Production of biofuels from C1-gases with Clostridium and related bacteria—Recent advances

Clostridium spp. are suitable for the bioconversion of C₁-gases (e.g., CO₂, CO and syngas) into different bioproducts. These products can be used as biofuels and are reviewed here, focusing on ethanol, butanol and hexanol, mainly. The production of higher alcohols (e.g., butanol and hexanol) has hardly been reviewed. Parameters affecting the optimization of the bioconversion process and bioreactor performance are addressed as well as the pathways involved in these bioconversions. New aspects, such as mixotrophy and sugar versus gas fermentation, are also reviewed. In addition, Clostridia can also produce higher alcohols from the integration of the Wood-Ljungdahl pathway and the reverse ß-oxidation pathway, which has also not yet been comprehensively reviewed. In the latter process, the acetogen uses the reducing power of CO/syngas to reduce C₄ or C₆ fatty acids, previously produced by a chain elongating microorganism (commonly Clostridium kluyveri), into the corresponding bioalcohol.     

Mevalonate production by engineered acetogen biocatalyst during continuous fermentation of syngas or CO2/H2 blend

Naturally mevalonate-resistant acetogen Clostridium sp. MT1243 produced only 425 mM acetate during syngas fermentation. Using Clostridium sp. MT1243 we engineered biocatalyst selectively producing mevalonate from synthesis gas or CO₂/H₂ blend. Acetate production and spore formation were eliminated from Clostridium sp. MT1243 using Cre-lox66/lox71-system. Cell energy released via elimination of phosphotransacetylase, acetate kinase and early stage sporulation genes powered mevalonate accumulation in fermentation broth due to expression of synthetic thiolase, HMG-synthase, and HMG-reductase, three copies of each, integrated using Tn7-approach. Recombinants produced 145 mM mevalonate in five independent single-step fermentation runs 25 days each in five repeats using syngas blend 60 % CO and 40 % H₂ (v/v) (p < 0.005). Mevalonate production was 97 mM if only CO₂/H₂ blend was fed instead of syngas (p < 0.005). Mevalonate from CO₂/H₂ blend might serve as a commercial route to mitigate global warming in proportion to CO₂ fermentation scale worldwide.     

Intracellular metabolite profiling and the evaluation of metabolite extraction solvents for Clostridium carboxidivorans fermenting carbon monoxide

Clostridium carboxidivorans ferments CO, CO₂, and H₂ via the Wood-Ljungdahl pathway. CO, CO_2, and H₂ are unique substrates, unlike other carbon sources like glucose, so it is necessary to analyze intracellular metabolite profiles for gas fermentation by C. carboxidivorans for metabolic engineering. Moreover, it is necessary to optimize the metabolite extraction solvent specifically for C. carboxidivorans fermenting syngas. In comparison with glucose media, the gas media allowed significant abundance changes of 38 and 34 metabolites in the exponential and stationary phases, respectively. Especially, C. carboxidivorans cultivated in the gas media showed changes of fatty acid metabolism and higher levels of intracellular fatty acid synthesis possibly due to cofactor imbalance and slow metabolism. Meanwhile, the evaluation of extraction solvents revealed the mixture of water-isopropanol-methanol (2:2:5, v/v/v) to be the best extraction solvent, which showed a higher extraction capability and reproducibility than pure methanol, the conventional extraction solvent. This is the first metabolomic study to demonstrate the unique intracellular metabolite profiles of the gas fermentation compared to glucose fermentation, and to evaluate water-isopropanol-methanol as the optimal metabolite extraction solvent for C. carboxidivorans on gas fermentation.     

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.     

