Acetic acid, growth rate, and mass transfer govern shifts in CO metabolism of Clostridium autoethanogenum.

Syngas fermentation is a leading microbial process for the conversion of carbon monoxide, carbon dioxide, and hydrogen to valuable biochemicals. Clostridium autoethanogenum stands as a model organism for this process, showcasing its ability to convert syngas into ethanol industrially with simultaneous fixation of carbon and reduction of greenhouse gas emissions. A deep understanding on the metabolism of this microorganism and the influence of operational conditions on fermentation performance is key to advance the technology and enhancement of production yields. In this work, we studied the individual impact of acetic acid concentration, growth rate, and mass transfer rate on metabolic shifts, product titres, and rates in CO fermentation by C. autoethanogenum. Through continuous fermentations performed at a low mass transfer rate, we measured the production of formate in addition to acetate and ethanol. We hypothesise that low mass transfer results in low CO concentrations, leading to reduced activity of the Wood-Ljungdahl pathway and a bottleneck in formate conversion, thereby resulting in the accumulation of formate. The supplementation of the medium with exogenous acetate revealed that undissociated acetic acid concentration increases and governs ethanol yield and production rates, assumedly to counteract the inhibition by undissociated acetic acid. Since acetic acid concentration is determined by growth rate (via dilution rate), mass transfer rate, and working pH, these variables jointly determine ethanol production rates. These findings have significant implications for process optimisation as targeting an optimal undissociated acetic acid concentration can shift metabolism towards ethanol production. KEY POINTS: • Very low CO mass transfer rate leads to leaking of intermediate metabolite formate. • Undissociated acetic acid concentration governs ethanol yield on CO and productivity. • Impact of growth rate, mass transfer rate, and pH were considered jointly.

Biomass-specific rates as key performance indicators: A nitrogen balancing method for biofilm-based electrochemical conversion.

Microbial electrochemical technologies (METs) employ microorganisms utilizing solid-state electrodes as either electron sink or electron source, such as in microbial electrosynthesis (MES). METs reaction rate is traditionally normalized to the electrode dimensions or to the electrolyte volume, but should also be normalized to biomass amount present in the system at any given time. In biofilm-based systems, a major challenge is to determine the biomass amount in a non-destructive manner, especially in systems operated in continuous mode and using 3D electrodes. We developed a simple method using a nitrogen balance and optical density to determine the amount of microorganisms in biofilm and in suspension at any given time. For four MES reactors converting CO2 to carboxylates, >99% of the biomass was present as biofilm after 69 days of reactor operation. After a lag phase, the biomass-specific growth rate had increased to 0.12-0.16 days-1. After 100 days of operation, growth became insignificant. Biomass-specific production rates of carboxylates varied between 0.08-0.37 molC molX -1d-1. Using biomass-specific rates, one can more effectively assess the performance of MES, identify its limitations, and compare it to other fermentation technologies.

Membrane bioreactors for syngas permeation and fermentation.

Syngas fermentation to biofuels and chemicals is an emerging technology in the biobased economy. Mass transfer is usually limiting the syngas fermentation rate, due to the low aqueous solubilities of the gaseous substrates. Membrane bioreactors, as efficient gas-liquid contactors, are a promising configuration for overcoming this gas-to-liquid mass transfer limitation, so that sufficient productivity can be achieved. We summarize the published performances of these reactors. Moreover, we highlight numerous parameters settings that need to be used for the enhancement of membrane bioreactor performance. To facilitate this enhancement, we relate mass transfer and other performance indicators to the type of membrane material, module, and flow configuration. Hollow fiber modules with dense or asymmetric membranes on which biofilm might form seem suitable. A model-based approach is advocated to optimize their performance.

A General Model for Biofilm-Driven Microbial Electrosynthesis of Carboxylates From CO2.

