Investigation of microorganisms involved in kefir biofilm formation

Kefir is a natural fermentation agent composed of various microorganisms. To address the mechanism of kefir grain formation, we investigated the microbial role in forming kefir biofilms. The results showed that a biofilm could be formed in kefir-fermented milk and the biofilm forming ability reached the maximum at 13 days. The strains Kluyveromyces marxianus, Lactococcus lactis, Leuconostoc mesenteroides, Lactobacillus kefiri, Lactobacillus sunkii and Acetobacter orientalis were isolated from kefir biofilms by the streak-plate method. These microorganisms were analysed with respect to biofilm forming properties, including their surface characterisation (hydrophobicity and zeta potentials) and the microbial aggregation. The results indicated that Klu. marxianus possessed the strongest biofilm forming properties with the strongest hydrophobicity, lowest zeta potential and greatest auto-aggregation ability. When Klu. marxianus and Ac. orientalis were co-cultured with kefir LAB strains respectively, it was found that mixing Klu. marxianus with Lb. sunkii produced the highest co-aggregation ability. These results elucidated the mechanism of kefir biofilm formation and the microorganisms involved.     

Enhancement of 2,3-butanediol production from Jerusalem artichoke tuber extract by a recombinant <Emphasis Type="Italic">Bacillus</Emphasis> sp. strain BRC1 with increased inulinase activity

A Bacillus sp. strain named BRC1 is capable of producing 2,3-butanediol (2,3-BD) using hydrolysates of the Jerusalem artichoke tuber (JAT), a rich source of the fructose polymer inulin. To enhance 2,3-BD production, we undertook an extensive analysis of the Bacillus sp. BRC1 genome, identifying a putative gene (sacC) encoding a fructan hydrolysis enzyme and characterizing the activity of the resulting recombinant protein expressed in and purified from Escherichia coli. Introduction of the sacC gene into Bacillus sp. BRC1 using an expression vector increased enzymatic activity more than twofold. Consistent with this increased enzyme expression, 2,3-BD production from JAT was also increased from 3.98 to 8.10 g L^?1. Fed-batch fermentation of the recombinant strain produced a maximal level of 2,3-BD production of 28.6 g L^?1, showing a high theoretical yield of 92.3%.     

Traditional fermented beverages from Mexico as a potential probiotic source

Fermentation is one of the oldest ways of processing food. Some fermented food is produced industrially, but can also be produced in an artisanal way by certain ethnic groups, called traditional fermented foods. In Mexico, there are a variety of traditional fermented beverages which are produced in an artisanal way. They include those made with maize (atole agrio, pozol, and tesgüino), fruit (tepache and colonche), and obtained by plant fermentation (pulque, tuba, and taberna). These beverages have been used since ancient times for religious and medicinal purposes. The medicinal effect may be due to fermented microorganisms. The presence of beneficial microorganisms known as probiotics provides beneficial effects to consumer health, improving the balance of intestinal host, and reducing the risk of gastrointestinal diseases, mainly. Most probiotics belong to the genus Lactobacillus, but Bifidobacterium, Bacillus, and yeast are also found. Therefore, it is important that the microbiological diversity of the beverages is studied and documented. This review includes information on the microbial diversity and probiotic potential of the most important traditional fermented beverages from Mexico.     

Polymorphism of lactose genes in the dairy yeasts <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>, potential probiotic microorganisms

Molecular karyotyping and Southern blot hybridization were used to investigate chromosomal polymorphism of the LAC genes controlling lactose fermentation in Kluyveromyces marxianus strains isolated from various dairy products and natural sources in Russia and CIS countries. Profound polymorphism of karyotype patterns and accumulation of LAC genes were observed in dairy K. marxianus strains. K. marxianus strains isolated from dairy products intensively fermented lactose at 37°C after one day of cultivation, while non-dairy strains exhibited delayed lactose fermentation or did not ferment it at all. Based on the fermentation tests, twelve K. marxianus strains were selected, which are of interest as potential probiotic microorganisms suitable for further molecular genetic studies and breeding.     

