Heterofermentative Lactic Acid Bacteria Research Articles

The role of homofermentative and heterofermentative lactic acid bacteria for alfalfa silage: a meta-analysis

AbstractLactic acid bacteria (LAB) are usually employed as alfalfa silage inoculants to obtain high-quality feed for animal husbandry. However, the effects of these inoculants are still unclear and need to be studied extensively. Therefore, the objective of this meta-analysis was to quantitatively summarize published research studies that assess the effects of homofermentative (HoLAB) and heterofermentative lactic acid bacteria (HeLAB) on fermentation parameters, nutritive value, microbiological composition and aerobic stability of alfalfa silage. PubMed, ScienceDirect and Scopus have been screened for articles published from 1980 to 2018. The criteria for inclusion were: randomized and controlled trials using alfalfa silage and published in peer-reviewed journals. It was found that inoculation with LAB decreased silage pH, neutral detergent fibre, acid detergent fibre and ammoniacal nitrogen, while it increased dry matter and crude protein compared to control in the pooled raw mean difference random-effect model. Additionally, LAB inoculation decreased acetate, propionate, ethanol and butyrate concentrations, whereas it increased lactate. In addition, inoculants reduced the counts of yeasts and moulds. Lastly, LAB inoculation improved aerobic stability. To the best of our knowledge, this is the first meta-analysis that aims at comparing the application of HoLAB and HeLAB for alfalfa silage. In the pool estimate, positive effects attributable to the application of microbial silage inoculants were found in most of the evaluated parameters; supporting the importance of applying both types of inoculants to improve forage preservation for the livestock industry.

Effects of Heterofermentative Lactic Acid Bacteria on the Ruminal Fermentation of Italian Ryegrass and Whole Crop Barley Silage

Effects of Heterofermentative Lactic Acid Bacteria on the Ruminal Fermentation of Italian Ryegrass and Whole Crop Barley Silage

Genetic Determinants of Hydroxycinnamic Acid Metabolism in Heterofermentative Lactobacilli.

Phenolic acids are among the most abundant phenolic compounds in edible parts of plants. Lactic acid bacteria (LAB) metabolize phenolic acids, but the enzyme responsible for reducing hydroxycinnamic acids to phenylpropionic acids (HcrB) was only recently characterized in Lactobacillus plantarum In this study, heterofermentative LAB species were screened for their hydroxycinnamic acid metabolism. Data on strain-specific metabolism in combination with comparative genomic analyses identified homologs of HcrB as putative phenolic acid reductases. Par1 and HcrF both encode putative multidomain proteins with 25% and 63% amino acid identity to HcrB, respectively. Of these genes, par1 in L. rossiae and hcrF in L. fermentum were overexpressed in response to hydroxycinnamic acids. The deletion of par1 in L. rossiae led to the loss of phenolic acid metabolism. The strain-specific metabolism of phenolic acids was congruent with the genotype of lactobacilli; however, phenolic acid reductases were not identified in strains of Weissella cibaria that reduced hydroxycinnamic acids to phenylpropionic acids. Phylogenetic analysis of major genes involved in hydroxycinnamic acid metabolism in strains of the genus Lactobacillus revealed that Par1 was found to be the most widely distributed phenolic acid reductase, while HcrB was the least abundant, present in less than 9% of Lactobacillus spp. In conclusion, this study increased the knowledge on the genetic determinants of hydroxycinnamic acid metabolism, explaining the species- and strain-specific metabolic variations in lactobacilli and providing evidence of additional enzymes involved in hydroxycinnamic acid metabolism of lactobacilli.IMPORTANCE The metabolism of secondary plant metabolites, including phenolic compounds, by food-fermenting lactobacilli is a significant contributor to the safety, quality, and nutritional quality of fermented foods. The enzymes mediating hydrolysis, reduction, and decarboxylation of phenolic acid esters and phenolic acids in lactobacilli, however, are not fully characterized. The genomic analyses presented here provide evidence for three novel putative phenolic acid reductases. Matching comparative genomic analyses with phenotypic analysis and quantification of gene expression indicates that two of the three putative phenolic acid reductases, Par1 and HcrF, are involved in reduction of hydroxycinnamic acids to phenylpropionic acids; however, the activity of Par2 may be unrelated to phenolic acids and recognizes other secondary plant metabolites. These findings expand our knowledge on the metabolic potential of lactobacilli and facilitate future studies on activity and substrate specificity of enzymes involved in metabolism of phenolic compounds.