Up to now, computational modeling of microbial electrosynthesis (MES) has been underexplored, but is necessary to achieve breakthrough understanding of the process-limiting steps. Here, a general framework for modeling microbial kinetics in a MES reactor is presented. A thermodynamic approach is used to link microbial metabolism to the electrochemical reduction of an intracellular mediator, allowing to predict cellular growth and current consumption. The model accounts for CO2 reduction to acetate, and further elongation to n-butyrate and n-caproate. Simulation results were compared with experimental data obtained from different sources and proved the model is able to successfully describe microbial kinetics (growth, chain elongation, and product inhibition) and reactor performance (current density, organics titer). The capacity of the model to simulate different system configurations is also shown. Model results suggest CO2 dissolved concentration might be limiting existing MES systems, and highlight the importance of the delivery method utilized to supply it. Simulation results also indicate that for biofilm-driven reactors, continuous mode significantly enhances microbial growth and might allow denser biofilms to be formed and higher current densities to be achieved.

Highly selective recovery of medium chain carboxylates from co-fermented organic wastes using anion exchange with carbon dioxide expanded methanol desorption.

The aim of this work was to recover a mixture of carboxylates ranging from 2 to 7 carbon atoms using a strong anion exchange resin, followed by desorption with CO2-expanded methanol. Medium chain carboxylates hexanoate and heptanoate adsorbed better than acetate, and the corresponding medium chain carboxylic acids desorbed easier than acetic acid. Consequently, hexanoate and heptanoate were concentrated up to 14.6 and 20.7 times, respectively. These findings will enable effective separation and purification of the produced carboxylic acids. Notably, the presence of inorganic ions in the sample, such as chloride, decreased the adsorption affinity compared to a synthetic mixture only of carboxylates.

Grand Research Challenges for Sustainable Industrial Biotechnology.

Future manufacturing will focus on new, improved products as well as on new and enhanced production methods. Recent biotechnological and scientific advances, such as CRISPR/Cas and various omic technologies, pave the way to exciting novel biotechnological research, development, and commercialization of new sustainable products. Rigorous mathematical descriptions of microbial cells and consortia thereof will enable deeper biological understanding and lead to powerful in silico cellular models. Biological engineering, namely model-based design together with synthetic biology, will accelerate the construction of robust and high-performing microorganisms. Using these organisms, and ambitions towards zero-concepts with respect to emissions and excess resources in bioprocess engineering, industrial biotechnology is expected to become highly integrated into sustainable generations of technology systems.

Integrated Vacuum Stripping and Adsorption for the Efficient Recovery of (Biobased) 2-Butanol.

Biobased 2-butanol offers high potential as biofuel, but its toxicity toward microbial hosts calls for efficient techniques to alleviate product inhibition in fermentation processes. Aiming at the selective recovery of 2-butanol, the feasibility of a process combining in situ vacuum stripping followed by vapor adsorption has been assessed using mimicked fermentation media. The experimental vacuum stripping of model solutions and corn stover hydrolysate closely aligned with mass transfer model predictions. However, the presence of lignocellulosic impurities affected 2-butanol recovery yields resulting from vapor condensation, which decreased from 96 wt % in model solutions to 40 wt % using hydrolysate. For the selective recovery of 2-butanol from a vapor mixture enriched in water and carbon dioxide, silicalite materials were the most efficient, particularly at low alcohol partial pressures. Integrating in situ vacuum stripping with vapor adsorption using HiSiv3000 proved useful to effectively concentrate 2-butanol above its azeotropic composition (>68 wt %), facilitating further product purification.

Microbial Hydrocarbon Formation from Biomass.

Fossil carbon sources mainly contain hydrocarbons, and these are used on a huge scale as fuel and chemicals. Producing hydrocarbons from biomass instead is receiving increased attention. Achievable yields are modest because oxygen atoms need to be removed from biomass, keeping only the lighter carbon and hydrogen atoms. Microorganisms can perform the required conversions, potentially with high selectivity, using metabolic pathways that often end with decarboxylation. Metabolic and protein engineering are used successfully to achieve hydrocarbon production levels that are relevant in a biorefinery context. This has led to pilot or demo processes for hydrocarbons such as isobutene, isoprene, and farnesene. In addition, some non-hydrocarbon fermentation products are being further converted into hydrocarbons using a final chemical step, for example, ethanol into ethene. The main advantage of direct microbial production of hydrocarbons, however, is their potentially easy recovery because they do not dissolve in fermentation broth.

