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Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG [Abstract] Volatile fatty acids (VFAs) can be considered as low-cost carbon substrates for lipid accumulation by oleaginous yeasts. This study demonstrates that a common mixture of VFAs, typically obtained from the anaerobic fermentation of C1-gases by some acetogenic bacteria, can be used in a second aerobic fermentation with the yeast Yarrowia lipolytica to obtain lipids as precursors of biodiesel. In the batch experiments, the preference of Yarrowia lipolytica W29 for acetic acid over butyric and caproic acids was demonstrated, with the highest consumption rate reaching 0.664 g/L·h. In the bioreactor experiments, the amount initial biomass inoculated, as well as the initial acid concentration, were found to have a significant influence on the process. Though the lipid content was relatively low, it can be optimized and further improved. Oleic, linoleic and palmitic acids accounted for about 80 % of the fatty acids in the lipids, which makes them suitable for biodiesel. This research is part of a project funded through the Spanish Ministry of Science and Innovation and European FEDER funds (PID2020-117805RB-I00). Funding for Open access publication provided by Universidade da Coruña-CISUG. The manuscript is based on research related to COST action Yeast4Bio. The authors, belonging to the BIOENGIN group, thank Xunta de Galicia for financial support to Competitive Reference Research Groups (ED431C 2021/55) Xunta de Galicia; ED431C 2021/55

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG [Abstract] Developing innovative bioprocess strategies for carbon capture and utilization has been a major focus of research over the past decade as a way of creating a more resilient world. In this study, zero-valent iron (ZVI) was utilized to generate hydrogen, which was then utilized as an electron donor for CO2 reduction by either Clostridium aceticum or Clostridium carboxidivorans. For this bioprocess, an early acidic condition in the reactors facilitated more hydrogen production and thereby more acetic acid accumulation, in contrast to later acidification, elucidating the role of pH in ZVI-CO2 bioconversion. Acetic acid was produced as single end product of the process in both batch bottle studies with no pH control as well as in reactor studies with pH regulation. This is, to the best of our knowledge, the first report on selective pure CO2 bioconversion to acetic acid by clostridia, with ZVI. In order to minimize costs, scrap iron rather than pure ZVI can be used for scaling-up. By lowering the pH to 6.6 for C. aceticum, in reactor studies with 75 g/L ZVI, some ethanol production (125 mg/L) was also observed, besides acetic acid. In reactor studies, a maximum acetic acid concentration of about 2 g/L was obtained at ZVI concentrations of 50 and 75 g/L. Thus, ZVI dosage and medium pH have a major effect on the metabolites generated during CO2 utilization. This research was partly financed by the Spanish Ministry of Science and Innovation Through Project PID2020-117805RB-I00 and European FEDER funds. BB thanks Xunta de Galicia for funding her predoctoral fellowship (ED481A-2020/226) to undertake research at the BIOENGIN group. The BIOENGIN group also thanks Xunta de Galicia for financial support to Competitive Reference Research Groups (ED431C 2021/55). Funding for open access charge provided by Universidade da Coruña/CISUG Xunta de Galicia; ED481A-2020/226 Xunta de Galicia; ED431C 2021/55

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG [Abstract] The capability of four genetically modified Acetobacterium woodii strains for improved production of acetone from CO2 and hydrogen was tested. The acetone biosynthesis pathway was constructed by combining genes from Clostridium acetobutylicum and Clostridium aceticum. Expression of acetone production genes was demonstrated in all strains. In bioreactors with continuous gas supply, all produced acetic acid, acetone, and, surprisingly, isopropanol. The production of isopropanol was caused by an endogenous secondary alcohol dehydrogenase (SADH) activity at low gas-feeding rate. Although high amounts of the natural end product acetic acid of A. woodii were formed,14.5 mM isopropanol and 7.6 mM acetone were also detected, showing that this is a promising approach for the production of new solvents from C1 gases. The highest acetic acid, acetone, and isopropanol production was detected in the recombinant A. woodii [pJIR750_ac1t1] strain, with final concentrations of 438 mM acetic acid, 7.6 mM acetone, and 14.5 mM isopropanol. The engineered strain A. woodii [pJIR750_ac1t1] was found to be the most promising strain for acetone production from a gas mixture of CO2 and H2 and the formation of isopropanol in A. woodii was shown for the first time. Research in Professor Peter Dürre's lab was funded by the ERA-IB7 project OBAC (FKZ 031B0274B). Research in Professor Christian Kennes' lab was also funded by the collaborative European ERA-IB7 project OBAC as well as PCIN2016-148. The authors in the latter laboratory, belonging to the BIOENGIN group, thank Xunta de Galicia for financial support to Competitive Reference Research Groups (ED431C 2021/55). Funding for open access charge is provided by Universidade da Coruña/CISUG Xunta de Galicia; ED431C 2021/55 Alemania. Bundesministerium für Bildung und Forschung (BMBF); 031B0274B