Reconstruction of the spatial structure of inulinase from <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> to find regulatory pathways of its catalytic activity

Reconstruction of the spatial structure of inulinase (EC 3.2.1.7) from Kluyveromyces marxianus (an enzyme that hydrolyzes inulin and other fructose-based polymers to fructose) was carried out by highthroughput computational modeling. A structural model of a closely related homologous protein, viz., invertase from yeast Saccharomyces cerevisiae (PDB-ID: 4EQV), was used as a template. The reconstructed model can be used for computer calculations for optimizing the biotechnological feasibility of inulinase.     

Low-Input Fermentations of <Emphasis Type="Italic">Agave tequilana</Emphasis> Leaf Juice Generate High Returns on Ethanol Yields

During tequila production, up to 75 % w/w of the Agave plant is discarded when leaves are removed from the stem. The discarded leaves represent an extensive amount of unexploited biomass that was used here for bioethanol production in no-input fermentations, where no acid or enzymatic hydrolysis, supplementation of nutrients or standardization of carbohydrate content occur. Ethanol yield from Agave leaf juice is unaffected by sterilization but reduced if fermentation is reliant solely on endogenous microorganisms. Non-Saccharomyces yeasts, including Kluyveromyces marxianus and Candida akabanensis, proved to be more robust than standard Saccharomyces spp. and yielded up to 88 % of the theoretical maximum ethanol from leaf juice. Combining leaf and stem juice, as from a whole plant, was predicted to maximize yield at up to 19,439 L/ha of ethanol from mature plants.     

Model-based biotechnological potential analysis of <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> central metabolism

The non-conventional yeast Kluyveromyces marxianus is an emerging industrial producer for many biotechnological processes. Here, we show the application of a biomass-linked stoichiometric model of central metabolism that is experimentally validated, and mass and charge balanced for assessing the carbon conversion efficiency of wild type and modified K. marxianus. Pairs of substrates (lactose, glucose, inulin, xylose) and products (ethanol, acetate, lactate, glycerol, ethyl acetate, succinate, glutamate, phenylethanol and phenylalanine) are examined by various modelling and optimisation methods. Our model reveals the organism’s potential for industrial application and metabolic engineering. Modelling results imply that the aeration regime can be used as a tool to optimise product yield and flux distribution in K. marxianus. Also rebalancing NADH and NADPH utilisation can be used to improve the efficiency of substrate conversion. Xylose is identified as a biotechnologically promising substrate for K. marxianus.     

Rhamnose-Containing Cell Wall Glycopolymers from <Emphasis Type="Italic">Rathayibacter toxicus</Emphasis> VKM Ac-1600 and “<Emphasis Type="Italic">Rathayibacter tanaceti</Emphasis>” VKM Ac-2596

Structures of the cell wall glycopolymers from two representatives of the genus Rathayibacter were investigated using chemical, NMR spectroscopy, and optical methods. The R. toxicus VKM Ac-1600 strain contains two neutral glycopolymers–a linear rhamnomannan →2)-α-D-Rhap-(1→3)-α-D-Manp-(1→ and a branched polysaccharide containing in the repeating unit the residues of D-Manp, D-Glcp, and L-Rhap in the ratios of 2: 4: 1, respectively (the structure is presented in the text). The “Rathayibacter tanaceti” VKM Ac-2596 contains a rhamnomannan that is different from the above-described one by localization of glycosidic bonds on the residues of α-Rhap and α-Manp, i.e. →3)-α-D-Rhap (1→2)-α-D-Manp-(1→. The structures of all identified glycopolymers are described for the first time in actinobacteria. The data obtained make it possible to characterize representatives of the studied actinobacteria more fully and can be used to differentiate Rathayibacter species at the phenotype level.     

Isolation and characterization of marine biofilm forming bacteria from a ship’s hull

Background

Diverse aquatic microorganisms are capable of colonizing living and non-living surfaces leading to the formation of biofilms. Commonly visualized as a slimy layer, these biofilms are filled with hundreds of other microorganisms compared to free living planktonic cells. Microbial surface colonization and surface-associated metabolic activities also exert several macroscale deleterious effects, including biofouling, biocorrosion and the persistence and transmission of harmful or pathogenic microorganisms and virulence determinants. The present study deals with the isolation and screening of marine bacteria for biofilm formation. The screened isolates were characterized and identified as Pychrobacter celer, Pychrobacter alimentarius and Kocuria rhizophila by 16S rRNA sequencing.