Perspectives for the microbial production of methyl propionate integrated with product recovery.

A new approach was studied for bio-based production of methyl propionate, a precursor of methyl methacrylate. Recombinant E. coli cells were used to perform a cascade reaction in which 2-butanol is reduced to butanone using alcohol dehydrogenase, and butanone is oxidized to methyl propionate and ethyl acetate using a Baeyer-Villiger monooxygenase (BVMO). Product was removed by in situ stripping. The conversion was in line with a model comprising product formation and stripping kinetics. The maximum conversion rates were 1.14 g-butanone/(L h), 0.11 g-ethyl acetate/(L h), and 0.09 g-methyl propionate/(L h). The enzyme regioselectivity towards methyl propionate was 43% of total ester. Starting from biomass-based production of 2-butanol, full-scale ester production with conventional product purification was calculated to be competitive with petrochemical production if the monooxygenase activity and regioselectivity are enhanced, and the costs of bio-based 2-butanol are minimized.

Prospects and challenges for the recovery of 2-butanol produced by vacuum fermentation - a techno-economic analysis.

The conceptual design of a bio-based process for 2-butanol production is presented for the first time. Considering a hypothetical efficient producing strain, a vacuum fermentation is proposed to alleviate product toxicity, but the main challenge is the energy-efficient product recovery from the vapor. Three downstream scenarios were examined for this purpose: 1) multi-stage vapor recompression; 2) temperature swing adsorption; and 3) vapor absorption. The processes were simulated using Aspen Plus, considering a production capacity of 101 kton/yr. Process optimization was performed targeting the minimum selling price of 2-butanol. The feasibility of the different configurations was analyzed based on the global energy requirements and capital expenditure. The use of integrated adsorption and absorption minimized the energy duty required for azeotrope purification, which represents 11% of the total operational expenditure in Scenario 1. The minimum selling price of 2-butanol as commodity chemical was estimated as 1.05 $/kg, 1.21 $/kg, and 1.03 $/kg regarding the fermentation integrated with downstream scenarios 1), 2), and 3), respectively. Significant savings in 2-butanol production could be achieved in the suggested integrated configurations if more efficient microbial strains were engineered, and more selective adsorption and absorption materials were found for product recovery.

Esters production via carboxylates from anaerobic paper mill wastewater treatment.

This paper describes a new option for integrated recovery and esterification of carboxylates produced by anaerobic digestion at a pH above the pKa. The carboxylates (acetate, propionate, butyrate, valerate and lactate) are recovered using a strong anion exchange resin in the bicarbonate form, and the resin is regenerated using a CO2-expanded alcohol technique, which allows for low chemicals consumption and direct esterification. Paper mill wastewater was used to study the effect of pH and the presence of other inorganic anions and cations on the adsorption and desorption with CO2-expanded methanol. Calcium, which is present in paper mill wastewater, can cause precipitation problems, especially at high pH. Esters yields ranged from 1.08±0.04mol methyl acetate/mol of acetatein to 0.57±0.02mol methyl valerate/mol of valeratein.

Growth inhibition of S. cerevisiae, B. subtilis, and E. coli by lignocellulosic and fermentation products.