In silico metabolic engineering of Clostridium ljungdahlii for synthesis gas fermentation

Synthesis gas fermentation is one of the most promising routes to convert synthesis gas (syngas; mainly comprised of H₂ and CO) to renewable liquid fuels and chemicals by specialized bacteria. The most commonly studied syngas fermenting bacterium is Clostridium ljungdahlii, which produces acetate and ethanol as its primary metabolic byproducts. Engineering of C. ljungdahlii metabolism to overproduce ethanol, enhance the synthesize of the native byproducts lactate and 2,3-butanediol, and introduce the synthesis of non-native products such as butanol and butyrate has substantial commercial value. We performed in silico metabolic engineering studies using a genome-scale reconstruction of C. ljungdahlii metabolism and the OptKnock computational framework to identify gene knockouts that were predicted to enhance the synthesis of these native products and non-native products, introduced through insertion of the necessary heterologous pathways. The OptKnock derived strategies were often difficult to assess because increase product synthesis was invariably accompanied by decreased growth. Therefore, the OptKnock strategies were further evaluated using a spatiotemporal metabolic model of a syngas bubble column reactor, a popular technology for large-scale gas fermentation. Unlike flux balance analysis, the bubble column model accounted for the complex tradeoffs between increased product synthesis and reduced growth rates of engineered mutants within the spatially varying column environment. The two-stage methodology for deriving and evaluating metabolic engineering strategies was shown to yield new C. ljungdahlii gene targets that offer the potential for increased product synthesis under realistic syngas fermentation conditions.     

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.     

Genomic Analysis of Carbon Monoxide Utilization and Butanol Production by Clostridium carboxidivorans Strain P7T

Increasing demand for the production of renewable fuels has recently generated a particular interest in microbial production of butanol. Anaerobic bacteria, such as Clostridium spp., can naturally convert carbohydrates into a variety of primary products, including alcohols like butanol. The genetics of microorganisms like Clostridium acetobutylicum have been well studied and their solvent-producing metabolic pathways characterized. In contrast, less is known about the genetics of Clostridium spp. capable of converting syngas or its individual components into solvents. In this study, the type of strain of a new solventogenic Clostridium species, C. carboxidivorans, was genetically characterized by genome sequencing. C. carboxidivorans strain P7^T possessed a complete Wood-Ljungdahl pathway gene cluster, involving CO and CO₂ fixation and conversion to acetyl-CoA. Moreover, with the exception of an acetone production pathway, all the genetic determinants of canonical ABE metabolic pathways for acetate, butyrate, ethanol and butanol production were present in the P7^T chromosome. The functionality of these pathways was also confirmed by growth of P7^T on CO and production of CO₂ as well as volatile fatty acids (acetate and butyrate) and solvents (ethanol and butanol). P7^T was also found to harbour a 19 Kbp plasmid, which did not include essential or butanol production related genes. This study has generated in depth knowledge of the P7^T genome, which will be helpful in developing metabolic engineering strategies to improve C. carboxidivorans''s natural capacity to produce potential biofuels from syngas.     

Energy Generation from the CO Oxidation-Hydrogen Production Pathway in Rubrivivax gelatinosus

When incubated in the presence of CO gas, Rubrivivax gelatinosus CBS induces a CO oxidation-H₂ production pathway according to the stoichiometry CO + H₂O → CO₂ + H₂. Once induced, this pathway proceeds equally well in both light and darkness. When light is not present, CO can serve as the sole carbon source, supporting cell growth anaerobically with a cell doubling time of nearly 2 days. This observation suggests that the CO oxidation reaction yields energy. Indeed, new ATP synthesis was detected in darkness following CO additions to the gas phase of the culture, in contrast to the case for a control that received an inert gas such as argon. When the CO-to-H₂ activity was determined in the presence of the electron transport uncoupler carbonyl-cyanide m-chlorophenylhydrazone (CCCP), the rate of H₂ production from CO oxidation was enhanced nearly 40% compared to that of the control. Upon the addition of the ATP synthase inhibitor N,N′-dicyclohexylcarbodiimide (DCCD), we observed an inhibition of H₂ production from CO oxidation which could be reversed upon the addition of CCCP. Collectively, these data strongly suggest that the CO-to-H₂ reaction yields ATP driven by a transmembrane proton gradient, but the detailed mechanism of this reaction is not yet known. These findings encourage additional research aimed at long-term H₂ production from gas streams containing CO.     