Butanol is a potential renewable fuel. To increase the selectivity for butanol during CO fermentation, exogenous acetic acid and ethanol, exogenous butyric acid or endogenous butyric acid from glucose fermentation have been investigated using CO as reducing power, with a highly enriched

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG [Abstract] The recent unprecedented increase in energy demand has led to a growing interest in emerging alternatives such as the production of microbial lipids with high energy density and environmentally-friendly characteristics. Oleaginous yeasts represent a versatile and attractive tool for the accumulation of such lipids, also known as single cell oils (SCOs), used to manufacture biofuels (e.g., biodiesel, aviation fuel) and bioproducts. This review provides an overview of the most common oleaginous species, analysing the viability of typical feedstocks and their effect on lipid accumulation. The best results in terms of lipid content using glucose, glycerol, lignocellulose, or acetic acid as substrates are 81.4, 70, 68.2 and 73.4% (w/w), respectively. Besides, an analysis of the parameters that can affect lipid production is also presented. For instance, the optimum conditions for lipid accumulation are usually a C/N ratio between 100 and 200, pH between 5 and 6 (being more alkaline if acids are used as substrates) and temperature around 30 °C. Besides, genetic modifications generally allow to increase the lipid yield, even by up to 400%. Finally, some cost analysis is provided for scaling-up, with feedstock costs estimated at 50–80%, followed by fermenter costs, and downstream costs estimated at around 13%. This study was funded by the Spanish Ministry of Science and Innovation and European FEDER funds (PID2020-117805RB-I00). The manuscript is based on research related to COST action Yeast4Bio. RR thanks the Ministry of Science and Innovation for financial support of his doctoral research (E-15-2019-0344365). CFB (ED481A-2020/028) thanks Xunta de Galicia for financing her doctoral research. The authors, belonging to the BIOENGIN group, thank Xunta de Galicia for financial support to Competitive Reference Research Groups (ED431C 2021/55) Xunta de Galicia; ED481A-2020/028 Xunta de Galicia; ED431C 2021/55

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG [Abstract] Acetobacterium woodii was able to produce high concentrations of acetic acid, i.e., > 20 g/L, from CO2, in the presence of H2 as an energy source, which was favoured by maintaining a near-optimal pH of 7.8 in an automated bioreactor. This allows the mitigation of CO2 emissions for their conversion to acetic acid, which was then further used to produce lipids (single cell oils) by Rhodosporidium toruloides in the next assay. The yeast, grown on acetic acid, efficiently accumulated lipids in A. woodii’s medium, and further improved bioconversion would result in a highly promising process. Acetic acid inhibitory studies performed with R. toruloides, at different initial concentrations of the acid, using the fermented broth of A. woodii grown on CO2, showed that the yeast maintained a constant growth rate and substrate consumption rate up to acid concentrations of 15 g/L. Both rates remained roughly constant at higher initial acetic acid concentrations; except for a more extended lag phase observed in batch assays before the yeast entered in its exponential growth phase. This research is part of a project funded through the Spanish Ministry of Science and Innovation and European FEDER funds (PID2020-117805RB-I00). RR thanks that Ministry for financial support of his doctoral contract (E-15-2019-0344365). The authors, belonging to the BIOENGIN group, thank Xunta de Galicia for financial support to Competitive Reference Research Groups (ED431C 2021/55). Funding for open access charge provided by Universidade da Coruña/CISUG Xunta de Galicia; ED431C 2021/55

Clostridium carboxidivorans produces alcohols from C1 gases (CO, CO2), converting them first into fatty acids, and subsequently into alcohols. This research identified conditions that allow to selectively produce either fatty acids or alcohols. The conversion of gases into acids and then into alcohols is catalysed by metalloenzymes, stimulated by specific trace metals. Therefore, different bioreactors were set-up, either with or without addition of tungsten (W) or selenium (Se) and at different pHs. Combining the presence of those trace metals with a low pH (5.0) allowed to accumulate high amounts of alcohols as major end products (8038 mg/L total alcohols; 3027 mg/L total acids). Instead, maintaining a higher pH (6.2), in the absence of those trace metals, allowed to selectively produce organic acids (9577 mg/L) and almost no alcohols (676 mg/L). Omitting W, but not Se, at high pH (6.2), led to a still higher concentration of acids (11303 mg/L).