Isolation, characterisation and exploitation of lactic acid bacteria capable of efficient conversion of sugars to mannitol

The demand for sugar reduction in products across the food and beverage industries has evoked the development of novel processes including the application of fermentation with lactic acid bacteria. Heterofermentative lactic acid bacteria (LAB) are diverse in their ability to utilise fermentable sugars and can also convert fructose into the sweet tasting polyol, mannitol. The sourdough microbiota has long been recognised as an ecological niche for a range of homofermentative and heterofermentative lactic acid bacteria. A leading determinant in the biodiversity of sourdough microbial populations is the type of flour used. Ten non-wheat flours were used and back-slopped for 7 days resulting in the isolation of 52 mannitol producing isolates which spanned six heterofermentative species of the genera Lactobacillus, Leuconostoc and Weissella. Assessment of mannitol productivity in fructose concentrations up to 100 g/L found Leuconostoc citreum TR116, to have the best mannitol producing characteristics, consuming 95% of available fructose and yielding 0.68 g of mannitol per gram of fructose consumed which equates to the maximal theoretical yield. Investigation of the effects of initial pH on mannitol production and other fermentation parameters in the isolates found pH 7 to be best for isolates Lactobacillus brevis TR052, Leuconostoc fallax TR111, Leuconostoc citreum TR116, Leuconostoc mesenteroides TR154 and Weissella paramesenteroides TR212, while pH 6 was optimal for Leuconostoc pseudomesenteroides TR080. The fermentation of apple juice with each isolate resulted in sugar reduction ranging from 30.3–74.0 g/L (34–72%). When apple juice fermentation with Leuconostoc citreum TR116 was scaled up to 1 L bioreactor a reduction in sugar of 98.6 g/L (83%) was achieved along with the production of 61.6 g/L mannitol. This demonstrates a fermentative process for sugar reduction in fruit juice with concomitant production of the sweet metabolite mannitol to create a fermentate that is suitable for further development as a low sugar fruit juice alternative.

Microbial degradation of wastewater

Pollution of the pond and lakes where waste water is discharged has a negative impact on the state of its flora and fauna. The purpose of the work was to study the species composition of bacteria involved in the process of industrial wastewater treatment in the Leningrad region. Biological treatment, which is based on microbiological processes, is the most cost-effective and affordable. Amount of bacteria was from 1091012 CFU / cm3. Biological wastewater treatment is the result of the active sludge - waste water system. The quality of wastewater treatment is determined by the content of coliform bacteria, oliphages in treated wastewater. For wastewater treatment, it is advisable to use bacteria that participate in the anaerobic oxidation of ammonium. Bacteria performing anammox belong to the phylum Planctomycetes. During cleaning of wastewater with a high content of carbohydrates and nitrogen deficiency, there was an intensive development of heterofermentative lactic acid bacteria of the genus Leuconostoc, which form a dextran capsule that makes it difficult to deposit silt in the secondary settling tank. With insufficient level of aeration anaerobic processes with the participation of microorganisms that perform butyric fermentation, sulfate reduction, denitrification, and the like were found. It is advisable to use nitrifying bacteria as indicators of the purification process, since their appearance in the treated water indicates the mineralization of the main part of organic substances.

