Acidogenic fermentation of food waste to generate electron acceptors and donors towards medium-chain carboxylic acids production.

This study investigated the optimum pH, temperature, and food-to-microorganisms (F/M) ratio for regulating the formation of electron acceptors and donors during acidogenic fermentation to facilitate medium-chain carboxylic acids (MCCAs) production from food waste. Mesophilic fermentation at pH 6 was optimal for producing mixed volatile fatty acids (719 ± 94 mg COD/g VS) as electron acceptors. Under mesophilic conditions, the F/M ratio (g VS/g VS) could be increased to 6 to generate 22 ± 2 g COD/L of electron acceptors alongside 2 ± 0 g COD/L of caproic acid. Thermophilic fermentation at pH 6 was the best condition for producing lactic acid as an electron donor. However, operating at F/M ratios above 3 g VS/g VS under thermophilic settings significantly reduced lactic acid yield. A preliminary techno-economic evaluation revealed that converting lactic acid and butyric acid generated during acidogenic fermentation to caproic acid was the most profitable food waste valorization scenario and could generate 442-468 €/t VS/y. The results presented in this study provide insights into how to tailor acidogenic fermentation reactions to desired intermediates and will help maximize MCCAs synthesis.

Effect of Addition of Pectins from Jujubes (Ziziphus jujuba Mill.) on Vitamin C Production during Heterolactic Fermentation.

Soluble fibers, including pectins from apple and lemon, are commonly used as prebiotic and to prepare functional foods. The present study aimed to investigate the physicochemical and functional properties of pectins extracted from jujubes (Ziziphus jujuba Mill.). Pectins were extracted from jujubes at three stages of harvesting and characterized by FTIR and SEM analyses. Whole milk inoculated with kefir grains was supplemented by 0.25 mg·mL-1 of pectins. The pH value and vitamin C content were evaluated after 24 and 48 h of fermentation. Pectins from jujubes at the first harvesting stage (PJ1K) showed the lowest methoxylation degree. The addition of pectins enhanced the production of vitamin C during heterolactic process. This result was found to depend on jujube harvesting stage as PJ1K stimulated the growth of yeasts in kefir grains yielding to the highest amount of vitamin C (0.83 ± 0.01 µg·mL-1) compared to other samples (0.53-0.60 µg·mL-1) at 24 h. Lactic acid bacteria diminish pH rapidly with respect to control (4.13 ± 0.05), according to the stage of maturation, reducing its initial value by 38.3% in PJ1K. Besides being an excellent prebiotic, pectins from jujubes could be used to enrich kefir with vitamin C.

Constitutive expression of phosphoketolase, a key enzyme for metabolic shift from homo- to heterolactic fermentation in Enterococcus mundtii QU 25.

Phosphoketolase (PK) is responsible for heterolactic fermentation; however, the PK gene of Enterococcus mundtii QU 25, xfpA, is transcribed constitutively, even under homolactic fermentation conditions. In order to deduce the regulatory mechanisms of PK activity in QU 25, XfpA levels in QU 25 cells under hetero- and homolactic fermentation conditions were tested using western blotting. The results showed that the XfpA protein expression was similar under both conditions and that the expression products formed complexes, most likely homodimers, indicating that the regulation of PK activity is downstream of translation.

A genome-scale metabolic network of the aroma bacterium Leuconostoc mesenteroides subsp. cremoris.

Leuconostoc mesenteroides subsp. cremoris is an obligate heterolactic fermentative lactic acid bacterium that is mostly used in industrial dairy fermentations. The phosphoketolase pathway (PKP) is a unique feature of the obligate heterolactic fermentation, which leads to the production of lactate, ethanol, and/or acetate, and the final product profile of PKP highly depends on the energetics and redox state of the organism. Another characteristic of the L. mesenteroides subsp. cremoris is the production of aroma compounds in dairy fermentation, such as in cheese production, through the utilization of citrate. Considering its importance in dairy fermentation, a detailed metabolic characterization of the organism is necessary for its more efficient use in the industry. To this aim, a genome-scale metabolic model of dairy-origin L. mesenteroides subsp. cremoris ATCC 19254 (iLM.c559) was reconstructed to explain the energetics and redox state mechanisms of the organism in full detail. The model includes 559 genes governing 1088 reactions between 1129 metabolites, and the reactions cover citrate utilization and citrate-related flavor metabolism. The model was validated by simulating co-metabolism of glucose and citrate and comparing the in silico results to our experimental results. Model simulations further showed that, in co-metabolism of citrate and glucose, no flavor compounds were produced when citrate could stimulate the formation of biomass. Significant amounts of flavor metabolites (e.g., diacetyl and acetoin) were only produced when citrate could not enhance growth, which suggests that flavor formation only occurs under carbon and ATP excess. The effects of aerobic conditions and different carbon sources on product profiles and growth were also investigated using the reconstructed model. The analyses provided further insights for the growth stimulation and flavor formation mechanisms of the organism.