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG [Abstract] In recent years, the possibility of merging technologies for waste recovery such as those based on syngas fermentation and chain elongation has been studied for the production of medium chain fatty acids (MCFAs) and bioalcohols, in an attempt to integrate the concept of circular economy in the industry. Nevertheless, one of the main issues of this approach is the pH mismatch between acetogens and chain elongating microorganisms. This work reports, for the first time, the suitability of a co-culture of C. aceticum and C. kluyveri metabolizing syngas at near neutral pH in stirred tank bioreactors. For this purpose, bioreactor studies were carried out with continuous syngas supply. In the first experiment, maximum concentrations of 𝑛-butyrate and 𝑛-caproate of 7.0 and 8.2 g/L, respectively, were obtained. In the second experiment, considerable amounts of 𝑛-butanol were produced as a result of the reduction, by C. aceticum, of the carboxylates already formed in the broth. In both experiments, ethanol was used as an exogenous electron agent at some point. Finally, batch bottle assays were performed with a pure culture of C. aceticum grown on CO in presence of n-butyrate to assess and confirm its ability to produce 𝑛-butanol, reaching concentrations up to 951 mg/L, with a 𝑛-butyrate conversion efficiency of 96%, which had never been reported before in this species. Therefore, this work contributes to the state of the art, presenting a novel system for the bioproduction of MCFAs by combining syngas fermentation and chain elongation at near neutral pH, as opposed to the acidic pH range used in all previously reported literature. This research was funded through the Spanish Ministry of Science and Innovation and European FEDER funds (PID2020-117805RB-I00). CFB thanks Xunta de Galicia for her doctoral contract (ED481A-2020/028). The authors, belonging to the BIOENGIN group, thank Xunta de Galicia for financial support to Competitive Reference Research Groups (ED431C 2021/55). Funding for open access charge provided by Universidade da Coruña/CISUG Xunta de Galicia; ED481A-2020/028 Xunta de Galicia; ED431C 2021/55

The influence of the carbon source on the metabolism and growth of Clostridium aceticum was investigated, supplying either CO or fructose as sole carbon source. The acid and solvent production patterns were determined under either autotrophic or heterotrophic conditions, elucidating the effect of pH on the substrate's bioconversion pattern. The highest maximum specific growth rate was observed with CO, under the organism's optimal growth conditions, reaching 0.052 h

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG [Abstract] The main aim of this work was to evaluate the efficiency of producing caproic acid and other volatile fatty acids using a co-digestion between cheese whey and sewage sludge in a continuous reactor. The effect of two different feeding regimes (one and two per day) and three hydraulic retention times (HRT) (15, 10 and 6 days) on the organic acids production were studied. The optimal conditions for the process were 10 days HRT, 2 feeding cycles per day, reaching a maximum degree of acidification of 44%. Under these conditions, the most abundant organic acid was caproic acid. The analysis of the microbial community dynamics in the reactor during the HRT changes revealed a microbiome enriched in organisms involved in caproic acid production. Additionally, the production of polyhydroxyalkanoates using the organic acids stream as feeding was verified in a fed-batch experiment obtaining a copolymer formed by hydroxybutyrate, hydroxyvalerate and hydroxyhexanoate. This study received funding from the European FEDER framework program CTQ2017-88292-R and from the Spanish Ministry of Economy and Competitiveness (MINECO), also by Xunta de Galicia under the grant agreement for Competitive Reference Research Groups (GRC) (ED431C 2017/66). The authors would like to thank the companies EMALCSA S.A., and INNOLACT S.L. for their contribution Xunta de Galicia; ED431C 2017/66

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG [Abstract] Syngas fermentation is a promising technology for bioalcohols production even though some produced volatile fatty acids (VFA) may remain unconsumed in the effluent. The present research explored the production of polyhydroxyalkanoates (PHA) from the remaining VFA of the syngas fermentation effluents in fed-batch bioreactors so that bioalcohols and biopolymers could be obtained in a combined way. In order to perform these PHA accumulation tests, two different syngas fermentation effluents, composed mainly of a mixture of VFA and alcohols, were used as substrate. Those effluents were characterized by different nitrogen availabilities, one being N-rich and the other N-limited. A mixed microbial culture (MMC) with a maximum PHA storage ability of 53.6% was used as inoculum. The microorganisms of the MMC mainly consumed the VFA rather than the alcohols, allowing the latter to accumulate as end product, besides PHA. However, when the N-rich effluent was used as substrate, the consumption rate of alcohols was 20 times higher (0.040 Cmmol-Alcohol Cmmol-X-1 h-1) compared to the N-limited effluent (0.002 Cmmol-Alcohol Cmmol-X-1 h-1). Despite not observing large differences in the maximum amount of accumulated PHA (40.5 – 41.5%), there was a decrease in the PHA content after reaching its maximum peak when the N-rich effluent was used as substrate. This trend was not observed with the N-limited effluent, in which the maximum PHA peak exactly matched with the end of the experiment. The present work has been financially supported by the Spanish Ministry of Science and Innovation and European FEDER funds (project PID2020–117805RB-I00). The authors, belonging to the BIOENGIN group, thank Xunta de Galicia for financial support to Competitive Reference Research Groups (GRC) (ED431C 2021/55). Besides, the authors would also like to acknowledge the support of INNOLACT S. L. Funding for open access charge provide by Universidade da Coruña/CISUG Xunta de Galicia; ED431C 2021/55