This paper describes the effect of several inhibiting components on three potential hosts for the bio-based production of methyl propionate, namely, wild-type Escherichia coli and Bacillus subtilis, and evolved Saccharomyces cerevisiae IMS0351. The inhibition by the lignocellulose-derived products 5-hydroxymethyl-2-furaldehyde, vanillin, and syringaldehyde and the fermentation products 2-butanol, 2-butanone, methyl propionate, and ethyl acetate has been assessed for these strains in defined medium. Multiple screenings were performed using small-scale cultures in both shake flasks and microtiter plates. Technical drawbacks revealed the limited applicability of the latter in this study. The microbial growth was characterized by means of a lag-time model, and the inhibitory thresholds were determined using product-inhibition models. The lignocellulose-derived products were found to be highly inhibitory, and none of the strains could grow in the presence of 2.0 g L-1 of product. From the fermentation products tested, methyl propionate had the most severe impact resulting in complete inhibition of all the strains when exposed to concentrations in the range of 12-18 g L-1. In general, S. cerevisiae and B. subtilis were comparatively more tolerant than E. coli to all the fermentation products, despite E. coli's lower sensitivity towards vanillin. The results suggest that, overall, the strains investigated have good potential to be engineered and further established as hosts for the bio-based production of methyl esters.

Recovery of carboxylic acids produced by fermentation.

Carboxylic acids such as citric, lactic, succinic and itaconic acids are useful products and are obtained on large scale by fermentation. This review describes the options for recovering these and other fermentative carboxylic acids. After cell removal, often a primary recovery step is performed, using liquid-liquid extraction, adsorption, precipitation or conventional electrodialysis. If the carboxylate is formed rather than the carboxylic acid, the recovery process involves a step for removing the cation of the formed carboxylate. Then, bipolar electrodialysis and thermal methods for salt splitting can prevent that waste inorganic salts are co-produced. Final carboxylic acid purification requires either distillation or crystallization, usually involving evaporation of water. Process steps can often be combined synergistically. In-situ removal of carboxylic acid by extraction during fermentation is the most popular approach. Recovery of the extractant can easily lead to waste inorganic salt formation, which counteracts the advantage of the in-situ removal. For industrial production, various recovery principles and configurations are used, because the fermentation conditions and physical properties of specific carboxylic acids differ.

Simultaneous Clostridial fermentation, lipase-catalyzed esterification, and ester extraction to enrich diesel with butyl butyrate.

The recovery of 1-butanol from fermentation broth is energy-intensive since typical concentrations in fermentation broth are below 20 g L(-1). To prevent butanol inhibition and high downstream processing costs, we aimed at producing butyl esters instead of 1-butanol. It is shown that it is possible to perform simultaneously clostridial fermentation, esterification of the formed butanol to butyl butyrate, and extraction of this ester by hexadecane. The very high partition coefficient of butyl butyrate pulls the esterification towards the product side even at fermentation pH and relatively low butanol concentrations. The hexadecane extractant is a model diesel compound and is nontoxic to the cells. If butyl butyrate enriched diesel can directly be used as car fuel, no product recovery is required. A proof-of-principle experiment for the one-pot bio-ester production from glucose led to 5 g L(-1) butyl butyrate in the hexadecane phase. The principle may be extended to a wide range of esters, especially to longer chain ones.

Pervaporation of ethanol from lignocellulosic fermentation broth.

Pervaporation can be applied in ethanol production from lignocellulosic biomass. Hydrophobic pervaporation, using a commercial PDMS membrane, was employed to concentrate the ethanol produced by fermentation of lignocellulosic hydrolysate. To our knowledge, this is the first report describing this. Pervaporation carried out with three different lignocellulosic fermentation broths reduced the membrane performance by 17-20% as compared to a base case containing only 3 wt.% ethanol in water. The membrane fouling caused by these fermentation broths was irreversible. Solutions containing model lignocellulosic components were tested during pervaporation at the same conditions. A total flux decrease of 12-15%, as compared to the base case, was observed for each component except for furfural. Catechol was found to be most fouling component whereas furfural permeated through the membrane and increased the total flux. The membrane selectivity increased in the presence of fermentation broth but remained unchanged for all selected components.

Production of fumaric acid by fermentation.