A metabolic reconstruction of Lactobacillus reuteri JCM 1112 and analysis of its potential as a cell factory

BackgroundLactobacillus reuteri is a heterofermentative Lactic Acid Bacterium (LAB) that is commonly used for food fermentations and probiotic purposes. Due to its robust properties, it is also increasingly considered for use as a cell factory. It produces several industrially important compounds such as 1,3-propanediol and reuterin natively, but for cell factory purposes, developing improved strategies for engineering and fermentation optimization is crucial. Genome-scale metabolic models can be highly beneficial in guiding rational metabolic engineering. Reconstructing a reliable and a quantitatively accurate metabolic model requires extensive manual curation and incorporation of experimental data.ResultsA genome-scale metabolic model of L. reuteri JCM 1112T was reconstructed and the resulting model, Lreuteri_530, was validated and tested with experimental data. Several knowledge gaps in the metabolism were identified and resolved during this process, including presence/absence of glycolytic genes. Flux distribution between the two glycolytic pathways, the phosphoketolase and Embden–Meyerhof–Parnas pathways, varies considerably between LAB species and strains. As these pathways result in different energy yields, it is important to include strain-specific utilization of these pathways in the model. We determined experimentally that the Embden–Meyerhof–Parnas pathway carried at most 7% of the total glycolytic flux. Predicted growth rates from Lreuteri_530 were in good agreement with experimentally determined values. To further validate the prediction accuracy of Lreuteri_530, the predicted effects of glycerol addition and adhE gene knock-out, which results in impaired ethanol production, were compared to in vivo data. Examination of both growth rates and uptake- and secretion rates of the main metabolites in central metabolism demonstrated that the model was able to accurately predict the experimentally observed effects. Lastly, the potential of L. reuteri as a cell factory was investigated, resulting in a number of general metabolic engineering strategies.ConclusionWe have constructed a manually curated genome-scale metabolic model of L. reuteri JCM 1112T that has been experimentally parameterized and validated and can accurately predict metabolic behavior of this important platform cell factory.

Comparative genome analysis reveals metabolic traits associated with probiotics properties in Lactobacillus reuteri KUB-AC5

Lactobacillus reuteri KUB-AC5 is a heterofermentative lactic acid bacteria that was isolated from the chicken intestine. Due to L. reuteri KUB-AC5 has been often used as probiotics, there is still largely unknown on its genes and functions in relation to probiotic properties. This study therefore aimed to perform a genome sequencing of L. reuteri KUB-AC5 and further accomplished comparative analysis with overall identifying its unique/shared metabolic genes and functions across L. reuteri genomes. Accordingly, a genome sequence of the L. reuteri KUB-AC5 has a total length of 2,187,995 base pairs with a GC content of 39.29%. Based metabolic annotation, out of 2196 protein-encoding genes, 477 genes were mostly found to be involved in the metabolism, especially in carbohydrates, cofactors and vitamins. Of these, interestingly, cobalamin (vitamin B12) and folate (vitamin B9) were proposed to be in relation to probiotic properties of L. reuteri KUB-AC5. Considering gene clusters identification, putative unique exopolysaccharide (EPS) biosynthetic gene cluster was also identified in this KUB-AC5 strain. In context of bacteriocin-producing strain, the L.reuteri KUB-AC5 genome promisingly displays to have its genes involved in functional roles of immunity, regulator and transporter. Our study serves for guiding on how L. reuteri KUB-AC5 is used as probiotics for impact to host health. This study shows a broader impact for designing efficient probiotics for industrial foods and feeds.

The influence of different lactic acid bacteria on sourdough flavor and a deep insight into sourdough fermentation through RNA sequencing

Ten lactic acid bacteria (LAB) strains of different species isolated from Chinese traditional sourdough were investigated on their volatile compounds in sourdough fermentation. Sourdoughs fermented with different LAB species were analyzed by principal component analysis (PCA) based on their volatiles. Furthermore, the in situ gene expressions of Lactobacillus sanfranciscensis during sourdough fermentation were investigated by using RNA sequencing for the first time. PCA could discriminate between the sourdoughs fermented by homofermentative and heterofermentative LAB, the former containing more aldehydes and ketones with more than 6 carbon atoms, while the latter featuring ethanol and esters. The Lactobacillus plantarum-fermented sourdough was characterized by high C4–C6 volatiles content, while L. sanfranciscensis showed a unique volatile profile without explicit explanatory compounds. The RNA sequencing suggested that, compared to the sourdough fermented for 6 h, L. sanfranciscensis enhanced carbohydrate metabolism and self-protection activities, but decreased cell proliferation in sourdough at 12 h.

