The ruminal microbiome, comprising large numbers of bacteria, ciliate protozoa, archaea and fungi, responds to diet and dietary additives in a complex way. The aim of this study was to investigate the benefits of increasing the depth of the community analysis in describing and explaining responses to dietary changes. Quantitative PCR, ssu rRNA amplicon based taxa composition, diversity and co-occurrence network analyses were applied to ruminal digesta samples obtained from four multiparous Nordic Red dairy cows fitted with rumen cannulae. The cows received diets with forage:concentrate ratio either 35:65 (diet H) or 65:35 (L), supplemented or not with sunflower oil (SO) (0 or 50 g/kg diet dry matter), supplied in a 4 × 4 Latin square design with a 2 × 2 factorial arrangement of treatments and four 35-day periods. Digesta samples were collected on days 22 and 24 and combined. QPCR provided a broad picture in which a large fall in the abundance of fungi was seen with SO in the H but not the L diet. Amplicon sequencing showed higher community diversity indices in L as compared to H diets and revealed diet specific taxa abundance changes, highlighting large differences in protozoal and fungal composition. Methanobrevibacter ruminantium and Mbb. gottschalkii dominated archaeal communities, and their abundance correlated negatively with each other. Co-occurrence network analysis provided evidence that no microbial domain played a more central role in network formation, that some minor-abundance taxa were at nodes of highest centrality, and that microbial interactions were diet specific. Networks added new dimensions to our understanding of the diet effect on rumen microbial community interactions.

So far, the inability to establish viable Lactobacillus surface layer (S-layer) null mutants has hampered the biotechnological applications of Lactobacillus S-layers. In this study, we demonstrate the utilization of Lactobacillus brevis S-layer subunits (SlpA) for the surface display of foreign antigenic epitopes. With an inducible expression system, L. brevis strains producing chimeric S-layers were obtained after testing of four insertion sites in the slpA gene for poliovirus epitope VP1, that comprises 10 amino acids. The epitope insertion site allowing the best surface expression was used for the construction of an integration vector carrying the gene region encoding the c-Myc epitopes from the human c-myc proto-oncogene, which is composed of 11 amino acids. A gene replacement system was optimized for L. brevis and used for the replacement of the wild-type slpA gene with the slpA-c-myc construct. A uniform S-layer, displaying on its surface the desired antigen in all of the S-layer protein subunits, was obtained. The success of the gene replacement and expression of the uniform SlpA-c-Myc recombinant S-layer was confirmed by PCR, Southern blotting MALDI-TOF mass spectrometry, whole-cell enzyme-linked immunosorbent assay, and immunofluorescence microscopy. Furthermore, the integrity of the recombinant S-layer was studied by electron microscopy, which indicated that the S-layer lattice structure was not affected by the presence of c-Myc epitopes. To our knowledge, this is the first successful expression of foreign epitopes in every S-layer subunit of a Lactobacillus S-layer while still maintaining the S-layer lattice structure.Many organisms from the domains Bacteria and Archaea possess a surface layer (S-layer) as the outermost structure of the cell envelope. S-layers are composed of regularly arranged proteinaceous subunits of a single protein or glycoprotein species with molecular masses ranging from 40 to 170 kDa (43, 48). S-layer proteins represent 10 to 15% of the total protein of the bacterial cell, and S-layer lattices cover the cell surface during all stages of growth, which indicates that efficient gene expression, S-layer protein synthesis, and secretion take place (4). A high content of hydrophobic and acidic amino acids and a low theoretical isoelectric point (pI) are typical features of S-layer proteins (48). In contrast, very high pI values have been described for the S-layer proteins from various lactobacilli (4, 54) and Methanothermus fervidus (6). In general, S-layers have been considered to function as cell shape determinants, protective coats, promoters for cell adhesion and surface recognition, and molecular and ion traps; however, no general function found in all S-layers has been recognized (47, 48).S-layers have been found from many species of the genus Lactobacillus (31, 56). However, the S-layer protein genes have been cloned and sequenced only from Lactobacillus brevis ATCC 8287 (54) and from the closely related species L. acidophilus (3), L. helveticus (8), and L. crispatus (46). Th...

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The potential of various biomasses for the production of green chemicals is currently one of the key topics in the field of the circular economy. Volatile fatty acids (VFAs) are intermediates in the methane formation pathway of anaerobic digestion and they can be produced in similar reactors as biogas to increase the productivity of a digestion plant, as VFAs have more varying end uses compared to biogas and methane. In this study, the aim was to assess the biogas and VFA production of food waste (FW) and cow slurry (CS) using the anaerobic biogas plant inoculum treating the corresponding substrates. The biogas and VFA production of both biomasses were studied in identical batch scale laboratory conditions while the process performance was assessed with chemical and microbial analyses. As a result, FW and CS were shown to have different chemical performances and microbial dynamics in both VFA and biogas processes. FW as a substrate showed higher yields in both processes (435 ml CH4/g VSfed and 434 mg VFA/g VSfed) due to its characteristics (pH, organic composition, microbial communities), and thus, the vast volume of CS makes it also a relevant substrate for VFA and biogas production. In this study, VFA profiles were highly dependent on the substrate and inoculum characteristics, while orders Clostridiales and Lactobacillales were connected with high VFA and butyric acid production with FW as a substrate. In conclusion, anaerobic digestion supports the implementation of the waste management hierarchy as it enables the production of renewable green chemicals from both urban and rural waste materials.

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A cassette based on the expression signals of the Lactobacillus brevis surface (S)-layer protein gene (slpA) was constructed. The low-copy-number vector pKTH2095, derived from pGK12, was used as the cloning vector. The efficiency of slpA promoters in intracellular protein production was studied using three reporter genes, beta-glucuronidase (gusA), luciferase (luc) and aminopeptidase N (pepN) in three different lactic acid bacteria hosts: Lactococcus lactis, Lactobacillus plantarum and Lactobacillus gasseri. The S-layer promoters were recognized in each strain and especially L. lactis and Lb. plantarum exhibited high levels of transcripts. The production kinetics of reporter proteins was studied as a function of growth. The GusA, Luc and PepN activities varied considerably among the lactic acid bacterial strains studied. The highest levels of beta-glucuronidase and luciferase activity were obtained in L. lactis. The level of GusA obtained in L. lactis corresponded to over 15% of the total cellular proteins. The highest level of aminopeptidase N activity was achieved in Lb. plantarum where PepN corresponded up to 28% of the total cellular proteins at the late exponential phase of growth. This level of PepN activity is 30-fold higher than that in Lb. helveticus, which is the species from which the pepN gene originates.

Lactobacillus brevis possesses a surface layer protein (SlpA) with tightly regulated synthesis. The slpA gene is expressed by two adjacent promoters, P1 and P2. The level of P2-derived transcripts was approximately 10 times higher than that of P1-derived transcripts throughout the entire growth of L. brevis. The half-lives of slpA transcripts were shown to be exceptionally long (14 min).

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