Fermentative fumaric acid production from renewable resources may become competitive with petrochemical production. This will require very efficient processes. So far, using Rhizopus strains, the best fermentations reported have achieved a fumaric acid titer of 126 g/L with a productivity of 1.38 g L(-1) h(-1) and a yield on glucose of 0.97 g/g. This requires pH control, aeration, and carbonate/CO(2) supply. Limitations of the used strains are their pH tolerance, morphology, accessibility for genetic engineering, and partly, versatility to alternative carbon sources. Understanding of the mechanism and energetics of fumaric acid export by Rhizopus strains will be a success factor for metabolic engineering of other hosts for fumaric acid production. So far, metabolic engineering has been described for Escherichia coli and Saccharomyces cerevisiae.

Fermentative production of isobutene.

Isobutene (2-methylpropene) is one of those chemicals for which bio-based production might replace the petrochemical production in the future. Currently, more than 10 million metric tons of isobutene are produced on a yearly basis. Even though bio-based production might also be achieved through chemocatalytic or thermochemical methods, this review focuses on fermentative routes from sugars. Although biological isobutene formation is known since the 1970s, extensive metabolic engineering is required to achieve economically viable yields and productivities. Two recent metabolic engineering developments may enable anaerobic production close to the theoretical stoichiometry of 1isobutene + 2CO(2) + 2H(2)O per mol of glucose. One relies on the conversion of 3-hydroxyisovalerate to isobutene as a side activity of mevalonate diphosphate decarboxylase and the other on isobutanol dehydration as a side activity of engineered oleate hydratase. The latter resembles the fermentative production of isobutanol followed by isobutanol recovery and chemocatalytic dehydration. The advantage of a completely biological route is that not isobutanol, but instead gaseous isobutene is recovered from the fermenter together with CO(2). The low aqueous solubility of isobutene might also minimize product toxicity to the microorganisms. Although developments are at their infancy, the potential of a large scale fermentative isobutene production process is assessed. The production costs estimate is 0.9 Euro kg(-1), which is reasonably competitive. About 70% of the production costs will be due to the costs of lignocellulose hydrolysate, which seems to be a preferred feedstock.

Development of a low pH fermentation strategy for fumaric acid production by Rhizopus oryzae.

Dicarboxylic acids that are produced from renewable resources are becoming attractive building blocks for the polymers industry. In this respect, fumaric acid is very interesting. Its low aqueous solubility facilitates product recovery. To avoid excessive waste salt production during downstream processing, a low pH for fumaric acid fermentation will be beneficial. Studying the influence of pH, working volume and shaking frequency on cell cultivation helped us to identify the best conditions to obtain appropriate pellet morphologies of a wild type strain of Rhizopus oryzae. Using these pellets, the effects of pH and CO(2) addition were studied to determine the best conditions to produce fumaric acid in batch fermentations under nitrogen-limited conditions with glucose as carbon source. Decreasing either the fermentation pH below 5 or increasing the CO(2) content of the inlet air above 10% was unfavourable for the cell-specific productivity, fumaric acid yield, and fumaric acid titer. However, switching off the pH control late in the batch phase did not affect these performance parameters and allowed achieving pH of 3.6. A concentration of 20 gL(-1) of fumaric acid was obtained at pH 3.6 while the average cell mass specific productivity and fumaric acid yield were the same as at pH 5.0. Consequently, relatively modest amounts of inorganic base were required for pH control, while recovery of the acid should be relatively easy at pH 3.6.

Purification, characterization, and cloning of a bifunctional molybdoenzyme with hydratase and alcohol dehydrogenase activity.

A bifunctional hydratase/alcohol dehydrogenase was isolated from the cyclohexanol degrading bacterium Alicycliphilus denitrificans DSMZ 14773. The enzyme catalyzes the addition of water to α,ß-unsaturated carbonyl compounds and the subsequent alcohol oxidation. The purified enzyme showed three subunits in SDS gel, and the gene sequence revealed that this enzyme belongs to the molybdopterin binding oxidoreductase family containing molybdopterins, FAD, and iron-sulfur clusters.