Suppression of lactate production of Lactobacillus reuteri JCM1112 by co-feeding glycerol with glucose

Lactate produced by lactic acid bacteria inhibits their growth. To suppress lactate production, it is necessary to regenerate NAD+ consumed by glycolysis with alternative pathways other than lactate dehydrogenase. In a heterofermentative lactic acid bacterium, Lactobacillus reuteri JCM1112, suppression of lactate production by regenerating NAD+ when producing 1,3-propanediol from glycerol was investigated. The bacterium produced lactate with a yield of 4.7±0.8g·g-cell-1 in a batch culture using glucose as the sole carbon source. When glycerol was added to glucose at a molar ratio (rGly/Glc) of three in the batch culture, the bacterium produced 1,3-propanediol at 1.6±0.7g·g-cell-1·h-1 and the lactate yield decreased to 3.6±0.5g·g-cell-1. When glycerol was co-fed with glucose exponentially to give a target specific growth rate of 0.1 h-1 (rGly/Glc=3), the lactate yield decreased to 1.5±0.2g·g-cell-1. The lactate production when glycerol was added together with glucose was reduced to one-third of that observed in the batch culture using glucose as a carbon source.

Microbial diversity of Livanjski cheese with the emphasis on lactic acid bacteria based on culture‐dependent and sequencing method

The microbial diversity of artisanal Livanjski cheese from traditional farms and dairies was analysed. The polyphasic approach included the isolation of microorganisms by using culture‐dependent methods and the identification of viable isolates by sequencing methods. In total, 159 isolates were identified, including five strains of yeast and 15 strains of fungi. Twenty‐two species of homofermentative and heterofermentative lactic acid bacteria were classified. The Shannon–Wiener index of diversity ranged from 0.462 to 2.51 within the cheese samples from the farms and dairies. The hygiene conditions during cheese production and the age of the cheese significantly influenced the counts and diversity of microorganisms.

Влияние технологических характеристик различных видов сырья на вкус и аромат кислых элей

Sour ales, traditionally produced in Belgium, Britain, and Germany, have gained wide popularity in America, Europe, and Russia. The paper provides generalized and systematized scientific data related to the technological and biotechnological characteristics of raw materials used in sour ales. The study featured malt, grain products (wheat, corn, rice, barley, rye, and oats), hop, Lactobacillus lactic-acid bacteria, Saccharomyces cerevisiae brewer’s yeast, and Brettanomyces yeast. Sour ales are usually prepared from a combination of different malts, such as Pilsner, Vienna, and Munich. Pilsner is used for malt type, while Munich is mostly employed as a basisfor dark varieties of sour ales. The review presents some of the flavor characteristics of malt types, as well as their recommended content. Unmalted grain products have a significant impact on the technological process and the organoleptic characteristics of acid ales. Unmalted wheat is used for Belgian sour ales, such as Lambic, Fruit Lambic, and Gueuze, in the amount of 30–40%, while 50 % are used in the German variety of Berliner Weiss. In general, the degree of wort pitching for sour ales should not exceed 8-15 IBU, since hop acids kill lactic-acid bacteria. Common hop varieties with a low or medium content of α-acids were found optimal for sour ale production. For example, for Flanders Red Ale, producers most often use one of the best traditional European varieties, namely Saazer (Zatetsky).The following types of homo- and heterofermentative lactic-acid bacteria are recommended for ale production technology: Lactobacillus delbrueckii, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus fermentum, and Lactobacillus plantarum. The aromatic characteristics of the wort fermented with lactic acid bacteria are described in the following terms: bread, yeast, honey, oil, cider, etc. Lactic acid bacteria are used in sour ales production, as well as Brettanomyces yeasts. Currently, brewing widely employstwo types of yeast: Brettanomyces bruxellensis and Brettanomyces anomalus. They give the drink a specific taste and aroma, due to hydroxycinnamic acids (HCAs) and esters (ethyl acetate, ethyl lactate, phenylacetate, etc.). The paper contains some practical recommendations on the use of specific types of raw materials to produce sour ales with a given sensory profile.

