Influence of nickel on butanol production by Clostridium beijerinckii using hydrolyzate from green coconut shell

The improvement of biotechnological processes capable of transforming agro-industrial waste into products with high added value has stood out in the area of renewable energies, promoting positive impacts to the environment. Thus, the present work evaluated the influence of nickel on the conversion of fermentable sugars, present in the green coconut shell hydrolyzate (GCSH), into butanol and other products. Fermentation assays were performed at 37 °C, starting with 19.4 g.L-1 of sugars and 1.0 g.L-1 of inoculum (C. beijerinckii). The GCSH was supplemented with tryptone, yeast extract, ammonium acetate, minerals and phosphate buffer. Two conditions were tested: with and without addition of nickel. Concentrations of sugars (glucose and xylose), intermediate products (organic acids), acetone, butanol, and ethanol were determined by high performance liquid chromatography (HPLC). The results show that the butanol production was higher from GCSH without addition of nickel, reaching a concentration of 2.14 g.L-1 of butanol. Therefore, the presence of nickel in the hydrolyzate was not favorable in the production of butanol under the studied process conditions.

Dark Fermentation Process Response to the Use of Undiluted Tequila Vinasse without Nutrient Supplementation

The technical feasibility of valorizing tequila vinasse (TV), a wastewater with high pollution potential, through the production of biogenic hydrogen via dark fermentation, has long been proven in diverse lab-scale reactors that were operated either in batch or continuous mode. However, such systems have mainly been tested with diluted streams and nutrient supplementation, hindering the techno-economic attractiveness of the TV-to-hydrogen concept at large scale. In this study, the feasibility of producing hydrogen from high-strength undiluted TV with no added extra nutrients was evaluated under batch mesophilic conditions. Additionally, the use of two different acidogenic inocula obtained either by heat or heat-aeration pretreatment was investigated to get a greater understanding of the effect of inoculum type on the process. The results obtained showed that the TV utilized herein contained macro- and micro-nutrients high enough to support the hydrogenogenic activity of both cultures, entailing average hydrogen yields of 2.4–2.6 NL H2/L vinasse and maximum hydrogen production rates of 1.4–1.9 NL H2/L-d. Interestingly, the consumption of lactate and acetate with the concomitant production of butyrate was observed as the main hydrogen-producing route regardless of the inoculum, pointing out the relevance of the lactate-driven dark fermentative process. Clostridium beijerinckii was ascertained as key bacteria, but only in association with microorganisms belonging to the genera Enterobacter and Klebsiella, as revealed by phylogenetic analyses.

Improving sustainable hydrogen production from green waste: [FeFe]-hydrogenases quantitative gene expression RT-qPCR analysis in presence of autochthonous consortia

Background Bio-hydrogen production via dark fermentation of low-value waste is a potent and simple mean of recovering energy, maximising the harvesting of reducing equivalents to produce the cleanest fuel amongst renewables. Following several position papers from companies and public bodies, the hydrogen economy is regaining interest, especially in combination with circular economy and the environmental benefits of short local supply chains, aiming at zero net emission of greenhouse gases (GHG). The biomasses attracting the largest interest are agricultural and urban green wastes (pruning of trees, collected leaves, grass clippings from public parks and boulevards), which are usually employed in compost production, with some concerns over the GHG emission during the process. Here, an alternative application of green wastes, low-value compost and intermediate products (partially composted but unsuitable for completing the process) is studied, pointing at the autochthonous microbial consortium as an already selected source of implementation for biomass degradation and hydrogen production. The biocatalysts investigated as mainly relevant for hydrogen production were the [FeFe]-hydrogenases expressed in Clostridia, given their very high turnover rates. Results Bio-hydrogen accumulation was related to the modulation of gene expression of multiple [FeFe]-hydrogenases from two strains (Clostridium beijerinckii AM2 and Clostridium tyrobutyricum AM6) isolated from the same waste. Reverse Transcriptase quantitative PCR (RT-qPCR) was applied over a period of 288 h and the RT-qPCR results showed that C. beijerinckii AM2 prevailed over C. tyrobutyricum AM6 and a high expression modulation of the 6 different [FeFe]-hydrogenase genes of C. beijerinckii in the first 23 h was observed, sustaining cumulative hydrogen production of 0.6 to 1.2 ml H2/g VS (volatile solids). These results are promising in terms of hydrogen yields, given that no pre-treatment was applied, and suggested a complex cellular regulation, linking the performance of dark fermentation with key functional genes involved in bio-H2 production in presence of the autochthonous consortium, with different roles, time, and mode of expression of the involved hydrogenases. Conclusions An applicative outcome of the hydrogenases genes quantitative expression analysis can be foreseen in optimising (on the basis of the acquired functional data) hydrogen production from a nutrient-poor green waste and/or low added value compost, in a perspective of circular bioeconomy.

Comparative genome analysis of Clostridium beijerinckii strains isolated from pit mud of Chinese strong flavor baijiu ecosystem

Clostridium beijerinckii is a well-known anaerobic solventogenic bacterium which inhabits a wide range of different niches. Previously, we isolated five butyrate-producing C. beijerinckii strains from pit mud (PM) of strong-flavor baijiu (SFB) ecosystems. Genome annotation of the five strains showed that they could assimilate various carbon sources as well as ammonium to produce acetate, butyrate, lactate, hydrogen, and esters but did not produce the undesirable flavours isopropanol and acetone, making them useful for further exploration in SFB production. Our analysis of the genomes of an additional 233 C. beijerinckii strains revealed an open pangenome based on current sampling and will likely change with additional genomes. The core genome, accessory genome, and strain-specific genes comprised 1567, 8851, and 2154 genes, respectively. A total of 298 genes were found only in the five C. beijerinckii strains from PM, among which only 77 genes were assigned to Clusters of Orthologous Genes (COG) categories. In addition, 15 transposase and 12 phage integrase families were found in all five C. beijerinckii strains from PM. Between 18 and 21 genome islands (GIs) were predicted for the five C. beijerinckii genomes. The existence of a large number of MGEs indicated that the genomes of the five C. beijerinckii strains evolved with the loss or insertion of DNA fragments in the PM of SFB ecosystems. This study presents a genomic framework of C. beijerinckii strains from PM that could be used for genetic diversification studies and further exploration of these strains.

Enhanced sucrose fermentation by introduction of heterologous sucrose transporter and invertase into Clostridium beijerinckii for acetone–butanol–ethanol production

A heterologous pathway for sucrose transport and metabolism was introduced into Clostridium beijerinckii to improve sucrose use for n -butanol production. The combined expression of StSUT1 , encoding a sucrose transporter from potato ( Solanum tuberosum ), and SUC2 , encoding a sucrose invertase from Saccharomyces cerevisiae , remarkably enhanced n -butanol production. With sucrose, sugarcane molasses and sugarcane juice as substrates, the C. beijerinckii strain harbouring StSUT1 and SUC2 increased acetone–butanol–ethanol production by 38.7%, 22.3% and 52.8%, respectively, compared with the wild-type strain. This is the first report to demonstrate enhanced sucrose fermentation due to the heterologous expression of a sucrose transporter and invertase in Clostridium . The metabolic engineering strategy used in this study can be widely applied in other microorganisms to enhance the production of high-value compounds from sucrose-based biomass.