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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.

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. Electronic supplementary material The online version of this article (10.1007/s10295-020-02319-y) contains supplementary material, which is available to authorized users.

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.

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.

In this study, continuous cultivations of C.carboxidivorans to study heterotrophic and mixotrophic conversion of glucose and H

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.

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.

In this study, the impact of gas composition (i.e. CO, CO

Photosynthetic Poly-β-hydroxybutyrate (PHB) productivity in cyanobacteria needs to be increased to make cyanobacterial derived bioplastics economically feasible and competitive with petroleum-based plastics. In this study, high PHB yielding mutants of Synechocystis sp. PCC 6714 have been generated by random mutagenesis, using UV light as a mutagen. The selection of strains was based on PHB content induced by nitrogen and phosphorus starvation. The fast growing mutant MT_a24 exhibited more than 2.5-fold higher PHB productivity than that of the wild-type, attaining values of 37 ± 4% dry cell weight PHB. The MT_a24 was characterized for phenotypes, CO2 uptake rate and gene expression levels using quantitative PCR. Genome sequencing showed that UV mutagenesis treatment resulted in a point mutation in the ABC-transport complex, phosphate-specific transport system integral membrane protein A (PstA). The MT_a24 shows potential for industrial production of PHB and also for carbon capture from the atmosphere or point sources.

Adaptation to highly saline conditions required halophilic organisms to develop unique features. The mounting interest in those features has resulted in a number of potential applications for moderate and extreme halophiles. However, industrial exploitation has been reported only for few instances. With the aim to bring concepts from laboratory to pilot-scale a customized bioreactor for halophilic cultures was developed. The setup combined a bubble column reactor (BCR) with a membrane module for cell retention. The BCR was compared to a conventional stirred tank reactor (STR) in terms of physiological and hydro-dynamical parameters. The results showed that the BCR is preferable in terms of energy efficiency. The BCR reached a maximum kLa of 84 h−1 at ambient pressure, which equals an oxygen transfer rate (OTR) of 6 mmol/(L*h) in medium with 150 g/L NaCl. To reach this mass transfer a STR required more than 3-fold the amount of energy. Cultivation of the extreme halophilic archaeon Haloferax mediterranei showed suitability of the BCR for continuous halophilic processes. To reduce cost and ecological impact of this process a highly saline industrial waste stream from a chemical production process was used as source for NaCl. The BCR presented in this study allows continuous and competitive cultivation of halophilic organisms at high volumetric rates and high biomass productivities as required for large scale industrial applications.

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.

The aim of this study was to investigate if the high substrate turnover rates of Vibrio natriegens enable 2,3-butanediol production at high productivities. Introduction of a heterologous pathway led to 2,3-butanediol production at yields comparable to Escherichia coli using glucose, sucrose and sugar beet molasses. Microaerobic fed-batch cultivations maintained at 0–1% dissolved oxygen yielded a combined diol titer of 49.9 g L−1 of 2,3-butanediol and acetoin and a production rate of 3.9 g L−1 h−1. Optimization of the oxygen supply in chemostat cultivations increased the product yield to 70% of the theoretical maximum at volumetric and specific production rates of 2 g L−1 h−1 and 0.66 g g−1 h−1, respectively. In conclusion, this study could for the first time demonstrate V. natriegens as novel host for 2,3-butanediol production with high volumetric and specific productivity, underlining its potential for microbial chemical production.

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.

In this study, we developed a toolbox for genetic manipulation of Lactobacillus diolivorans, a promising production organism for 1,3-propanediol from glycerol. Two major findings play a key role for successful transformation of this organism: (1) the absence of a native plasmid, because a native plasmid is a major obstacle for transformation of L. diolivorans, and (2) the absence of DNA methylation. A suitable expression plasmid, pSHM, for homologous and heterologous protein expression in L. diolivorans was constructed. This plasmid is based on the replication origin repA of L. diolivorans. The native glyceraldehyde-3-phosphate dehydrogenase promoter is used for constitutive expression of the genes of interest. Functional expression of genes in L. diolivorans was shown with two examples: production of green fluorescent protein resulted in a 40- to 60-fold higher fluorescence of the obtained clones compared with the wild-type strain. Finally, the homologous overexpression of a putatively NADPH-dependent 1,3-propanediol oxidoreductase improved 1,3-propanediol production by 20% in batch cultures.

Synthetic biology is mainly focused on the design and construction of new biological parts. Assembly of these parts aids with the creation of synthetic biological pathways. This ultimately leads to an overlap of disciplines in which the synthetic parts are applied to a microbial production host and metabolic engineering for production of value-added compounds. This chapter intends to highlight the supportive input of synthetic biology parts, devices and modules to metabolic pathway engineering for the production of chemicals and fuels. Synthetic bricks find applications on the genome, transcriptome and proteome levels. By exemplifying several applications on the different levels, this chapter intends to give a brief overview of recent developments in the field; furthermore, upcoming developments in protein scaffolding and spatial organisation of pathways in bacterial microcompartments are discussed.

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 β-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., Highlights • Recombinant protein production in P. pastoris affects the central metabolism. • A genome scale metabolic model can predict these metabolic flux changes. • Mutations in central metabolic genes enhanced recombinant protein yield up to 40%. • These beneficial mutations were predicted by the metabolic model with high accuracy.

Lactobacillus diolivorans was evaluated as a potential organism for production of 1,3-propanediol under industrially relevant conditions. Crude glycerol of different origins has been tested and showed no inhibitory effects on growth or production. Using crude glycerol from biodiesel production from palm oil 85 g/l 1,3-propanediol have been obtained with a productivity of 0.45 g/l h in a fed-batch cultivation. Sugar necessary for the formation of biomass was replaced with a hydrolysate from lignocellulosic material resulting in 75 g/l 1,3-propanediol and a productivity of 0.36 g/l h. Lignocellulosic hydrolysate contained the potential inhibitors furfural and 5-hydroxymethylfurfural at concentrations of 0.7 and 0.3 g/l, respectively. Addition of furfural and 5-hydroxymethylfurfural to batch cultures in said concentrations did not show inhibitory effects on growth or 1,3-propanediol production.

The aim of this study was to evaluate the natural producer Lactobacillus diolivorans as potential production organism of 1,3-propanediol from glycerol. Different cultivation parameters, such as oxygen supply, feeding-strategy, or medium composition have been tested in batch and fed-batch cultivations. The 1,3-propanediol concentration obtained in batch cultivations was 41.7 g/l. This could be increased to 73.7 g/l in a fed-batch co-feeding glucose and glycerol with a molar ratio of 0.1. Yeast extract as part of the MRS cultivation medium could be replaced by nicotinic acid and riboflavin. Furthermore, the addition of Vitamin B(12) to the culture medium increased production by 15% to a final titer of 84.5 g/l 1,3-propanediol.