A Novel D-Galacturonate Fermentation Pathway in Lactobacillus suebicus Links Initial Reactions of the Galacturonate-Isomerase Route With the Phosphoketolase Pathway.

D-galacturonate, a key constituent of pectin, is a ubiquitous monomer in plant biomass. Anaerobic, fermentative conversion of D-galacturonate is therefore relevant in natural environments as well as in microbial processes for microbial conversion of pectin-containing agricultural residues. In currently known microorganisms that anaerobically ferment D-galacturonate, its catabolism occurs via the galacturonate-isomerase pathway. Redox-cofactor balancing in this pathway strongly constrains the possible range of products generated from anaerobic D-galacturonate fermentation, resulting in acetate as the predominant organic fermentation product. To explore metabolic diversity of microbial D-galacturonate fermentation, anaerobic enrichment cultures were performed at pH 4. Anaerobic batch and chemostat cultures of a dominant Lactobacillus suebicus strain isolated from these enrichment cultures produced near-equimolar amounts of lactate and acetate from D-galacturonate. A combination of whole-genome sequence analysis, quantitative proteomics, enzyme activity assays in cell extracts, and in vitro product identification demonstrated that D-galacturonate metabolism in L. suebicus occurs via a novel pathway. In this pathway, mannonate generated by the initial reactions of the canonical isomerase pathway is converted to 6-phosphogluconate by two novel biochemical reactions, catalyzed by a mannonate kinase and a 6-phosphomannonate 2-epimerase. Further catabolism of 6-phosphogluconate then proceeds via known reactions of the phosphoketolase pathway. In contrast to the classical isomerase pathway for D-galacturonate catabolism, the novel pathway enables redox-cofactor-neutral conversion of D-galacturonate to ribulose-5-phosphate. While further research is required to identify the structural genes encoding the key enzymes for the novel pathway, its redox-cofactor coupling is highly interesting for metabolic engineering of microbial cell factories for conversion of pectin-containing feedstocks into added-value fermentation products such as ethanol or lactate. This study illustrates the potential of microbial enrichment cultivation to identify novel pathways for the conversion of environmentally and industrially relevant compounds.

Primary souring: A novel bacteria-free method for sour beer production.

In the beverage fermentation industry, especially at the craft or micro level, there is a movement to incorporate as many local ingredients as possible to both capture terroir and stimulate local economies. In the case of craft beer, this has traditionally only encompassed locally sourced barley, hops, and other agricultural adjuncts. The identification and use of novel yeasts in brewing lags behind. We sought to bridge this gap by bio-prospecting for wild yeasts, with a focus on the American Midwest. We isolated 284 different strains from 54 species of yeast and have begun to determine their fermentation characteristics. During this work, we found several isolates of five species that produce lactic acid and ethanol during wort fermentation: Hanseniaspora vineae, Lachancea fermentati, Lachancea thermotolerans, Schizosaccharomyces japonicus, and Wickerhamomyces anomalus. Tested representatives of these species yielded excellent attenuation, lactic acid production, and sensory characteristics, positioning them as viable alternatives to lactic acid bacteria (LAB) for the production of sour beers. Indeed, we suggest a new LAB-free paradigm for sour beer production that we term "primary souring" because the lactic acid production and resultant pH decrease occurs during primary fermentation, as opposed to kettle souring or souring via mixed culture fermentation.

Characterization of the microbial diversity in yacon spontaneous fermentation at 20 °C.