Lactic acid bacteria modulate organic acid production during early stages of food waste composting

Lactic acid bacteria are observed during early stages of almost all food waste composting. Among them, 2 types of lactic acid bacteria, Pediococcus (homofermentative lactic acid bacterium) and Weissella (heterofermentative lactic acid bacterium) have been often reported. In this study, the roles of these 2 types of lactic acid bacteria in the composting were tried to elucidate. It has been pointed out that Pediococcus accelerates the composting process by producing lactic acid which prevented acetic acid generation, thus activating indigenous composting microorganisms. On the other hand, this study elucidated that Weissella produced acetic acid of 20 mg g−1 DS, which is harmful to composting microorganisms, resulting in the inhibition of vigorous organic matter degradation. When these 2 coexist in the starting material, whether the composting succceeds or not depends on the ratio of these 2 lactic acid bacteria. If Pediococcus and Weissella ratio was higher than 101.5, acetic acid level was almost 3 times lower than that observed in the composting with their lower ratios of 1 and 10−1, probably because of the interaction of Pediococcus and Weissella resulting in the suppression of Weissella activity, and thus composting was accelerated.

Synergetic effect of combined ensiling of freshly harvested and excessively wilted maize stover for efficient biogas production

This study investigated the synergetic effects of ensiling freshly harvested maize stover (FHM) and excessively wilted maize stover (EWM) on biogas production. FHM and EWM were mixed in various proportions to obtain dry matter (DM) contents of 30%, 35% and 40%. For reference, FHM alone was ensiled and stored in open-air. Successful storage performance was obtained by the ensiling treatments, and the organic matter loss of 1.1–2.2% was far lower than in open-air storage (63.1%). An initial water-soluble carbohydrate (WSC) of 5% DM is adequate for the combined ensiling of maize stover with the highest WSC degradation rate of 81.2%. Combined ensiling enhanced the activity of Weissella, a genus of heterofermentative lactic acid bacteria, under relatively high pH conditions. Therefore, the combined ensiling can preserve FHM and enhance the digestibility of EWM (theoretical specific methane yield increased 16.5%), which would be a promising storage strategy for efficient biogas production.

Uncovering carbohydrate metabolism through a genotype-phenotype association study of 56 lactic acid bacteria genomes

Owing to their unique potential to ferment carbohydrates, both homo- and heterofermentative lactic acid bacteria (LAB) are widely used in the food industry. Deciphering the genetic basis that determine the LAB fermentation type, and hence carbohydrate utilization, is paramount to optimize LAB industrial processes. Deep sequencing of 24 LAB species and comparison with 32 publicly available genome sequences provided a comparative data set including five major LAB genera for further analysis. Phylogenomic reconstruction confirmed Leuconostoc and Pediococcus species as independently emerging from the Lactobacillus genus, within one of the three phylogenetic clades identified. These clades partially grouped LABs according to their fermentation types, suggesting that some metabolic capabilities were independently acquired during LAB evolution. In order to apply a genome-wide association study (GWAS) at the multigene family level, utilization of 49 carbohydrates was also profiled for these 56 LAB species. GWAS results indicated that obligately heterofermentative species lack 1-phosphofructokinase, required for d-mannose degradation in the homofermentative pathway. Heterofermentative species were found to often contain the araBAD operon, involved in l-arabinose degradation, which is important for heterofermentation. Taken together, our results provide helpful insights into the genetic determinants of LAB carbohydrate metabolism, and opens for further experimental research, aiming at validating the role of these candidate genes for industrial applications.