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG [Abstract] The nature of microbial populations plays an essential role in the production of volatile fatty acids (VFA) during acidogenesis, the first stage in polyhydroxyalkanoates (PHA) production using mixed cultures. However, the composition of microbial communities is generally affected by substrate alterations. This work aimed to unravel the microbial dynamics in response to a gradual change in the feedstock composition in an acidogenic reactor, with subsequent PHA production. To achieve this, co-digestion of cheese whey and brewery wastewater (BW) was carried out for the production of VFA, in which the ratio of these feedstocks was varied by gradually increasing the proportion of BW from 0 up to 50% of the organic content. Bacteria such as Megasphaera, Bifidobacterium or Caproiciproducens were the most abundant in the first stages of the co-digestion. However, when BW reached 25% of the organic load, new taxa emerged and displaced the former ones; like Selenomonas, Ethanoligenens or an undefined member of the Bacteroidales order. Accordingly, the production of butyric acid dropped from 52 down to 27%, while the production of acetic acid increased from 36 up to 52%. Furthermore, the gradual increase of the BW ratio led to a progressive drop in the degree of acidification, from 72 down to 57%. In a subsequent approach, the VFA-rich streams, obtained from the co-digestion, were used as substrates in PHA accumulation tests. All the tests yielded similar PHA contents, but with slightly different monomeric composition. The overall results confirmed that the microbiome was altered by a gradual change in the feedstock composition and, consequently, the VFA profile and the monomeric composition of the biopolymer also did. The current investigation has been financially supported by both the Spanish Ministry of Science and Innovation and European FEDER funds (PID2020-117805RB-I00 project). The authors, belongin to the BIOENGIN group, thank Xunta de Galicia for financial support to Competitive Reference Research Groups(ED431C 2021/55). B. Lagoa-Costa would also like to thank the financial contribution of INDITEX Group and the technical support of Dr. Ben Allen and Dr. Tom P. Curtis, from Newcastle University, during his internship. Funding for open access charge was provided by Universidade da Coruña/CISUG Xunta de Galicia; ED431C 2021/55

The quantification of the SARS-CoV-2 load in wastewater has emerged as a useful method to monitor COVID-19 outbreaks in the community. This approach was implemented in the metropolitan area of A Coruña (NW Spain), where wastewater from the treatment plant of Bens was analyzed to track the epidemic’s dynamic in a population of 369,098 inhabitants. We developed statistical regression models that allowed us to estimate the number of infected people from the viral load detected in the wastewater with a reliability close to 90%. This is the first wastewater-based epidemiological model that could potentially be adapted to track the evolution of the COVID-19 epidemic anywhere in the world, monitoring both symptomatic and asymptomatic individuals. It can help to understand with a high degree of reliability the true magnitude of the epidemic in a place at any given time and can be used as an effective early warning tool for predicting outbreaks.

The treatment of waste gas (WG) containing dichloromethane (DCM) using advanced oxidation processes (AOPs) [UV and UV-TiO

The present work explored the production of volatile fatty acids through the anaerobic co-digestion of sewage sludge (SS) and cheese whey (CW). Two batch experiments were conducted to evaluate the effect of the substrate mixing ratio (SS%:CW% of total COD of feedstock) and the initial pH on the acidogenic fermentation of SS with CW at different temperatures. The first batch experiment showed that a decrease of the SS proportion in the co-digestion with CW led to a higher degree of acidification observing a synergistic effect at a SS:CW mixing ratio of 25:75 (SS25:CW75). In the second batch experiment, three temperatures (30 °C, 38 °C and 50 °C) and two initial pH (5.5 and 9) were studied at SS60:CW40 and SS25:CW75 substrate mixing ratios. Maximum degrees of acidification of 56% and 73% were achieved, at 50 °C and initial pH of 5.5, for the SS60:CW40 and SS25:CW75 substrate mixing ratios, respectively. Finally, the performance of a semi-continuous reactor was demonstrated at laboratory scale reactor. Different hydraulic retention times (HRT) (10 and 20 days), pH (uncontrolled, 5.5 and 9) and the effect of a thermal pre-treatment of the SS was studied. The maximum degree of acidification in the lab-scale reactor was 45% at 37 °C, HRT of 20 days and pH of 5.5. Under these conditions, the volatile fatty acids (VFA) profile was dominated by butyric and acetic acids.

Agroindustry generates huge amounts of wastes leading to environmental problems in the zones where they are disposed. One of the strategies for the valorization of these wastes is the acidogenic fe...

