Bioaugmented Mixed Culture by Clostridium aceticum to Manipulate Volatile Fatty Acids Composition From the Fermentation of Cheese Production Wastewater

Production of targeted volatile fatty acid (VFA) composition by fermentation is a promising approach for upstream and post-stream VFA applications. In the current study, the bioaugmented mixed microbial culture by Clostridium aceticum was used to produce an acetic acid dominant VFA mixture. For this purpose, anaerobic sequencing batch reactors (bioaugmented and control) were operated under pH 10 and fed by cheese processing wastewater. The efficiency and stability of the bioaugmentation strategy were monitored using the production and composition of VFA, the quantity of C. aceticum (by qPCR), and bacterial community profile (16S rRNA Illumina Sequencing). The bioaugmented mixed culture significantly increased acetic acid concentration in the VFA mixture (from 1170 ± 18 to 122 ± 9 mgCOD/L) compared to the control reactor. Furthermore, the total VFA production (from 1254 ± 11 to 5493 ± 36 mgCOD/L) was also enhanced. Nevertheless, the bioaugmentation could not shift the propionic acid dominancy in the VFA mixture. The most significant effect of bioaugmentation on the bacterial community profile was seen in the relative abundance of the Thermoanaerobacterales Family III. Incertae sedis, its relative abundance increased simultaneously with the gene copy number of C. aceticum during bioaugmentation. These results suggest that there might be a syntropy between species of Thermoanaerobacterales Family III. Incertae sedis and C. aceticum. The cycle analysis showed that 6 h (instead of 24 h) was adequate retention time to achieve the same acetic acid and total VFA production efficiency. Biobased acetic acid production is widely applicable and economically competitive with petroleum-based production, and this study has the potential to enable a new approach as produced acetic acid dominant VFA can replace external carbon sources for different processes (such as denitrification) in WWTPs. In this way, the higher treatment efficiency for WWTPs can be obtained by recovered substrate from the waste streams that promote a circular economy approach.

Is reduced ferredoxin the physiological electron donor for MetVF-type methylenetetrahydrofolate reductases in acetogenesis? A hypothesis

The methylene-tetrahydrofolate reductase (MTHFR) is a key enzyme in acetogenic CO2 fixation. The MetVF-type enzyme has been purified from four different species and the physiological electron donor was hypothesized to be reduced ferredoxin. We have purified the MTHFR from Clostridium ljungdahlii to apparent homogeneity. It is a dimer consisting of two of MetVF heterodimers, has 14.9 ± 0.2 mol iron per mol enzyme, 16.2 ± 1.0 mol acid-labile sulfur per mol enzyme, and contains 1.87 mol FMN per mol dimeric heterodimer. NADH and NADPH were not used as electron donor, but reduced ferredoxin was. Based on the published electron carrier specificities for Clostridium formicoaceticum, Thermoanaerobacter kivui, Eubacterium callanderi, and Clostridium aceticum, we provide evidence using metabolic models that reduced ferredoxin cannot be the physiological electron donor in vivo, since growth by acetogenesis from H2 + CO2 has a negative ATP yield. We discuss the possible basis for the discrepancy between in vitro and in vivo functions and present a model how the MetVF-type MTHFR can be incorporated into the metabolism, leading to a positive ATP yield. This model is also applicable to acetogenesis from other substrates and proves to be feasible also to the Ech-containing acetogen T. kivui as well as to methanol metabolism in E. callanderi.

Energy Conservation in the Acetogenic Bacterium Clostridium aceticum

In times of global warming caused by the extensive use of fossil fuels, the need to capture gaseous carbon compounds is growing bigger. Several groups of microorganisms can fix the greenhouse gas CO2. Out of these, acetogenic bacteria are role models in their ability to reduce CO2 with hydrogen to acetate, which makes acetogens prime candidates for genetic modification towards biotechnological production of value-added compounds from CO2, such as biofuels. However, growth of acetogens on gaseous substrates is strongly energy-limited, and successful metabolic engineering requires a detailed knowledge of the bioenergetics. In 1939, Clostridium aceticum was the first acetogen to be described. A recent genomic study revealed that this organism contains cytochromes and therefore may use a proton gradient in its respiratory chain. We have followed up these studies and will present data that C. aceticum does not use a H+ but a Na+ gradient for ATP synthesis, established by a Na+-Rnf. Experimental data and in silico analyses enabled us to propose the biochemistry and bioenergetics of acetogenesis from H2 + CO2 in C. aceticum.

The Complete Genome Sequence of Clostridium aceticum: a Missing Link between Rnf- and Cytochrome-Containing Autotrophic Acetogens

ABSTRACTClostridium aceticumwas the first isolated autotrophic acetogen, converting CO2plus H2or syngas to acetate. Its genome has now been completely sequenced and consists of a 4.2-Mbp chromosome and a small circular plasmid of 5.7 kbp. Sequence analysis revealed major differences from other autotrophic acetogens.C. aceticumcontains an Rnf complex for energy conservation (via pumping protons or sodium ions). Such systems have also been found inC. ljungdahliiandAcetobacterium woodii. However,C. aceticumalso contains a cytochrome, as doesMoorella thermoacetica, which has been proposed to be involved in the generation of a proton gradient. Thus,C. aceticumseems to represent a link between Rnf- and cytochrome-containing autotrophic acetogens. InC. aceticum, however, the cytochrome is probably not involved in an electron transport chain that leads to proton translocation, as no genes for quinone biosynthesis are present in the genome.IMPORTANCEAutotrophic acetogenic bacteria are receiving more and more industrial focus, as CO2plus H2as well as syngas are interesting new substrates for biotechnological processes. They are both cheap and abundant, and their use, if it results in sustainable products, also leads to reduction of greenhouse gases.Clostridium aceticumcan use both gas mixtures, is phylogenetically not closely related to the commonly used species, and may thus become an even more attractive workhorse. In addition, its energy metabolism, which is characterized here, and the ability to synthesize cytochromes might offer new targets for improving the ATP yield by metabolic engineering and thus allow use ofC. aceticumfor production of compounds by pathways that currently present challenges for energy-limited acetogens.