Incorporating hydrodynamics into spatiotemporal metabolic models of bubble column gas fermentation

Gas fermentation has emerged as a technologically and economically attractive option for producing renewable fuels and chemicals from carbon monoxide (CO) rich waste streams. LanzaTech has developed a proprietary strain of the gas fermentating acetogen Clostridium autoethanogenum as a microbial platform for synthesizing ethanol, 2,3-butanediol, and other chemicals. Bubble column reactor technology is being developed for the large-scale production, motivating the investigation of multiphase reactor hydrodynamics. In this study, we combined hydrodynamics with a genome-scale reconstruction of C. autoethanogenum metabolism and multiphase convection–dispersion equations to compare the performance of bubble column reactors with and without liquid recycle. For both reactor configurations, hydrodynamics was predicted to diminish bubble column performance with respect to CO conversion, biomass production, and ethanol production when compared with bubble column models in which the gas phase was modeled as ideal plug flow plus axial dispersion. Liquid recycle was predicted to be advantageous by increasing CO conversion, biomass production, and ethanol and 2,3-butanediol production compared with the non-recycle reactor configuration. Parametric studies performed for the liquid recycle configuration with two-phase hydrodynamics showed that increased CO feed flow rates (more gas supply), smaller CO gas bubbles (more gas–liquid mass transfer), and shorter column heights (more gas per volume of liquid per time) favored ethanol production over acetate production. Our computational results demonstrate the power of combining cellular metabolic models and two-phase hydrodynamics for simulating and optimizing gas fermentation reactors.     

The role of ethanol oxidation during carboxydotrophic growth of Clostridium autoethanogenum

The Wood–Ljungdahl pathway is an ancient metabolic route used by acetogenic carboxydotrophs to convert CO into acetate, and some cases ethanol. When produced, ethanol is generally seen as an end product of acetogenic metabolism, but here we show that it acts as an important intermediate and co-substrate during carboxydotrophic growth of Clostridium autoethanogenum. Depending on CO availability, C. autoethanogenum is able to rapidly switch between ethanol production and utilization, hereby optimizing its carboxydotrophic growth. The importance of the aldehyde ferredoxin:oxidoreductase (AOR) route for ethanol production in carboxydotrophic acetogens is known; however, the role of the bifunctional alcohol dehydrogenase AdhE (Ald–Adh) route in ethanol metabolism remains largely unclear. We show that the mutant strain C. autoethanogenum ∆adhE1a, lacking the Ald subunit of the main bifunctional aldehyde/alcohol dehydrogenase (AdhE, CAETHG_3747), has poor ethanol oxidation capabilities, with a negative impact on biomass yield. This indicates that the Adh–Ald route plays a major role in ethanol oxidation during carboxydotrophic growth, enabling subsequent energy conservation via substrate-level phosphorylation using acetate kinase. Subsequent chemostat experiments with C. autoethanogenum show that the wild type, in contrast to ∆adhE1a, is more resilient to sudden changes in CO supply and utilizes ethanol as a temporary storage for reduction equivalents and energy during CO-abundant conditions, reserving these ‘stored assets’ for more CO-limited conditions. This shows that the direction of the ethanol metabolism is very dynamic during carboxydotrophic acetogenesis and opens new insights in the central metabolism of C. autoethanogenum and similar acetogens.     

Online measurement of dissolved carbon monoxide concentrations reveals critical operating conditions in gas fermentation experiments