Financiado para publicación en acceso aberto: Universidade da Coruña/CISUG [Abstract] An anaerobic mixed culture able to grow on pure carbon monoxide (CO) as well as syngas (CO, CO2 and H2), that produced unusual high concentrations of butanol, was enriched in a bioreactor with intermittent CO gas feeding. At pH 6.2, it mainly produced acids, generally acetic and butyric acid. After adaptation, under stress conditions of CO exposure at a partial pressure of 1.8 bar and low pH (e.g., 5.7), the enrichment accumulated ethanol, but also high amounts of butanol, up to 6.8 g/L, never reported before, with a high butanol/butyric acid molar ratio of 12.6, highlighting the high level of acid to alcohol conversion. At the end of the assay, both the acetic acid and ethanol concentrations decreased, with concomitant butyric acid production, suggesting C2 to C4 acid bioconversion, though this was not a dominant bioconversion process. The reverse reaction of ethanol oxidation to acetic acid was observed in the presence of CO2 produced during CO fermentation. Interestingly, butanol oxidation with simultaneous butyric acid production occurred upon production of CO2 from CO, which has to the best of our knowledge never been reported. Although the sludge inoculum contained a few known solventogenic Clostridia, the relative taxonomic abundance of the enriched sludge was diverse in Clostridia and Bacilli classes, containing known solventogens, e.g., Clostridium ljungdhalii, Clostridium ragsdalei and Clostridium coskatii, confirming their efficient enrichment. The relative abundance of unassigned Clostridium species amounted to 27% with presumably novel ethanol/butanol producers. This publication has emanated from research supported by Science Foundation Ireland (SFI) through the SFI Research Professorship Programme entitled Innovative Energy Technologies for Biofuels, Bioenergy and a Sustainable Irish Bioeconomy (IETSBIO3; grant number 15/RP/2763) and the Research Infrastructure research grant Platform for Biofuel Analysis (Grant Number 16/RI/3401). This research was partly funded by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) through project CTQ2017-88292-R and European FEDER funds. The BIOENGIN group thanks Xunta de Galicia for financial support to its Competitive Reference Research Group (ED431C 2021/55). Funding for open access charge provided by Universidade da Coruña/CISUG Irlanda. Science Foundation Ireland; 15/RP/2763 Irlanda. Science Foundation Ireland; 16/RI/3401 Xunta de Galicia; ED431C 2021/55

This research was funded by the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) through project CTQ2017-88292-R and European FEDER funds, and through European ERA-IB7 project OBAC and PCIN2016-148. The authors, belonging to the BIOENGIN group, thank Xunta de Galicia for financial support to Competitive Reference Research Groups (ED431C 2017/66)

Fish canning industries generate a significant amount of solid waste that can be digested anaerobically into volatile fatty acids (VFA). The aim of this research was to study the effect of various pHs, ranging from 5.0 to 10.0, and percentage of total solids on the anaerobic digestion of tuna waste into VFA, both in batch assays and continuous reactor. The production of VFA was affected by pH and was significantly higher under alkaline conditions. At pH 8.0, the VFA production reached 30,611mgCOD/L. The VFA mainly consisted of acetic, propionic, n-butyric and i-valeric acids. Acetic acid was the main product at all the pHs tested. In terms of total solids (TS) the best results were obtained with 2.5% total solids, reaching 0.73gCODVFA/gCODwaste. At higher TS concentrations (5 and 8% TS) lower yields were reached probably due to inhibition at high VFA concentration.

BACKGROUND Syngas and some waste gases are composed mainly of carbon monoxide, carbon dioxide and hydrogen, which can be used by some acetogenic bacteria to produce ethanol, butanol or hexanol, and represents an attractive alternative to the conventional ABE (acetone–butanol–ethanol) fermentation. Experiments were carried out in bioreactors under different conditions (pH 5.75 and 4.75) with continuous supply of a mixture of CO/CO2/H2/N2 converted by Clostridium carboxidivorans into hexanol, butanol and ethanol (H-B-E fermentation). RESULTS Applying different pH control strategies will affect the syngas fermentation pattern, among others in terms of bioconversion rates as well as final concentrations of acids and alcohols. The highest concentrations of alcohols were obtained at pH 5.75, i.e. 2.7 g L−1 ethanol, 1.9 g L−1 butanol and 0.85 g L−1 hexanol, whereas the maximum production rates were observed at pH 4.75, reaching 0.048 g-ethanol h−1 g−1-biomass, 0.037 g-butanol h−1 g−1-biomass, and 0.026 g-hexanol h−1 g−1-biomass. However, a low pH negatively affects growth and acids production in the first metabolic step, with lower growth and acids production at pH 4.75 than 5.75. Growth rates reached 0.0057 h−1 and 0.072 h−1, respectively, at pH 4.75 and 5.75. CONCLUSIONS Maintaining initially a higher pH of 5.75 allows accumulating higher concentrations of acids than when natural acidification takes place. Such higher concentrations of acids allow the production of higher amounts of alcohols as end metabolites, showing the importance of pH on bioconversion and biomass growth. © 2017 Society of Chemical Industry

