial community consisting of three bacterial genera. To further improve process economics and productivity, an optimization of the co-substrate feeding strategy for glycerol is required, as results indicated a potential correlation between glycerol feeding and formate degradation rates. To that end, decoupling of the glycerol feeding from the residual process brine feed is a potential way to increase process control options and allow for easy adaptation of the process to changing residual process brine compositions. Ultimately, the process described here could be a promising alternative for chemical or physical methods of treating residual process brine and once more underlines the potential to exploit natural microbial diversity for industrial purposes.

Mixotrophic co-utilization of glucose and carbon monoxide boosts ethanol and butanol productivity of continuous Clostridium carboxidivorans cultures.

In this study, continuous cultivations of C.carboxidivorans to study heterotrophic and mixotrophic conversion of glucose and H2, CO2, and CO were established. Glucose fermentations at pH 6 showed a high ratio of alcohol-to-acid production of 2.79 mol mol-1. While H2 or CO2 were not utilized together with glucose, CO feeding drastically increased the combined alcohol titer to 9.1 g l-1. Specifically, CO enhanced acetate (1.9-fold) and ethanol (1.7-fold) production and triggered chain elongation to butanol (1.5-fold) production but did not change the alcohol:acid ratio. Flux balance analysis showed that CO served both as a carbon and energy source, and CO mixotrophy displayed a carbon and energy efficiency of 45 and 77%, respectively. This study expands the knowledge on physiology and metabolism of C.carboxidivorans and can serve as the starting point for rational engineering and process intensification to establish efficient production of alcohols and acids from carbon waste.

Blending industrial blast furnace gas with H2 enables Acetobacterium woodii to efficiently co-utilize CO, CO2 and H2.

In this study, the impact of gas composition (i.e. CO, CO2 and H2 partial pressures) on CO2 utilization, growth, and acetate production was investigated in batch and continuous cultures of A. woodii. Based on an industrial blast furnace gas, H2 blending was used to study the impact of H2 availability on CO2 fixation alone and together with CO using idealized gas streams. With H2 available as an additional energy source, net CO2 fixation and CO, CO2 and H2 co-utilization was achieved in gas-limited fermentations. Using industrial blast furnace gas, up to 15.1 g l-1 acetate were produced in continuous cultures. Flux balance analysis showed that intracellular fluxes and total ATP production were dependent on the availability of H2 and CO. Overall, H2 blending was shown to be a suitable control strategy for gas fermentations and demonstrated that A. woodii is an interesting host for CO2 fixation from industrial gas streams.

Microbial upgrading of acetate into 2,3-butanediol and acetoin by E. coli W.

BACKGROUND: Acetate is an abundant carbon source and its use as an alternative feedstock has great potential for the production of fuel and platform chemicals. Acetoin and 2,3-butanediol represent two of these potential platform chemicals. RESULTS: The aim of this study was to produce 2,3-butanediol and acetoin from acetate in Escherichia coli W. The key strategies to achieve this goal were: strain engineering, in detail the deletion of mixed-acid fermentation pathways E. coli W ΔldhA ΔadhE Δpta ΔfrdA 445_Ediss and the development of a new defined medium containing five amino acids and seven vitamins. Stepwise reduction of the media additives further revealed that diol production from acetate is mediated by the availability of aspartate. Other amino acids or TCA cycle intermediates did not enable growth on acetate. Cultivation under controlled conditions in batch and pulsed fed-batch experiments showed that aspartate was consumed before acetate, indicating that co-utilization is not a prerequisite for diol production. The addition of aspartate gave cultures a start-kick and was not required for feeding. Pulsed fed-batches resulted in the production of 1.43 g l-1 from aspartate and acetate and 1.16 g l-1 diols (2,3-butanediol and acetoin) from acetate alone. The yield reached 0.09 g diols per g acetate, which accounts for 26% of the theoretical maximum. CONCLUSION: This study for the first time showed acetoin and 2,3-butanediol production from acetate as well as the use of chemically defined medium for product formation from acetate in E. coli. Hereby, we provide a solid base for process intensification and the investigation of other potential products.

Soft Sensor-Based Monitoring and Efficient Control Strategies of Biomass Concentration for Continuous Cultures of Haloferax mediterranei and Their Application to an Industrial Production Chain.

Continuous bioprocessing using cell retention allows the achievement of high space-time yields for slow-growing organisms such as halophiles. However, the lack of efficient methods for monitoring and control limits the application of biotechnological processes in the industry. The aim of this study was to implement a control and online monitoring strategy for biomass in continuous cultures. For the first time, a feedforward cultivation strategy in a membrane-based cell retention system allowed to control the biomass concentration of the extreme halophilic Haloferax mediterranei at defined levels. Moreover, soft sensor-based biomass estimation allowed reliable monitoring of biomass online. Application of the combined monitoring and control strategy using industrial process water containing formate, phenol, aniline and 4,4'-methylenedianiline could for the first time demonstrate high throughput degradation in this extremophilic bioremediation process, obtaining degradation efficiencies of up to 100%. This process demonstrates the usefulness of continuous halophilic cultures in a circular economy application.

Towards biobased industry: acetate as a promising feedstock to enhance the potential of microbial cell factories.

A broad range of different chemical and pharmaceutical compounds have been produced in microbial cell factories. To compete with traditional crude oil based production processes, the use of complex alternative raw materials such as lignocellulosic biomass, waste streams and utilization of CO2 in gas fermentations has been suggested. All of these streams contain acetate, a cheap and potentially interesting carbon source for microbial production processes. Acetate (co-)utilization remains challenging, which is the reason for extensive research on the use of acetate for the production of value-added compounds. For industrial implementation of microbial conversion processes using acetate as a feedstock gaining a deeper insight into acetate metabolism of microorganisms is essential. Systems level analyses and manipulation of potential host organisms should be applied to achieve full utilization of this prospective substrate.