Syngas fermentation is one possible contributor to the reduction of greenhouse gas emissions. The conversion of industrial waste gas streams containing CO or H₂, which are usually combusted, directly reduces the emission of CO₂ into the atmosphere. Additionally, other carbon‐containing waste streams can be gasified, making them accessible for microbial conversion into platform chemicals. However, there is still a lack of detailed process understanding, as online monitoring of dissolved gas concentrations is currently not possible. Several studies have demonstrated growth inhibition of Clostridium ljungdahlii at high CO concentrations in the headspace. However, growth is not inhibited by the CO concentration in the headspace, but by the dissolved carbon monoxide tension (DCOT). The DCOT depends on the CO concentration in the headspace, CO transfer rate, and biomass concentration. Hence, the measurement of the DCOT is a superior method to investigate the toxic effects of CO on microbial fermentation. Since CO is a component of syngas, a detailed understanding is crucial. In this study, a newly developed measurement setup is presented that allows sterile online measurement of the DCOT. In an abiotic experiment, the functionality of the measurement principle was demonstrated for various CO concentrations in the gas supply (0%–40%) and various agitation rates (300–1100 min^?1). In continuous stirred tank reactor fermentation experiments, the measurement showed reliable results. The production of ethanol and 2,3‐butanediol increased with increasing DCOT. Moreover, a critical DCOT was identified, leading to the inhibition of the culture. Thus, the reported online measurement method is beneficial for process understanding. In future processes, it can be used for closed‐loop fermentation control.     

Influence of process parameters on growth of Clostridium ljungdahlii and Clostridium autoethanogenum on synthesis gas

Effects of initial medium pH and gas flow rate on Clostridium ljungdahlii and Clostridium autoethanogenum in liquid batch, continuous gas fermentations were investigated. Synthesis gas components were supplied at varying flow rates (5, 7.5 and 10 mL/min) for C. ljungdahlii (pH 6.8 and 5.5) and C. autoethanogenum (pH 6.0). Growth on synthesis gas was slower than growth on sugars. For C. ljungdahlii, higher cell densities were achieved at pH 6.8 (579 mg/L) compared to pH 5.5 (378 mg/L). The ethanol concentration at pH 6.8 was also 110% greater than that at pH 5.5. The interaction of flow rate and pH was statistically significant with the greatest acetate production in the 10 mL/min, pH 6.8 treatment. The ethanol to acetate ratios were smaller at lower pH levels and higher flow rates. In C. autoethanogenum fermentations, higher flow rates resulted in greater end product formation with no significant effect on product ratios.     

Ethanol and acetate production by <Emphasis Type="Italic">Clostridium ljungdahlii</Emphasis> and <Emphasis Type="Italic">Clostridium autoethanogenum</Emphasis> using resting cells

Combined gasification and fermentation technologies can potentially produce biofuels from renewable biomass. Gasification generates synthesis gas consisting primarily of CO, CO₂, H₂, N₂, with smaller amounts of CH₄, NO_x, O₂, C₂ compounds, ash and tars. Several anaerobic bacteria species can ferment bottled mixtures of pure synthesis gas constituents. However, there are challenges to maintaining culture viability of synthesis gas exposed cells. This study was designed to enhance culture stability and improve ethanol-to-acetate ratios using resting (non-growing) cells in synthesis gas fermentation. Resting cell states were induced in autotrophic Clostridium ljungdahlii cultures with minimal ethanol and acetate production due to low metabolic activity compared to growing cell production levels of 5.2 and 40.1 mM of ethanol and acetate. Clostridium autoethanogenum cultures were not induced into true resting states but did show improvement in total ethanol production (from 5.1 mM in growing cultures to 9.4 in one nitrogen-limited medium) as well as increased shifts in ethanol-to-acetate production ratios.     

Dynamic modeling of syngas fermentation in a continuous stirred-tank reactor: Multi-response parameter estimation and process optimization

Syngas fermentation is one of the bets for the future sustainable biobased economies due to its potential as an intermediate step in the conversion of waste carbon to ethanol fuel and other chemicals. Integrated with gasification and suitable downstream processing, it may constitute an efficient and competitive route for the valorization of various waste materials, especially if systems engineering principles are employed targeting process optimization. In this study, a dynamic multi-response model is presented for syngas fermentation with acetogenic bacteria in a continuous stirred-tank reactor, accounting for gas–liquid mass transfer, substrate (CO, H₂) uptake, biomass growth and death, acetic acid reassimilation, and product selectivity. The unknown parameters were estimated from literature data using the maximum likelihood principle with a multi-response nonlinear modeling framework and metaheuristic optimization, and model adequacy was verified with statistical analysis via generation of confidence intervals as well as parameter significance tests. The model was then used to study the effects of process conditions (gas composition, dilution rate, gas flow rates, and cell recycle) as well as the sensitivity of kinetic parameters, and multiobjective genetic algorithm was used to maximize ethanol productivity and CO conversion. It was observed that these two objectives were clearly conflicting when CO-rich gas was used, but increasing the content of H₂ favored higher productivities while maintaining 100% CO conversion. The maximum productivity predicted with full conversion was 2 g·L⁻¹·hr⁻¹ with a feed gas composition of 54% CO and 46% H₂ and a dilution rate of 0.06 hr⁻¹ with roughly 90% of cell recycle.     