CO, H2, and CO2 are major components of syngas and some industrial CO-rich waste gases (e.g. waste gases from steel industries), besides some additional minor compounds. It was recently shown that those gases can be bioconverted, by acetogenic/solventogenic bacteria, into ethanol and higher alcohols such as butanol, but also hexanol, through the so-called HBE fermentation. That process presents some advantages over existing chemical conversion processes. This paper reviews HBE fermentation from C1-gases after briefly describing the more conventional ABE (acetone-butanol-ethanol) fermentation from carbohydrates by Clostridium acetobutylicum, in order to allow for comparison of both processes. Although acetone may appear in carbohydrate fermentation, alcohols are the only major end-metabolites in the HBE process with Clostridium carboxidivorans. The few acetogenic bacteria known to metabolize C1-gases and produce butanol or higher alcohols are described. Clostridium carboxidivorans has been used in most cases. Bioconversion of the gaseous substrates takes place in two stages, namely acidogenesis (production of acids) followed by solventogenesis (production of alcohols), characterized by different optimal fermentation conditions. Major parameters affecting each bioconversion stage as well as the overall fermentation process are analyzed. Although it has been claimed that acidification is required in ABE fermentation to initiate the solventogenic stage, strong acidification seems to some extent not to be a prerequisite for solventogenesis in the HBE process. Bioreactors potentially suitable for this type of bioconversion process are described as well. © 2017 Society of Chemical Industry

The effect of trace metals, namely tungsten and selenium, on the production of acids and alcohols through gas fermentation by a CO-enriched anaerobic sludge in a continuous gas-fed bioreactor was investigated. The CO-enriched sludge was first supplied with a tungsten-deficient medium (containing selenium) and in a next assay, a selenium-deficient medium (containing tungsten) was fed to the bioreactor, at a CO gas flow rate of 10 mL/min. In the absence of tungsten (tungstate), an initial pH of 6.2 followed by a pH decrease to 4.9 yielded 7.34 g/L acetic acid as the major acid during the high pH period. Subsequently, bioconversion of the acids at a lower pH of 4.9 yielded only 1.85 g/L ethanol and 1.2 g/L butanol in the absence of tungsten (tungstate). A similar follow up assay in the same bioreactor with two consecutive periods at different pH values (i.e., 6.2 and 4.9) with a selenium deficient medium yielded 6.6 g/L acetic acid at pH 6.2 and 4 g/L ethanol as well as 1.88 g/L butanol at pH 4.9. The results from the microbial community analysis showed that the only known CO fixing microorganism able to produce alcohols detected in the bioreactor was Clostridium autoethanogenum, both in the tungsten and the selenium deprived media, although that species has so far not been reported to be able to produce butanol. No other solventogenic acetogen was detected.

Several anaerobic bioconversion technologies produce short chain volatile fatty acids and sometimes ethanol, which can together be elongated to hexanoic acid (C6 acid) by Clostridium kluyveri in a secondary fermentation process. Initiatives are needed to further optimize the process. Therefore, five strategies were tested aiming at elucidating their influence on hexanoic acid production from mixtures of acetic acid, butyric acid and ethanol. pH-regulated bioreactors, maintained at pH 7.5, 6.8 or 6.4 led to maximum C6 acid concentrations of, respectively, 19.4, 18.3 and 13.3 g L−1. At pH 6.8, yeast extract omission resulted in a decrease of the hexanoic acid concentration to 12.0 g L−1 while the addition of an inorganic carbon source, such as bicarbonate, for pH control, increased the C6 acid concentration up to 21.4 g L−1. This research provides guidelines for efficient improved production of hexanoic acid by pure cultures of C. kluyveri, contributing to the state of art.

[Abstract] Clostridium carboxidivorans can produce acids and/or alcohols through syngas fermentation. In that C1-gas fermentation process, the production of acids takes place at higher pH (acetogenesis) (e.g., around 6.00), while the conversion of accumulated acids into alcohols (solventogenesis) is more favourable at a lower pH (e.g., 4.75–5.00). The pH drop, when switching from acetogenesis to solventogenesis, can either be natural—and result from the production of acids—or artificial. In the latter case, for the acidification process, a strong acid (HCl) was added to a syngas fermenting bioreactor in this study, while NaOH was added to increase the pH whenever needed. Cycles of high and low pH were applied in order to switch from acetogenic to solventogenic stages. This pH adjustment procedure leads to the accumulation of salts. The possible inhibitory effect exerted by changes in salinity in the bioreactor was estimated in batch bottles assays, carried out with different salinities (media with different concentrations of sodium chloride) using C. carboxidivorans and CO as a carbon source. At NaCl concentrations below 9 g/L, maximum growth rates around 0.055 h−1 were obtained, whereas increasing the concentration of sodium chloride had a negative effect on bacterial growth and CO consumption. In the case of the most concentrated bottles, above 15 g/L NaCl no relevant growth was observed. Also, the IC50, i.e. concentration yielding 50% growth inhibition, was estimated, and reached a value of 11 g/L sodium chloride. Ministerio de Economía, Industria y Competitividad; CTQ2017-88292-R

The ethanol production capability of Clostridium aceticum was investigated and optimized, in order to evaluate the ability of that organism to produce high concentrations of fuel-ethanol. The results showed that C. aceticum can produce significant amounts of ethanol when a natural pH drop occurs in the fermentation broth as a consequence of acetic acid production in a first stage. Applying different pH-regulating strategies allowed to optimize ethanol production, which proved to be more efficient in case of natural acidification due to acetic acid, reaching up to 5.6 g/L ethanol, compared to artificial pH adjustment through the addition of hydrogen chloride. Playing with the pH value and the bioreactor operating conditions showed that, under specific conditions, C. aceticum is able to perform the reverse reaction as well and convert ethanol, produced at low pH, back to acetic acid, impeding, under those specific conditions, further accumulation of ethanol in the fermentation broth.