Methods

Biofilm forming bacteria were isolated by spread plate technique and subjected to screening by microtiter plate assay. The potent biofilm formers were identified by molecular characterization using 16S rRNA gene sequencing.

Results

Twelve bacterial isolates were obtained by pour plate technique and subjected to biofilm assay. Among the 12 isolates three isolates which showed maximum biofilm formation were subjected to molecular characterizationby 16S rRNA gene sequencing method. The isolates were identified as Pychrobacter celer, Pychrobacter alimentarius and Kocuria rhizophila. The EPS produced by the three biofilm forming bacteria was extracted and the protein and carbohydrate content determined.

Conclusion

Among the isolates screened, isolate 8 (Kocuria rhizophila) produced maximum protein and carbohydrate which was also in accordance with the results of microtiter plate assay.

     

Phylogenetic Diversity of Microorganisms from the Sludge of a Biogas Reactor Processing Oil-Containing and Municipal Waste

High-throughput sequencing of the 16S rRNA gene fragments was used to determine the phylogenetic diversity of prokaryotes, including human pathogens, in the liquid phase of the sludge of a biogas reactor processing oil-containing and municipal waste. A unique microbial community was found to develop in the sludge, which comprised the microorganisms of municipal wastewater (bacteria of human feces) and specific groups of aerobic and anaerobic microorganisms, which possibly arrived with oil-containing water. In the 16S rRNA gene library, the sequences of representatives of Firmicutes prevailed (54.9%), which belonged to anaerobic bacteria of the genera Gelria (26.6%), Syntrophomonas (6.0%), Lutispora (2.0%), and uncultured Clostridia (group MBA03, 11.1%). The Proteobacteria sequences (20.7%) belonged mostly to the metabolically diverse members of the genus Pseudomonas (13.8%). The phylum Bacteroidetes (7%) was represented by uncultured bacteria (VadinBC27 wastewater-sludge group), while members of the phylum Cloacimonetes were mainly syntrophic bacteria Candidatus Cloacamonas (7.5%). The sequences of bacteria commonly occurring in oilfields (Clostridia, Anaerolinea, Bacteroidetes, sulfate-reducing Deltaproteobacteria, members of the family Syntrophaceae, and of the genera Thauera, Pseudomonas, Dechloromonas, and Petrimonas) were revealed. No sequences of bacteria known to be pathogenic to humans were found. The cultured microorganisms were aerobic organotrophic and anaerobic fermenting, denitrifying, and methanogenic prokaryotes. Fermenting and methanogenic enrichments grew on a broad range of organic substrates (sucrose, glycerol, starch), producing volatile fatty acids (acetate, n-butyrate, and propionate), gases (Н₂, СО₂, and CH₄), and decreasing pH of the medium from 7.0 to 4.5–5.0. The possible application of the biogas reactor sludge as a source of fermenting and methanogenic anaerobic prokaryotes, as well as of aerobic hydrocarbonoxidizing bacteria for oilfield introduction and for production of new preparations for enhanced oil recovery and for bioremediation of oil contamination is discussed.     

Progress in the microbial production of <Emphasis Type="Italic">S</Emphasis>-adenosyl-<Emphasis Type="SmallCaps">l</Emphasis>-methionine

S-Adenosyl-l-methionine (SAM), which exists in all living organisms, serves as an activated group donor in a range of metabolic reactions, including trans-methylation, trans-sulfuration and trans-propylamine. Compared with its chemical synthesis and enzyme catalysis production, the microbial production of SAM is feasible for industrial applications. The current clinical demand for SAM is constantly increasing. Therefore, vast interest exists in engineering the SAM metabolism in cells for increasing product titers. Here, we provided an overview of updates on SAM microbial productivity improvements with an emphasis on various strategies that have been used to enhance SAM production based on increasing the precursor and co-factor availabilities in microbes. These strategies included the sections of SAM-producing microbes and their mutant screening, optimization of the fermentation process, and the metabolic engineering. The SAM-producing strains that were used extensively were Saccharomyces cerevisiae, Pichia pastoris, Candida utilis, Scheffersomyces stipitis, Kluyveromyces lactis, Kluyveromyces marxianus, Corynebacterium glutamicum, and Escherichia coli, in addition to others. The optimization of the fermentation process mainly focused on the enhancement of the methionine, ATP, and other co-factor levels through pulsed feeding as well as the optimization of nitrogen and carbon sources. Various metabolic engineering strategies using precise control of gene expression in engineered strains were also highlighted in the present review. In addition, some prospects on SAM microbial production were discussed.     