Switching the mode of sucrose utilization by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Overflow metabolism is an undesirable characteristic of aerobic cultures of Saccharomyces cerevisiae during biomass-directed processes. It results from elevated sugar consumption rates that cause a high substrate conversion to ethanol and other bi-products, severely affecting cell physiology, bioprocess performance, and biomass yields. Fed-batch culture, where sucrose consumption rates are controlled by the external addition of sugar aiming at its low concentrations in the fermentor, is the classical bioprocessing alternative to prevent sugar fermentation by yeasts. However, fed-batch fermentations present drawbacks that could be overcome by simpler batch cultures at relatively high (e.g. 20 g/L) initial sugar concentrations. In this study, a S. cerevisiae strain lacking invertase activity was engineered to transport sucrose into the cells through a low-affinity and low-capacity sucrose-H⁺ symport activity, and the growth kinetics and biomass yields on sucrose analyzed using simple batch cultures.     

Genome-wide identification of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> genes required for tolerance to acetic acid

Background  

Acetic acid is a byproduct of Saccharomyces cerevisiae alcoholic fermentation. Together with high concentrations of ethanol and other toxic metabolites, acetic acid may contribute to fermentation arrest and reduced ethanol productivity. This weak acid is also a present in lignocellulosic hydrolysates, a highly interesting non-feedstock substrate in industrial biotechnology. Therefore, the better understanding of the molecular mechanisms underlying S. cerevisiae tolerance to acetic acid is essential for the rational selection of optimal fermentation conditions and the engineering of more robust industrial strains to be used in processes in which yeast is explored as cell factory.     

Microbial tolerance to alcohols: role of the cell membrane

The rate of fermentative production of ethanol declines progressively as ethanol accumulates, increasing the time required to complete substrate conversion and limiting the final concentrations which are achieved. In commercial ethanol production, both these factors directly affect the size of the capital investment required for a given plant capacity and the cost of substrate conversion. Despite centuries of experience in ethanol production, we are only just beginning to understand the mechanisms of ethanol inhibition of fermentation. A basic understanding of ethanol actions on cellular metabolism and fermentation offers the potential for developing improved organisms for fermentation.     

Engineering industrial <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains for xylose fermentation and comparison for switchgrass conversion

Saccharomyces’ physiology and fermentation-related properties vary broadly among industrial strains used to ferment glucose. How genetic background affects xylose metabolism in recombinant Saccharomyces strains has not been adequately explored. In this study, six industrial strains of varied genetic background were engineered to ferment xylose by stable integration of the xylose reductase, xylitol dehydrogenase, and xylulokinase genes. Aerobic growth rates on xylose were 0.04–0.17 h⁻¹. Fermentation of xylose and glucose/xylose mixtures also showed a wide range of performance between strains. During xylose fermentation, xylose consumption rates were 0.17–0.31 g/l/h, with ethanol yields 0.18–0.27 g/g. Yields of ethanol and the metabolite xylitol were positively correlated, indicating that all of the strains had downstream limitations to xylose metabolism. The better-performing engineered and parental strains were compared for conversion of alkaline pretreated switchgrass to ethanol. The engineered strains produced 13–17% more ethanol than the parental control strains because of their ability to ferment xylose.     