Hydrogen photo-evolution upon S deprivation stepwise: an illustration of microalgal photosynthetic and metabolic flexibility and a step stone for future biotechnological methods of renewable H2 production

The metabolic flexibility of some photosynthetic microalgae enables them to survive periods of anaerobiosis in the light by developing a particular photofermentative metabolism. The latter entails compounds of the photosynthetic electron transfer chain and an oxygen-sensitive hydrogenase in order to reoxidize reducing equivalents and to generate ATP for maintaining basal metabolic function. This pathway results in the photo-evolution of hydrogen gas by the algae. A decade ago, Melis and coworkers managed to reproduce such a condition in a laboratory context by depletion of sulfur in the algal culture media, making the photo-evolution by the algae sustainable for several days (Melis et al. in Plant Physiol 122:127–136, 2000). This observation boosted research in algal H₂ evolution. A feature, which due to its transient nature was long time considered as a curiosity of algal photosynthesis suddenly became a phenomenon with biotechnological potential. Although the Melis procedure has not been developed into a biotechnological process of renewable H₂ generation so far, it has been a useful tool for studying microalgal metabolic and photosynthetic flexibility and a possible step stone for future H₂ production procedures. Ten years later most of the critical steps and limitations of H₂ production by this protocol have been studied from different angles particularly with the model organism Chlamydomonas reinhardtii, by introducing various changes in culture conditions and making use of mutants issued from different screens or by reverse genomic approaches. A synthesis of these observations with the most important conclusions driven from recent studies will be presented in this review.     

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.     

Improvement of fermentative hydrogen production: various approaches

Fermentation of biomass or carbohydrate-based substrates presents a promising route of biological hydrogen production compared with photosynthetic or chemical routes. Pure substrates, including glucose, starch and cellulose, as well as different organic waste materials can be used for hydrogen fermentation. Among a large number of microbial species, strict anaerobes and facultative anaerobic chemoheterotrophs, such as clostridia and enteric bacteria, are efficient producers of hydrogen. Despite having a higher evolution rate of hydrogen, the yield of hydrogen [mol H₂ (mol substrate^–1)] from fermentative processes is lower than that achieved using other methods; thus, the process is not economically viable in its present form. The pathways and experimental evidence cited in the literature reveal that a maximum of four mol of hydrogen can be obtained from substrates such as glucose. Modifications of the fermentation process, by redirection of metabolic pathways, gas sparging and maintaining a low partial pressure of hydrogen to make the reaction thermodynamically favorable, efficient product removal, optimum bioreactor design and integrating fermentative process with that of photosynthesis, are some of the ways that have been attempted to improve hydrogen productivity. This review briefly describes recent advances in these approaches towards improvement of hydrogen yield by fermentation.     

Arginine deiminase pathway provides ATP and boosts growth of the gas-fermenting acetogen Clostridium autoethanogenum

Acetogens are attractive organisms for the production of chemicals and fuels from inexpensive and non-food feedstocks such as syngas (CO, CO₂ and H₂). Expanding their product spectrum beyond native compounds is dictated by energetics, particularly ATP availability. Acetogens have evolved sophisticated strategies to conserve energy from reduction potential differences between major redox couples, however, this coupling is sensitive to small changes in thermodynamic equilibria. To accelerate the development of strains for energy-intensive products from gases, we used a genome-scale metabolic model (GEM) to explore alternative ATP-generating pathways in the gas-fermenting acetogen Clostridium autoethanogenum. Shadow price analysis revealed a preference of C. autoethanogenum for nine amino acids. This prediction was experimentally confirmed under heterotrophic conditions. Subsequent in silico simulations identified arginine (ARG) as a key enhancer for growth. Predictions were experimentally validated, and faster growth was measured in media containing ARG (t_D~4 h) compared to growth on yeast extract (t_D~9 h). The growth-boosting effect of ARG was confirmed during autotrophic growth. Metabolic modelling and experiments showed that acetate production is nearly abolished and fast growth is realised by a three-fold increase in ATP production through the arginine deiminase (ADI) pathway. The involvement of the ADI pathway was confirmed by metabolomics and RNA-sequencing which revealed a ~500-fold up-regulation of the ADI pathway with an unexpected down-regulation of the Wood-Ljungdahl pathway. The data presented here offer a potential route for supplying cells with ATP, while demonstrating the usefulness of metabolic modelling for the discovery of native pathways for stimulating growth or enhancing energy availability.     