Clostridium kluyveri was used for chain elongation of C2 C4 fatty acids in stirred tank bioreactors. The influence of different electron acceptors (acetic acid, butyric acid and the mixture of both) on C6 fatty acid production was evaluated in presence of ethanol using similar molar alcohol/acid ratios around 3.5. Bottle batch assays without pH regulation and with only acetic acid as electron acceptor yielded a final C6 fatty acid concentration of 6.8 ± 0.6 g L−1. Then, pH-regulated bioreactors were operated at constant pH of 6.8. Under such conditions, the maximum growth rate was 0.039 h−1 obtained using acetic acid and butyric acid as electron acceptors, whereas the lowest growth rate was 0.010 h−1 with only butyric acid as electron acceptor. The maximum growth rate with acetic acid only, was similar, though slightly lower, as with the mixture of C2 C4 fatty acids. Besides, the maximum productions of hexanoic acid were 11.8 g L−1, 13.1 g L−1 and 21.2 g L−1 using, respectively, acetic acid, butyric acid and the mixture of both acids as electron acceptors. Thus, the use of a mixture of acetic acid and butyric acid in presence of ethanol for chain elongation, at constant pH, proved to be efficient for hexanoic acid production.

Fermentation medium and fermentation pH are among the main parameters that control acidogenesis and solventogenesis in clostridia during CO bioconversion. The approach of cyclic pH shifts from high (pH 5.75) to low (pH 4.75) levels, in continuous gas-fed bioreactors, allowed Clostridium autoethanogenum to consume and convert CO and the produced acetic acid from CO to alcohols using one single bioreactor. Increasing amounts of ethanol accumulate after each pH shift cycle, while acids are used up and converted during each cycle. In this study, a total of three cyclic pH shifts along with partial medium replenishment, resulted in a high ethanol production, reaching a concentration of 7143 mg/L. The mixotrophic fermentation using xylose as additional carbon source besides carbon monoxide allowed to conclude that xylose is a preferred carbon source compared to CO and that it allows to increase the cell mass concentration (421 mg/L).

Ethanol production from C1 compounds, such as carbon monoxide, using acetogenic bacteria is an attractive alternative to produce renewable fuels. However, the process is generally associated with acetic acid accumulation, which is often produced in larger quantities than ethanol itself. This study shows the continuous production of ethanol and complete conversion of produced acetic acid to ethanol, which is possible through pH shifts from high pH to low pH using an optimized medium and one single gas-fed bioreactor. A first study with a single pH shift resulted in the production of 2408 mg/L of ethanol without any accumulation of acetic acid. In a next study a final ethanol concentration of 4260 mg/L was reached with just one pH shift and one partial medium renewal. These results indicate that maintaining an optimal growth pH at the beginning of the process in order to obtain a high cell mass, and later shifting the pH to a low value will improve ethanol productivity and avoid any acetic acid accumulation.

BACKGROUND Brewery wastewater is a potential feedstock for polyhydroxyalkanoates (PHA) production since it contains high concentrations of easily biodegradable organic matter. Activated sludge was submitted to aerobic dynamic feeding conditions in a sequencing batch reactor fed fermented brewery wastewater in order to select a microbial culture with high storage capacity. The effect of several operational parameters, such as substrate concentration (57, 79 and 109 Cmmol L−1), pH (7 and 9 and without pH control (8–9)) and temperature (15 and 30 °C and without T control (15–28 °C)) was investigated. RESULTS The concentration of PHA stored by the activated sludge increased with the substrate concentration, with 39% PHA content on a dry biomass basis obtained at 109 Cmmol L−1. The storage yield increased from 0.35 to 0.43 Cmmol HA Cmmol−1 VFA when the substrate concentration was raised from 57 to 79 Cmmol L−1. However, a further increase in substrate concentration to 109 Cmmol L−1 led to a storage yield decrease to 0.39 Cmmol HA Cmmol−1 VFA. Control of pH and temperature did not enhance PHA storage. CONCLUSIONS Brewery wastewater is a good feedstock for production of PHAs using mixed cultures enriched under feast and famine conditions. PHA production can be enhanced by manipulating the operational conditions. © 2016 Society of Chemical Industry