Metabolic control of Clostridium thermocellum via inhibition of hydrogenase activity and the glucose transport rate

Clostridium thermocellum has the ability to catabolize cellulosic biomass into ethanol, but acetic acid, lactic acid, carbon dioxide, and hydrogen gas (H₂) are also produced. The effect of hydrogenase inhibitors (H₂, carbon monoxide (CO), and methyl viologen) on product selectivity was investigated. The anticipated effect of these hydrogenase inhibitors was to decrease acetate production. However, shifts to ethanol and lactate production are also observed as a function of cultivation conditions. When the sparge gas of cellobiose-limited chemostat cultures was switched from N₂ to H₂, acetate declined, and ethanol production increased 350%. In resting cell suspensions, lactate increased when H₂ or CO was the inhibitor or when the cells were held at elevated hyperbaric pressure (6.8 atm). In contrast, methyl-viologen-treated resting cells produced twice as much ethanol as the other treatments. The relationship of chemostat physiology to methyl viologen inhibition was revealed by glucose transport experiments, in which methyl viologen decreased the rate of glucose transport by 90%. C. thermocellum produces NAD⁺ from NADH by H₂, lactate, and ethanol production. When the hydrogenases were inhibited, the latter two products increased. However, excess substrate availability causes fructose 1,6-diphosphate, the glycolytic intermediate that triggers lactate production, to increase. Compensatory ethanol production was observed when the chemostat fluid dilution rate or methyl viologen decreased substrate transport. This research highlights the complex effects of high concentrations of dissolved gases in fermentation, which are increasingly envisioned in microbial applications of H₂ production for the conversion of synthetic gases to chemicals.     

Tracing carbon monoxide uptake by Clostridium ljungdahlii during ethanol fermentation using 13C-enrichment technique

Conversion of synthesis gas (CO and H₂) to ethanol can be an alternative, promising technology to produce biofuels from renewable biomass. To distinguish microbial utilization of carbon source between fructose and synthesis gas CO and to evaluate biological production of ethanol from CO, we adopted the ¹³C-enrichment of the CO substrate and hypothesized that the residual increase in δ¹³C of the cell biomass would reflect the increased contribution of ¹³C-enriched CO. Addition of synthesis gas to live culture medium for ethanol fermentation by Clostridum ljungdahlii increased the microbial growth and ethanol production. Despite the high ¹³C-enrichment in CO (99 atom % ¹³C), however, microbial δ¹³C increased relatively small compared to the microbial growth. The uptake efficiency of CO estimated using the isotope mass balance equation was also very low: 0.0014 % for the low CO and 0.0016 % for the high CO treatment. Furthermore, the fast production of ethanol in the early stage indicated that the presence of sugar in fermentation medium would limit the utilization of CO as a carbon source by C. ljungdahlii.     

Feed rate control in fed‐batch fermentations based on frequency content analysis

A new strategy for controlling substrate feed in the exponential growth phase of aerated fed‐batch fermentations is presented. The challenge in this phase is typically to maximize specific growth rate while avoiding the accumulation of overflow metabolites which can occur at high substrate feed rates. In the new strategy, regular perturbations to the feed rate are applied and the proximity to overflow metabolism is continuously assessed from the frequency spectrum of the dissolved oxygen signal. The power spectral density for the frequency of the external perturbations is used as a control variable in a controller to regulate the substrate feed. The strategy was implemented in an industrial pilot scale fermentation set up and calibrated and verified using an amylase producing Bacillus licheniformis strain. It was shown that a higher biomass yield could be obtained without excessive accumulation of harmful overflow metabolites. The general applicability of the strategy was further demonstrated by implementing the controller in another process using a Bacillus licheniformis strain currently used in industrial production processes. In addition, in this case a higher growth rate and decreased accumulation of overflow metabolites in the exponential growth phase was achieved in comparison to the reference controller. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:817–824, 2013     

Fed-batch xylitol production with recombinantXYL-1-expressingSaccharomyees cerevisiae using ethanol as a co-substrate