Reaction of microorganisms to the digestive fluid of earthworms

The reaction of soil bacteria and fungi to the digestive fluid of the earthworm Aporrectodea caliginosa was studied. The fluid was obtained by centrifugation of the native enzymes of the digestive tract. The inhibition of growth of certain bacteria, spores, and fungal hyphae under the effect of extracts from the anterior and middle sections of the digestive tract of A. caliginosa was discovered for the first time. In bacteria, microcolony formation was inhibited as early as 20–30 s after the application of the gut extracts, which may indicate the nonenzymatic nature of the effect. The digestive fluid exhibited the same microbicidal activity whether the earthworms were feeding on soil or sterile sand. This indicates that the microbicidal agents are formed within the earthworm’s body, rather than by soil microorganisms. The effect of the digestive fluid from the anterior and middle divisions is selective in relation to different microorganisms. Of 42 strains of soil bacteria, seven were susceptible to the microbicidal action of the fluid (Alcaligenes faecalis 345-1, Microbacterium sp. 423-1, Arthrobacter sp. 430-1, Bacillus megaterium 401-1, B. megaterium 413-1, Kluyvera ascorbata 301-1, Pseudomonas reactans 387-2). The remaining bacteria did not die in the digestive fluid. Of 13 micromycetes, the digestive fluid inhibited spore germination in Aspergillus terreus and Paecilomyces lilacinus and the growth of hyphae in Trichoderma harzianum and Penicillium decumbens. The digestive fluid stimulated spore germination in Alternaria alternata and the growth of hyphae in Penicillium chrysogenum. The reaction of the remaining micromycetes was neutral. The gut fluid from the posterior division of the abdominal tract did not possess microbicidal activity. No relation was found between the reaction of microorganisms to the effects of the digestive fluid and the taxonomic position of the microorganisms. The effects revealed are similar to those shown earlier for millipedes and wood lice in the following parameters: quick action of the digestive fluid on microorganisms, and the selectivity of the action on microorganisms revealed at the strain level. The selective effect of the digestive gut fluid of the earthworms on soil microorganisms is important for animal feeding, maintaining the homeostasis of the gut microbial community, and the formation of microbial communities in soils.     

Growth,ethanol production,and inulinase activity on various inulin substrates by mutant <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> strains NRRL Y-50798 and NRRL Y-50799

Economically important plants contain large amounts of inulin. Disposal of waste resulting from their processing presents environmental issues. Finding microorganisms capable of converting inulin waste to biofuel and valuable co-products at the processing site would have significant economic and environmental impact. We evaluated the ability of two mutant strains of Kluyveromyces marxianus (Km7 and Km8) to utilize inulin for ethanol production. In glucose medium, both strains consumed all glucose and produced 0.40 g ethanol/g glucose at 24 h. In inulin medium, Km7 exhibited maximum colony forming units (CFU)/mL and produced 0.35 g ethanol/g inulin at 24 h, while Km8 showed maximum CFU/mL and produced 0.02 g ethanol/g inulin at 96 h. At 24 h in inulin + glucose medium, Km7 produced 0.40 g ethanol/g (inulin + glucose) and Km8 produced 0.20 g ethanol/g (inulin + glucose) with maximum CFU/mL for Km8 at 72 h, 40 % of that for Km7 at 36 h. Extracellular inulinase activity at 6 h for both Km7 and Km8 was 3.7 International Units (IU)/mL.     