The prebiotic fructooligosaccharide content of yacon makes this root an attractive alternative for the supplementation of a variety of food products. The preservation of yacon by fermentation has been proposed as an alternative to increase the probiotic content of the root concomitantly with its shelf life. Thus the fermented yacon could have significant functional content. The objective of this research was to characterize the biochemistry and microbiology of spontaneous yacon fermentation with 2% NaCl and define the viability of the proposed process. The biochemical analysis of spontaneous heterolactic fermentation of yacon showed a progressive drop in pH with increased lactic and acetic acids, and the production of mannitol during fermentation. The microbial ecology of yacon fermentation was investigated using culture-dependent and culture-independent methods. Bacterial cell counts revealed a dominance of lactic acid bacteria (LAB) over yeasts, which were also present during the first 2 days of the fermentation. Results showed that the heterofermentative LAB were primarily Leuconostoc species, thus it presents a viable method to achieve long term preservation of this root.

Produções de ácido acético, etanol e dos isômeros óticos do ácido lático por linhagens de Lactobacillus isoladas de fermentações alcoólicas industriais / Production of acetic acid, ethanol and optical isomers of lactic acid by Lactobacillus strains isolated from industrial ethanol fermentations

Avaliaram-se no presente trabalho, as produções de etanol e dos ácidos acético e lático, bem como das proporções dos isômeros óticos D(-) e L(+) desse último, por 17 linhagens de Lactobacillus isoladas de fermentações industriais de produção de etanol. As linhagens foram crescidas a 32ºC por 24 horas, em meio contendo 1 por cento de glucose, 1 por cento de frutose, 1 por cento de extrato de levedura, sais nutrientes (K, Mg e Mn) e tampão fosfato. Foram estimados os teores de ácido lático, ácido acético e etanol mediante cromatografia líquida de alta eficiência, assim como dos isômeros óticos D(-) e L(+) do ácido lático mediante espectrofotometria ao ultra-violeta, empregando desidrogenases láticas estereoespecíficas. O crescimento bacteriano foi inferido pela absorvância a 600 nm. Os resultados obtidos mostraram, pelos perfis de excreção dos metabólitos, a presença de 8 linhagens homofermentativas obrigatórias (produzindo unicamente ácido lático), 8 linhagens heterofermentativas obrigatórias (com produções de ácidos lático, acético e etanol) e 1 linhagem supostamente heterofermentativa facultativa. Observou-se também, em relação à formação dos estereoisômeros, que 12 linhagens foram incluídas no grupo DL, 4 no grupo L e 1 no grupo D. Os resultados permitem concluir que os Lactobacillus que contaminam processos fermentativos industriais de produção de etanol, podem se apresentar nos 3 biotipos fermentativos e produzindo as mais variadas proporções dos dois estereoisômeros do ácido lático, com relevantes implicações biotecnológicas. Este é o primeiro relato sobre as produções dos isômeros óticos do ácido lático por bactérias do gênero Lactobacillus isoladas de fermentações industriais baseadas na cana-de-açúcar.

The aim of the present work was to evaluate the metabolism type of 17 Lactobacillus strains isolated from industrial ethanol fermentation plants. The strains were grown at 32°C for 24 hours on a mixture of equal amounts of glucose and fructose as the carbon source, and supplemented with yeast extract, mineral nutrients and buffer. Bacterial growth was estimated by absorbance at 600nm and the main end products of bacterial metabolism (lactate, acetate and ethanol) were measured by high performance liquid chromatography, while the stereoisomers, D(-)- and L(+)-lactate, were assayed by an enzymatic methodology using stereospecific lactate-dehydrogenases. According to the results, all the three types of metabolism were found among the bacteria investigated: obligately homofermentative (8 strains), facultative heterofermentative (1 strain) and obligately heterofermentative (8 strains). The results have showed a predominance of DL strains regarding the stereoisomers production, but it was also found strains producing a single type of the isomeric form. These findings suggest the possibility to explore the lactobacilli biodiversity in fuel ethanol fermentation plants for lactate production of chemically pure optical isomers. This is the first report on lactic acid isomers formation by Lactobacillus strains isolated from sugar cane based-industrial fermentations.