The bioconversion of xylose into xylitol in fed-batch fermentation with a recombinantSaccharomyces cerevisiae strain, transformed with the xylose-reductase gene ofPichia stipitis, was studied. When only xylose was fed into the fermentor, the production of xylitol continued until the ethanol that had been produced during an initial growth phase on glucose, was depleted. It was concluded that ethanol acted as a redox-balance-retaining co-substrate. The conversion of high amounts of xylose into xylitol required the addition of ethanol to the feed solution. Under O₂-limited conditions, acetic acid accumulated in the fermentation broth, causing poisoning of the yeast at low extracellular pH. Acetic acid toxicity could be avoided by either increasing the pH from 4.5 to 6.5 or by more effective aeration, leading to the further metabolism of acetic acid into cell mass. The best xylitol/ethanol yield, 2.4 gg^–1 was achieved under O₂-limited conditions. Under anaerobic conditions ethanol could not be used as a co-substrate, because the cell cannot produce ATP for maintenance requirements from ethanol anaerobically. The specific rate of xylitol production decreased with increasing aeration. The initial volumetric productivity increased when xylose was added in portions rather than by continuous feeding, due to a more complete saturation of the transport system and the xylose reductase enzyme.     

Uncoupling glucose sensing from GAL metabolism for heterologous lactose fermentation in Saccharomyces cerevisiae

Objectives

Development of a system for direct lactose to ethanol fermentation provides a market for the massive amounts of underutilized whey permeate made by the dairy industry. For this system, glucose and galactose metabolism were uncoupled in Saccharomyces cerevisiae by deleting two negative regulatory genes, GAL80 and MIG1, and introducing the essential lactose hydrolase LAC4 and lactose transporter LAC12, from the native but inefficient lactose fermenting yeast Kluyveromyces marxianus.

Results

Previously, integration of the LAC4 and LAC12 genes into the MIG1 and NTH1 loci was achieved to construct strain AY-51024M. Low rates of lactose conversion led us to generate the Δmig1Δgal80 diploid mutant strain AY-GM from AY-5, which exhibited loss of diauxic growth and glucose repression, subsequently taking up galactose for consumption at a significantly higher rate and yielding higher ethanol concentrations than strain AY-51024M. Similarly, in cheese whey permeate powder solution (CWPS) during three, repeated, batch processes in a 5L bioreactor containing either 100 g/L or 150 g/L lactose, the lactose uptake and ethanol productivity rates were both significantly greater than that of AY-51024M, while the overall fermentation times were considerably lower.

Conclusions

Using the Cre-loxp system for deletion of the MIG1 and GAL80 genes to relieve glucose repression, and LAC4 and LAC12 overexpression to increase lactose uptake and conversion provides an efficient basis for yeast fermentation of whey permeate by-product into ethanol.

     

Ethanol and acetate synthesis from waste gas using batch culture of Clostridium ljungdahlii

Single inorganic carbon source was used for production of chemicals and fuels via fermentation processes. Clostridium ljungdahlii, a strictly anaerobic autotrophic bacterium, was grown on synthesis gas to produce acetate and ethanol from gaseous substrates. C. ljungdahlii was grown on a various concentrations of carbon monoxide with synthesis gas total pressures of 0.8–1.8 atm with an interval of 0.2 atm. The cell and product yields were 0.015 g cell/g CO and 0.41 g acetate/g CO, respectively. Formation of acetate was steady and the production trend was about the same for all of the gases initial pressure and at constant cell density. The ethanol concentration was enhanced by the initial presence of hydrogen and carbon dioxide in the liquid phase. There was no substrate inhibition while C. ljungdahlii was grown in the batch fermentation, even at high system pressure of 1.6 and 1.8 atm. A desired product molar ratio of ethanol:acetate (5:1) was achieved with total gas pressure of 1.6 and 1.8 atm.     