The molecular and phenotypic characterization of fructophilic lactic acid bacteria isolated from the guts of Apis mellifera L. derived from a Polish apiary

This paper describes taxonomic position, phylogeny, and phenotypic properties of 14 lactic acid bacteria (LAB) originating from an Apis mellifera guts. Based on the 16S rDNA and recA gene sequence analyses, 12 lactic acid bacteria were assigned to Lactobacillus kunkeei and two others were classified as Fructobacillus fructosus. Biochemically, all isolated lactic acid bacteria showed typical fructophilic features and under anaerobic conditions grew well on fructose, but poorly on glucose. Fast growth of bacteria on glucose was noted in the presence of oxygen or fructose as external electron acceptors. The residents of honeybee guts were classified as heterofermentative lactic acid bacteria. From glucose, they produced almost equimolar amounts of lactic acid, acetic acid, and trace amounts of ethanol. Furthermore, they inhibited the growth of the major honeybee pathogen, Paenibacillus larvae, meaning that the LAB studied may have the health-conferring properties of probiotics.

A meta-analysis on the effectiveness of homofermentative and heterofermentative lactic acid bacteria for corn silage.

This meta-analysis aims to assess the effect of lactic acid bacteria (LAB) inoculation on fermentation parameters, microbiological composition and aerobic stability of corn silage. Databases (PubMed, ScienceDirect and Scopus) were searched from 1980 to 2017. The criteria for inclusion were: randomized and controlled experiments using corn silage and published in peer-reviewed journals. The meta-analysis showed that LAB supplementation increased pH, acetate and propionate concentrations, and decreased acid detergent fibre, water-soluble carbohydrates and ammoniacal nitrogen (NH3 -N) compared to controls in the pooled raw mean difference random effect model. In addition, inoculation reduced counts of yeasts and moulds, increased LAB counts and markedly improved aerobic stability in corn silage. However, results indicated that the effect of inoculants may differ depending on the administration of homofermentative or heterofermentative LAB. For the development of functional bacterial inoculants, both types of LAB should be used. To our knowledge, this is the first meta-analysis to compare the application of homofermentative and heterofermentative LAB for corn silage.

Polyol-producing lactic acid bacteria isolated from sourdough and their application to reduce sugar in a quinoa-based milk substitute

The interest for plant-based dairy substitutes is expanding rapidly and consumers are opting for nutritious and healthy dairy alternatives. The reduction of sugar using different exogenous enzymes in combination with lactic acid fermentation in a quinoa-based milk substitute was explored in this study. Different amylolytic enzymes were used to release sugar from the raw material, which were further metabolised to mannitol, due to fermentation with two heterofermentative lactic acid bacteria. Using these two biotechnological techniques enables the reduction of sugar, while also preserving some of the sweetness. Leuconostoc citreum TR116, and Lactobacillus brevis TR055 were isolated from sourdough. Both strains showed high viable cell counts with L. citreum TR116 > 8.4 and L. brevis TR055 > 9.3 log cfu/mL, and a reduction in pH to 3.7 and 3.5 respectively. When fructose was available, mannitol was produced in conjunction with acetic acid in addition to lactic acid. Due to these processes, the original glucose value was reduced from 50 mmol/100 g to approximately 30 mmol/100 g, which equates to a glucose reduction of 40%. In respect to mannitol production, both strains performed well: L. citreum TR116 showed a conversion factor of 1:1 from fructose to mannitol, while L. brevis TR055 showed a lower yield, with a conversion factor of 1:0.8. Glycaemic load was reduced by more than a third, bringing it down to the low range with a value of about 10. Overall, enzymatic modification in conjunction with mannitol-producing lactic acid bacteria shows great potential for further possible application in the development of nutritious and sugar reduced plant-based milk substitutes.