Production of vineomycin A1 and chaetoglobosin A by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. PAL114

An actinobacteria strain PAL114, isolated from a Saharan soil in Algeria, produces bioactive compounds. Morphological and chemical studies indicated that this strain belongs to the genus Streptomyces. Analysis of the 16S rRNA gene sequence showed a similarity level of 99.8 % with S. griseoflavus LMG 19344^T, the most closely related species. Two bioactive compounds, named P44 and P40, were extracted by dichloromethane from the cell-free supernatant broth and were purified by HPLC. Minimum inhibitory concentrations (MIC) of the compounds were determined against pathogenic and toxigenic microorganisms, most of which are multiresistant to antibiotics. The P40 fraction showed a strong activity especially against Candida albicans, Bacillus subtilis, and Staphylococcus aureus and has lower MIC values than those of P44 against most microorganisms tested. Chemical structures of compounds were determined based on spectroscopic and spectrometric analyses (UV-visible, mass, ¹H, and ¹³C NMR spectra). The compounds P44 and P40 were identified as vineomycin A1 and chaetoglobosin A, respectively. Vineomycin A1 is known to be produced by some Streptomyces species. However, chaetoglobosin A is known to be produced only by fungi belonging to the genera Chaetomium, Penicillium, and Calonectria. This is the first time that chaetoglobosin A, known for its antimicrobial, anticancer, and cytotoxic effects, is reported in prokaryotes.     

The Molecular mechanism of the action of <Emphasis Type="Italic">Helianthus tuberosus</Emphasis> L. polysaccharide

Evidence for immunoadjuvant activity of the Helianthus tuberosus L. polysaccharide was obtained in an antibody-producing cell model. Dectin-1 and TLR-6 were identified as major receptors required for biological activity of polysaccharide in a TNF-α stimulation model. The CR3 receptor was not implicated in polysaccharide recognition in the same model. Enzyme treatment of the H. tuberosus L. polysaccharide demonstrated the presence of ß-(1 → 4) and ß-(1 → 3) glycosidic bonds.     

Hydrolytic activities of hydrolase enzymes from halophilic microorganisms

Biomass is normally processed using acidic or basic catalysts, which both have their drawbacks. One suitable alternative is the application of hydrolytic enzymes that can convert biomass into simpler molecules, which can be fermented and processed into biofuel. Hydrolytic enzymes include proteases, lipases, amylases, cellulases, mannanases, chitinases, and xylanases. To discover sources of these enzymes, 19 halophilic strains of microorganisms that are significantly resistant to high salt concentrations were analyzed. The objective of this research was to identify halophilic microorganisms that produce the target enzymes with high activities, and to characterize these enzymes according to their salt tolerances. The results obtained indicated that Pseudolateromonas phenolica, Micrococcus luteus, Pseudoalteromonas peptidolytica, Halomonas socia, Marinobacter maritimus, and Exiguobacterium aurantiacum strain 2 produced the highest protease, lipase, amylase, cellulase, mannanase, chitinase, and xylanase relative activities, respectively. Except for protease from P. phenolica, all the enzymes tested for salt resistance either maintained or increased their activities with increasing NaCl concentration.     

Isolation and molecular characterization of the fungal endophytic microbiome from conventionally and organically grown avocado trees in South Florida

Fungal endophytes are the most ubiquitous and highly diverse microorganisms that inhabit the interior of healthy plants. They are important in plant ecology and offer untapped potential to improve plant health and productivity in agroecosystems. The endophytic assemblage of avocado is poorly understood; therefore, surveys of fungal endophytes of Persea americana Mill. (Avocado) in South Florida organic and conventional orchards were conducted. A total of 17 endophytic fungal species were recovered from healthy avocado terminal branches. Endophytic fungal species were identified by rDNA sequencing of the internal transcribed spacer (ITS) region, using UNITE Species Hypotheses to reliably assign a taxon name, and determined as belonging to the genera Alternaria, Cladosporium, Colletotrichum, Corynespora, Diaporthe, Lasiodiplodia, Neofusicoccum, Neopestalotiopsis, Phyllosticta, and Strelitziana. Endophyte community assemblage differed between organic and conventional agroecosystems. This is the first report of Alternaria eichhorniae, Cladosporium tenuissimum, Corynespora cassiicola, Colletotrichum alatae, Diaporthe fraxini-angustifoliae, Lasiodiplodia gonubiensis, Neofusicoccum algeriense, Neofusicoccum andinum, Neopestalotiopsis foedans, Phyllosticta capitalensis, and Strelitziana africana as endophytes of avocado. Evaluation using pathogenicity tests on avocado leaves and terminal branches showed that endophytic fungal isolates did not cause disease symptoms.     