Application of acetate buffer in pH adjustment of sorghum mash and its influence on fuel ethanol fermentation

A 2 M sodium acetate buffer at pH 4.2 was tried to simplify the step of pH adjustment in a laboratory dry-grind procedure. Ethanol yields or conversion efficiencies of 18 sorghum hybrids improved significantly with 2.0–5.9% (3.9% on average) of relative increases when the method of pH adjustment changed from traditional HCl to the acetate buffer. Ethanol yields obtained using the two methods were highly correlated (R ² = 0.96, P < 0.0001), indicating that the acetate buffer did not influence resolution of the procedure to differentiate sorghum hybrids varying in fermentation quality. Acetate retarded the growth of Saccharomyces cerevisiae, but did not affect the overall fermentation rate. With 41–47 mM of undissociated acetic acid in mash of a sorghum hybrid at pH 4.7, rates of glucose consumption and ethanol production were inhibited during exponential phase but promoted during stationary phase. The maximum growth rate constants (μ _max) were 0.42 and 0.32 h⁻¹ for cells grown in mashes with pH adjusted by HCl and the acetate buffer, respectively. Viable cell counts of yeast in mashes with pH adjusted by the acetate buffer were 36% lower than those in mashes adjusted by HCl during stationary phase. Coupled to a 5.3% relative increase in ethanol, a 43.6% relative decrease in glycerol was observed, when the acetate buffer was substituted for HCl. Acetate helped to transfer glucose to ethanol more efficiently. The strain tested did not use acetic acid as carbon source. It was suggested that decreased levels of ATP under acetate stress stimulate glycolysis to ethanol formation, increasing its yield at the expense of biomass and glycerol production. Names are necessary to report factually on available data; however, the U.S. Department of Agriculture neither guarantees nor warrants the standard of the product, and use of the name by the U.S. Department of Agriculture implies no approval of the product to the exclusion of others that may also be suitable.     

Electron availability in CO2, CO and H2 mixtures constrains flux distribution,energy management and product formation in Clostridium ljungdahlii

Acetogens such as Clostridium ljungdahlii can play a crucial role reducing the human CO₂ footprint by converting industrial emissions containing CO₂, CO and H₂ into valuable products such as organic acids or alcohols. The quantitative understanding of cellular metabolism is a prerequisite to exploit the bacterial endowments and to fine-tune the cells by applying metabolic engineering tools. Studying the three gas mixtures CO₂ + H₂, CO and CO + CO₂ + H₂ (syngas) by continuously gassed batch cultivation experiments and applying flux balance analysis, we identified CO as the preferred carbon and electron source for growth and producing alcohols. However, the total yield of moles of carbon (mol-C) per electrons consumed was almost identical in all setups which underlines electron availability as the main factor influencing product formation. The Wood–Ljungdahl pathway (WLP) showed high flexibility by serving as the key NAD⁺ provider for CO₂ + H_2, whereas this function was strongly compensated by the transhydrogenase-like Nfn complex when CO was metabolized. Availability of reduced ferredoxin (Fd_red) can be considered as a key determinant of metabolic control. Oxidation of CO via carbon monoxide dehydrogenase (CODH) is the main route of Fd_red formation when CO is used as substrate, whereas Fd_red is mainly regenerated via the methyl branch of WLP and the Nfn complex utilizing CO₂ + H₂. Consequently, doubled growth rates, highest ATP formation rates and highest amounts of reduced products (ethanol, 2,3-butanediol) were observed when CO was the sole carbon and electron source.     

Influence of iron,phosphate and methyl viologen on glycerol fermentation of Clostridium butyricum

 The effect of methyl viologen addition, and iron and phosphate limitation on product distribution during glycerol fermentation of Clostridium butyricum DSM 5431 was investigated in continuous culture. Special attention was paid to the gaseous products H₂ and CO₂, which were measured on-line. In all three cases, an increased yield of 1,3-propanediol linked to a decreased hydrogen release was observed, indicating that a higher proportion of electrons was channelled from reduced ferredoxin towards NADH₂ production. The specific substrate consumption rates and the specific production rates revealed that this increase in propanediol yield was not obtained at the expense of glycolysis products but by an increased substrate conversion (overflow metabolism). The acetate/ butyrate ratio during glycerol fermentation was essentially influenced by the availability of iron. It was substantially increased when the culture turned from iron excess to iron-limited conditions. Therefore iron limitation proved to be a suitable means to achieve high 1,3-propanediol yields and to reduce butyrate formation. Received: 29 August 1995 / Accepted: 20 September 1995