Microbiology of synthesis gas fermentation for biofuel production

A significant portion of biomass sources like straw and wood is poorly degradable and cannot be converted to biofuels by microorganisms. The gasification of this waste material to produce synthesis gas (or syngas) could offer a solution to this problem, as microorganisms that convert CO and H2) (the essential components of syngas) to multicarbon compounds are available. These are predominantly mesophilic microorganisms that produce short-chain fatty acids and alcohols from CO and H2. Additionally, hydrogen can be produced by carboxydotrophic hydrogenogenic bacteria that convert CO and H2O to H2 and CO2. The production of ethanol through syngas fermentation is already available as a commercial process. The use of thermophilic microorganisms for these processes could offer some advantages; however, to date, few thermophiles are known that grow well on syngas and produce organic compounds. The identification of new isolates that would broaden the product range of syngas fermentations is desirable. Metabolic engineering could be employed to broaden the variety of available products, although genetic tools for such engineering are currently unavailable. Nevertheless, syngas fermenting microorganisms possess advantageous characteristics for biofuel production and hold potential for future engineering efforts.     

Biomass–oxygen gasification in a high-temperature entrained-flow gasifier

The technology associated with indirect biomass liquefaction is currently arousing increased attention, as it could ensure a supply of transportation fuels and reduce the use of petroleum. The characteristics of biomass–oxygen gasification in a bench-scale laminar entrained-flow gasifier were studied in the paper. Experiments were carried out to investigate the influence of some key factors, including reaction temperature, residence time and oxygen/biomass ratio, on the gasification. The results indicated that higher temperature favored H₂ and CO production. Cold gas efficiency was improved by > 10% when the temperature was increased from 1000 to 1400 °C. The carbon conversion increased and the syngas quality was improved with increasing residence time. A shorter residence resulted in incomplete gasification. An optimal residence time of 1.6 s was identified in this study. The introduction of oxygen to the gasifier strengthened the gasification and improved the carbon conversion, but lowered the lower heating value and the H₂/CO ratio of the syngas. The optimal oxygen/biomass ratio in this study was 0.4. The results of this study will help to improve our understanding of syngas production by biomass high-temperature gasification.     

Anthrahydroquinone-2,6,-disulfonate (AH<Subscript>2</Subscript>QDS) increases hydrogen molar yield and xylose utilization in growing cultures of <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

H₂ production and xylose utilization were investigated using the fermentative culture Clostridium beijerinckii NCIMB 8052. Adding anthrahydroquinone-2,6-disulfonate (AH₂QDS) increased the extent of xylose utilization by 56% and hydrogen molar yield by 24–37%. Enhanced hydrogen molar yield correlated with increased xylose utilization and increases in the acetate/butyrate product ratio. An electron balance indicated that AH₂QDS shifted the electrons from the butyric acid pathway (NADH-dependent pathway) to the acetic acid pathway (non-NADH-dependent pathway), putatively creating a surplus of reducing equivalents that were then available for hydrogen production. These data demonstrate that hydrogen yield and xylose utilization can be manipulated by amending redox active molecules into growing cultures. This will impact biohydrogen/biofuel production by allowing physiological manipulations of growing cells for increased (or decreased) output of selected metabolites using amendments that are not consumed during the reactions. Although the current yield increases are small, they suggest a target for cellular alterations. In addition, increased xylose utilization will be critical to the fermentation of pretreated lignocellulosic feedstocks, which may have higher xylose content.     

Process simulation of single-step dimethyl ether production via biomass gasification

In this study, we simulated the single-step process of dimethyl ether (DME) synthesis via biomass gasification using ASPEN Plus™. The whole process comprised four parts: gasification, water gas shift reaction, gas purification, and single-step DME synthesis. We analyzed the influence of the oxygen/biomass and steam/biomass ratios on biomass gasification and synthesis performance. The syngas H₂/CO ratio after water gas shift process was modulated to 1, and the syngas was then purified to remove H₂S and CO₂, using the Rectisol process. Syngas still contained trace amounts of H₂S and about 3% CO₂ after purification, which satisfied the synthesis demands. However, the high level of cold energy consumption was a problem during the purification process. The DME yield in this study was 0.37, assuming that the DME selectivity was 0.91 and that CO was totally converted. We performed environmental and economic analyses, and propose the development of a poly-generation process based on economic considerations.     