Characterization of extracellular yeast peptide factors and their stress-protective effect on probiotic lactic acid bacteria

Protective effect of the extracellular peptide fraction (reactivating factors, RF) produced by yeasts of various taxonomic groups (Saccharomyces cerevisiae, Kluyveromyces lactis, Candida utilis, and Yarrowia lipolytica) on probiotic lactic acid bacteria (LAB) Lactobacillus casei, L. acidophilus, and L. reuteri under bile salt (BS)-induced stress was shown. RF of all yeasts were shown to be of peptide nature; the active component of the S. cerevisiae RF was identified as a combination of low-molecular polypeptides with molecular masses of 0.6 to 1.5 kDa. The protective and reactivating effects of the yeast factors were not species-specific and were similar to those of the Luteococcus japonicus subsp. casei RF. In BS-treated cells of the tester bacteria, a protective effect was observed after 10-min preincubation of the LAB cell suspension with yeast RF: the number of surviving cells (CFU) was 2 to 4.5 times higher than in the control. The reactivating effect was observed when RF was added to LAB cell suspensions not later than 15 min after stress treatment. It was less pronounced than the protector effect, with the CFU number 1 to 3 times that of the control. Both the protector and the reactivating effects were most pronounced in the S. cerevisiae and decreased in the row C. utilis > K. lactis > Y. lipolytica. The efficiency of protective action of yeast RF was found to depend on the properties of recipient LAB cells, with the L. casei strain being most sensitive to BS treatment. In both variants, the highest protective effect of RF (increase in the CFU number) was observed for L. acidophilus, while the least pronounced one was observed for L. casei. The reasons for application of the LAB strains combining high stress resistance and high response to stress-protecting metabolites, including RF factors, as probiotics, is discussed.     

Culturable microorganisms from the earthworm digestive tract

The cultured aerobic copiotrophic bacteria and fungi from food-free digestive tracts of Aporrectodea caliginosa, Lumbricus terrestris, and Eisenia fetida earthworms, soil (compost), and fresh earthworm excrements were investigated. The microorganisms were isolated on nutrient media and identified by sequencing the fragments of bacterial 16S rRNA and fungal 28S rRNA (D1/D2 domain) gene sequences with subsequent phylogenetic analysis. Bacteria isolated from the digestive tracts of earthworms belonged to the families Aeromonadaceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae, Moraxellaceae, Pseudomonadaceae, and Sphingobacteriaceae (Bacteroidetes), as well as Actinobacteria. For five strains, namely Ochrobactrum sp. 341-2 (α-Proteobacteria), Massilia sp. 557-1 (β-Proteobacteria), Sphingobacterium sp. 611-2 (Bacteroidetes), Leifsonia sp. 555-1, and a bacterium from the family Microbacteriaceae, isolate 521-1 (Actinobacteria), the similarity to known 16S rRNA sequences was 93–97%; they therefore, probably belong to new species and genera. Bacterial groups isolated from the digestive tracts of earthworms were significantly different from those isolated from soil and excrements. Some bacterial taxa occurred in different sections of A. caliginosa intestine and in intestines of different earthworm species; however, the overall composition of bacterial communities in these objects is different. Existence of bacterial groupings symbiotically associated with intestines is proposed. Among the fungi, Bjerkandera adusta and Syspastospora parasitica were isolated from the cleaned digestive tracts as light-colored, sterile mycelium, as well as Geotrichum candidum, Acremonium murorum (A. murorum var. felina), Alternaria alternata, Aspergillus candidus, A. versicolor, Cladosporium cladosporioides, Rhizomucor racemosus, Mucor hiemalis, Fusarium (F. oxysporum, Fusarium sp.), and Penicillium spp. These fungi survive for a long time in the earthworm’s digestive environment. Investigation of the functional characteristics and role in the host organism is required to confirm the symbiotic status of the microorganisms associated with the earthworm digestive tract.