The present research explored the optimization of volatile fatty acids (VFA) production from cheese whey in an anaerobic sequencing batch reactor (AnSBR). For that purpose, changes of solid and hydraulic retention times (SRT and HRT) were applied. Moreover, the experiments were coupled to metagenomic analyses by 16S rRNA sequencing. The results showed an enhancement of the process effectiveness at longer SRT and shorter HRT. The degree of acidification (DA) improved from 0.73 to 0.83 when increasing the SRT from 5 to 15 days. It also increased from 0.79 to 0.83 when lowering the HRT from 3 to 1 day. The acidification yield (YVFA/S) improved from 0.78 to 0.87 and from 0.86 to 0.90 g COD-VFA g COD-Lactose−1 when increasing the SRT from 5 to 15 days and decreasing the HRT from 3 to 1 day, respectively. Hydrolytic bacteria dominated the microbial community at the shortest SRT, although they were replaced by acidogenic bacteria at longer SRT.

The effect of trace metals, namely tungsten and selenium, on the production of acids and alcohols through gas fermentation by a CO-enriched anaerobic sludge in a continuous gas-fed bioreactor was investigated. The CO-enriched sludge was first supplied with a tungsten-deficient medium (containing selenium) and in a next assay, a selenium-deficient medium (containing tungsten) was fed to the bioreactor, at a CO gas flow rate of 10 mL/min. In the absence of tungsten (tungstate), an initial pH of 6.2 followed by a pH decrease to 4.9 yielded 7.34 g/L acetic acid as the major acid during the high pH period. Subsequently, bioconversion of the acids at a lower pH of 4.9 yielded only 1.85 g/L ethanol and 1.2 g/L butanol in the absence of tungsten (tungstate). A similar follow up assay in the same bioreactor with two consecutive periods at different pH values (i.e., 6.2 and 4.9) with a selenium deficient medium yielded 6.6 g/L acetic acid at pH 6.2 and 4 g/L ethanol as well as 1.88 g/L butanol at pH 4.9. The results from the microbial community analysis showed that the only known CO fixing microorganism able to produce alcohols detected in the bioreactor was Clostridium autoethanogenum, both in the tungsten and the selenium deprived media, although that species has so far not been reported to be able to produce butanol. No other solventogenic acetogen was detected.

Production of biofuels and bioproducts from syngas or industrial waste gases, containing C1 compounds such as CO and CO 2 , is a promising alternative to the technologies used in conventional chemical industries and refineries. Various parameters and strategies that can improve the production process are addressed, focusing mainly on bioethanol. The advantages of fermentation over the thermochemical process using syngas as feedstock are discussed. Aspects of the Wood-Ljungdahl pathway and energy conservation mechanism in acetogens are included, and results from various batch assays and continuous studies performed in different bioreactor configurations to improve productivity are reviewed. Although interest in syngas fermentation has progressively increased in the recent years, research is needed to further optimize the process. Mass transfer of gas to liquid phase and the need to achieve higher biomass concentrations are still some main bottlenecks of the syngas fermentation process.

An anaerobic granular sludge was acclimatized to utilize CO in a continuously gas-fed stirred tank bioreactor by applying operating conditions expected to stimulate solventogenesis, i.e. the production of alcohols, and allowing to enrich for solventogenic populations. A cycle of high (6.2) and low (4.9) pH was applied in order to produce volatile fatty acids first at high pH, followed by their bioconversion into alcohols at low pH. The addition of yeast extract stimulated biomass growth, but not necessarily solventogenesis. The highest concentrations of metabolites achieved were 6.18 g/L acetic acid (30th day), 1.18 g/L butyric acid (28th day), and 0.423 g/L hexanoic acid (32nd day). Subsequently, acids were metabolized at lower pH, producing alcohols at concentrations of 11.1 g/L ethanol (43rd day), 1.8 g/L butanol (41st day) and 1.46 g/L hexanol (42nd day), confirming the successful enrichment strategy. Similarly, the enriched sludge could also convert syngas into acids and alcohols.

Clostridium carboxidivorans produces alcohols from C1 gases (CO, CO

BACKGROUND Recent studies have characterized the most effective absorbent polymers for the removal of volatile hydrophobic pollutants from air. This study evaluates the performance of a laboratory scale α-pinene-degrading biotrickling filter (BTF) packed with lava rock. A second solid phase, Hytrel® G3548L (5%, v/v), was added in order to check its effect on the performance of the system during long-term operation. RESULTS The biodegradation profile was similar in both bioreactor configurations, with or without solid polymers, reaching a maximum elimination capacity (ECmax) around 25.6 g m−3 h−1 when an inlet loading rate of 57.2 g m−3 h−1 was applied. Tracer studies were carried out in order to determine the effect of the liquid and gas velocities, and to analyze the gas distribution with the different packing materials. CONCLUSIONS Deviation from ideal flow caused by channelling of the fluid was observed from tracer studies. No better performance was observed under steady-state conditions in the presence of polymeric Hytrel material. Further considerations are needed for the selection of an appropriate absorbent polymer for α-pinene removal. © 2015 Society of Chemical Industry