The Interplay of Proton,Electron, and Metabolite Supply for Photosynthetic H2 Production in Chlamydomonas reinhardtii

To obtain a detailed picture of sulfur deprivation-induced H₂ production in microalgae, metabolome analyses were performed during key time points of the anaerobic H₂ production process of Chlamydomonas reinhardtii. Analyses were performed using gas chromatography coupled to mass spectrometry (GC/MS), two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GCxGC-TOFMS), lipid and starch analysis, and enzymatic determination of fermentative products. The studies were designed to provide a detailed metabolite profile of the solar Bio-H₂ production process. This work reports on the differential analysis of metabolic profiles of the high H₂-producing strain Stm6Glc4 and the wild-type cc406 (WT) before and during the H₂ production phase. Using GCxGC-TOFMS analysis the number of detected peaks increased from 128 peaks, previously detected by GC/MS techniques, to ∼1168. More detailed analysis of the anaerobic H₂ production phase revealed remarkable differences between wild-type and mutant cells in a number of metabolic pathways. Under these physiological conditions the WT produced up to 2.6 times more fatty acids, 2.2 times more neutral lipids, and up to 4 times more fermentation products compared with Stm6Glc4. Based on these results, specific metabolic pathways involving the synthesis of fatty acids, neutral lipids, and fermentation products during anaerobiosis in C. reinhardtii have been identified as potential targets for metabolic engineering to further enhance substrate supply for the hydrogenase(s) in the chloroplast.     

Physiological response of Clostridium carboxidivorans during conversion of synthesis gas to solvents in a gas-fed bioreactor

Clostridium carboxidivorans P7 is one of three microbial catalysts capable of fermenting synthesis gas (mainly CO, CO₂, and H₂) to produce the liquid biofuels ethanol and butanol. Gasification of feedstocks to produce synthesis gas (syngas), followed by microbial conversion to solvents, greatly expands the diversity of suitable feedstocks that can be used for biofuel production beyond commonly used food and energy crops to include agricultural, industrial, and municipal waste streams. C. carboxidivorans P7 uses a variation of the classic Wood–Ljungdahl pathway, identified through genome sequence‐enabled approaches but only limited direct metabolic analyses. As a result, little is known about gene expression and enzyme activities during solvent production. In this study, we measured cell growth, gene expression, enzyme activity, and product formation in autotrophic batch cultures continuously fed a synthetic syngas mixture. These cultures exhibited an initial phase of growth, followed by acidogenesis that resulted in a reduction in pH. After cessation of growth, solventogenesis occurred, pH increased and maximum concentrations of acetate (41 mM), butyrate (1.4 mM), ethanol (61 mM), and butanol (7.1 mM) were achieved. Enzyme activities were highest during the growth phase, but expression of carbon monoxide dehydrogenase (CODH), Fe‐only hydrogenases and two tandem bi‐functional acetaldehyde/alcohol dehydrogenases were highest during specific stages of solventogenesis. Several amino acid substitutions between the tandem acetaldehyde/alcohol dehydrogenases and the differential expression of their genes suggest that they may have different roles during solvent formation. The data presented here provide a link between the expression of key enzymes, their measured activities and solvent production by C. carboxidivorans P7. This research also identifies potential targets for metabolic engineering efforts designed to produce higher amounts of ethanol or butanol from syngas. Biotechnol. Bioeng. 2012; 109: 2720–2728. © 2012 Wiley Periodicals, Inc.     

Carbon monoxide gasing leads to alcohol production and butyrate uptake without acetone formation in continuous cultures ofClostridium acetobutylicum

Summary When continuous, steady-state, glucose-limited cultures ofClostridium acetobutylicum were sparged with CO, the completely or almost completely acidogenic fermentations became solventogenic. Alcohol (butanol and ethanol) and lactate production at very high specific production rates were initiated and sustained without acetone, and little or no acetate and butyrate formation. In one fermentation, strong butyrate uptake without acetone formation was observed. Growth could be sustained even with 100% inhibition of H₂ formation. Although CO gasing inhibited growth up to 50%, and H₂ formation up to 100%, it enhanced the rate of glucose uptake up to 300%. TheY _ATP was strongly affected and mostly reduced with respect to its steady-state value. The results support the hypothesis that solvent formation is triggered by an altered electron flow.