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Multifunctional reactor microbiomes can elongate short-chain carboxylic acids (SCCAs) to medium-chain carboxylic acids (MCCAs), such as n-caproic acid. However, it is unclear whether this microbiome biotechnology platform is stable enough during long operating periods to consistently produce MCCAs. During a period of 550 days, we improved the operating conditions of an anaerobic bioreactor for the conversion of complex yeast-fermentation beer from the corn kernel-to-ethanol industry into primarily n-caproic acid. We incorporated and improved in-line, membrane liquid-liquid extraction to prevent inhibition due to undissociated MCCAs at a pH of 5.5 and circumvented the addition of methanogenic inhibitors. The microbiome accomplished several functions, including hydrolysis and acidogenesis of complex organic compounds and sugars into SCCAs, subsequent chain elongation with undistilled ethanol in beer, and hydrogenotrophic methanogenesis. The methane yield was 2.40 ± 0.52% based on COD and was limited by the availability of carbon dioxide. We achieved an average n-caproate production rate of 3.38 ± 0.42 g L(-1) d(-1) (7.52 ± 0.94 g COD L(-1) d(-1)) with an n-caproate yield of 70.3 ± 8.81% and an n-caproate/ethanol ratio of 1.19 ± 0.15 based on COD for a period of ∼55 days. The maximum production rate was achieved by increasing the organic loading rates in tandem with elevating the capacity of the extraction system and a change in the complex feedstock batch.

High productivity and specificity in anaerobic digesters arise because complex microbiomes organize into a metabolic cascade to maximize energy recovery and to utilize the advantage that the gaseous end product methane freely bubbles out of the system. These lessons were applied to ascertain whether a reactor microbiome could be shaped to produce a different end product. The liquid product n-caproic acid was chosen, which is a 6-carbon-chain carboxylic acid that is valuable and that has a relatively low maximum solubility concentration for product recovery. Acetoclastic methanogenesis was inhibited by pH control and a route was provided for n-caproic acid extraction by implementing selective, in-line recovery. Next, ethanol was supplemented to promote chain elongation, which is a pathway in which short-chain carboxylic acids are elongated sequentially into medium-chain carboxylic acids with two-carbon units derived from ethanol. The reactor microbiome developed accordingly with the terminal process catalyzed by chain-elongating bacteria. As a result, n-caproic acid production rates increased to levels comparable to anaerobic digestion systems for solid waste treatment.

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BackgroundThe product of current syngas fermentation systems is an ethanol/acetic acid mixture and the goal is to maximize ethanol recovery. However, ethanol currently has a relatively low market value and its separation from the fermentation broth is energy intensive. We can circumvent these disadvantages of ethanol production by converting the dilute ethanol/acetic acid mixture into products with longer carbon backbones, which are of higher value and are more easily extracted than ethanol. Chain elongation, which is the bioprocess in which ethanol is used to elongate short-chain carboxylic acids to medium-chain carboxylic acids (MCCAs), has been studied with pure cultures and open cultures of microbial consortia (microbiomes) with several different substrates. While upgrading syngas fermentation effluent has been studied with open cultures, to our knowledge, no study exists that has performed this with pure cultures.ResultsHere, pure cultures of Clostridium kluyveri were used in continuous bioreactors to convert ethanol/acetic acid mixtures into MCCAs. Besides changing the operating conditions in regards to substrate loading rates and composition, the effect of in-line product extraction, pH, and the use of real syngas fermentation effluent on production rates were tested. Increasing the organic loading rates resulted in proportionally higher production rates of n-caproic acid, which were up to 40 mM day−1 (4.64 g L−1 day−1) at carbon conversion efficiencies of 90% or higher. The production rates were similar for bioreactors with and without in-line product extraction. Furthermore, a lower ethanol/acetic acid ratio (3:1 instead of 10:1) enabled faster and more efficient n-caproic acid production. In addition, n-caprylic acid production was observed for the first time with C. kluyveri (up to 2.19 ± 0.34 mM in batch). Finally, the use of real effluent from syngas fermentation, without added yeast extract, but with added defined growth factors, did maintain similar production rates. Throughout the operating period, we observed that the metabolism of C. kluyveri was inhibited at a mildly acidic pH value of 5.5 compared to a pH value of 7.0, while reactor microbiomes perform successfully at mildly acidic conditions.Conclusions Clostridium kluyveri can be used as a biocatalyst to upgrade syngas fermentation effluent into MCCAs at pH values above 5.5.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0764-6) contains supplementary material, which is available to authorized users.

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Here, we studied the microbiome succession and time-scale variability of four mesophilic anaerobic reactors in a co-digestion study with the objective to find links between changing environmental conditions and the microbiome composition. The changing environmental conditions were ensured by gradual increases in loading rates and mixing ratios of three co-substrates with a constant manure-feeding scheme during an operating period longer than 900 days. Each co-substrate (i.e., alkaline hydrolysate, food waste, and glycerol) was co-digested separately. High throughput 16S rRNA gene sequencing was used to examine the microbiome succession. The alkaline hydrolysate reactor microbiome shifted and adapted to high concentrations of free ammonia, total volatile fatty acids, and potassium to maintain its function. The addition of food waste and glycerol as co-substrates also led to microbiome changes, but to a lesser extent, especially in the case of the glycerol reactor microbiome. The divergence of the food waste reactor microbiome was primarily linked to increasing free ammonia levels in the reactor; though, these levels remained below previously reported inhibitory levels for acclimated biomass. The glycerol reactor microbiome succession included an increase in Syntrophomonadaceae family members, which have previously been linked to long-chain fatty acid degradation. The glycerol reactor exhibited rapid failure and limited adaptation at the end of the study.