Bacterial diversity and community structure in Chongqing radish paocai brines revealed using PacBio single-molecule real-time sequencing technology.

Traditional Chongqing radish paocai fermented with aged brine is considered to have the most intense flavor and authentic taste. Eight 'Yanzhi' (red, RRPB group) and 'Chunbulao' (white, WRPB) radish paocai brine samples were collected from Chongqing peasant households, and the diversity and community structures of bacteria present in these brines were determined using PacBio single-molecule real-time sequencing of their full-length 16S rRNA genes. In total, 30 phyla, 218 genera, and 306 species were identified from the RRPB group, with 20 phyla, 261 genera, and 420 species present in the WRPB group. Obvious differences in bacterial profiles between the RRPB and WRPB groups were found, with the bacterial diversity of the WRPB group shown to be greater than that of the RRPB group. This study revealed several characteristics of the bacteria composition, including the predominance of heterofermentative lactic acid bacteria, the species diversity of genus Pseudomonas, and the presence of three opportunistic pathogenic species. This study provides detailed information on the bacterial diversity and community structure of Chongqing radish paocai brine samples, and suggests it may be necessary to analyze paocai brine for potential sources of bacterial contamination and take appropriate measures to exclude any pathogenic species. © 2018 Society of Chemical Industry.

Effects of Storage Conditions, Total Solids Content and Silage Additives on Fermentation Profiles and Methane Preservation of Cattle Manure Before Anaerobic Digestion

The impacts of storage conditions, total solids content and silage additives were investigated at laboratory scale for cattle manure during up to 120 days. Wheat straw was used as co-substrate in the study of the effects of total solids content. Glucose and starch were used as model molecules of sugar-rich co-substrates. Single-handedly cattle manure lost 37% of its original methane potential after 120 days of anaerobic storage. This was essentially caused by the low total solids content and by the absence of water soluble carbohydrates in the feedstock. Wheat straw addition decreased moisture content and enabled cattle manure stabilization for a pH of 6.0. In contrast, extensive heterofermentative lactic acid bacteria fermentation occurred during co-ensiling with glucose and the pH quickly dropped to values below 4.0. Starch was used as substrate for fermentation but its hydrolysis limited bacterial activity and acidification. Finally, all the three co-substrates led to minimal methane potential losses after 120 days of storage. Co-ensiling will improve the energy efficiency of agricultural biogas plants that use stored cattle manure as feedstock.

Aerobic and respirative growth of heterofermentative lactic acid bacteria: A screening study

Heterofermentative lactic acid bacteria (76 strains) belonging to Lactobacillus, Leuconostoc and Weissella species which are important in fermentation, spoilage or as probiotics were screened in a factorial experiment for their ability to grow, produce catalase and consume oxygen in aerobiosis or in anaerobiosis, with or without supplementation with hemin and/or menaquinone in a medium containing glucose as a carbohydrate source. Aerobiosis improved growth with a few exceptions. The effect of supplementation with heme and/or menaquinone was strain specific and clear evidence of heme-boosted respiration was found in some cases. Heme-catalase was produced by strains of L. brevis, W. minor and Leuc. mesenteroides; some strains of the latter species produced non-heme catalase. Shaken flasks experiments showed that aerobic growth resulted in increased maximum growth rate and in a limited increase in biomass. Heme supplementation during aerobic growth resulted in a further increase in growth rate and final biomass only for a few strains; this was often related to catalase, which was also responsible for increased tolerance of H2O2. In both experiments we found evidence of heme toxicity, especially in anaerobiosis and in absence of menaquinone. Dose response curves for aerobic growth in the presence of combinations of hemin and menaquinone were non-monotonic, with growth stimulation at low doses of heme (<2.5 mg/l) and toxicity at higher doses. Menaquinone at 0.25–8 mg/l increased growth stimulation and partially reduced toxicity.