Applied Microbiology and Biotechnology

Biopreservatives including nisin and its derivatives are becoming more desirable in the food processing industry because of the growing demand for naturally preserved and minimally processed foods free from artificial preservatives. However, ensuring microbiological safety while meeting these consumer preferences remains a major challenge. This has necessitated the continuous investigation of potential new antimicrobial agents produced by naturally occurring microorganisms. Hence, this study explored the synthesis, characterisation, and optimisation of a bacteriocinogenic lactic acid bacterium and its antimicrobial product, possibly novel bacteriocin (Nisin 2A) from Lactococcus lactis isolated from commercial brined cheese. The isolation was achieved by screening for wild-type bacteriocin-producing lactic acid bacteria from dairy products using MRS media. Screening was performed using antagonism assays, yielding five producer organisms. Of these, the isolate whose metabolites exhibited the most potent antimicrobial activity was identified as Lactococcus lactis, which synthesised an active antimicrobial peptide designated as Nisin 2A, with a molecular mass of approximately 3.3 kDa as determined by UHPLC-MS and SDS-PAGE. Production of Nisin 2A was scaled up through fed-batch fermentation of Lactococcus lactis in modified MRS broth following process optimisation using a Plackett-Burman experimental design and purified by ammonium sulphate precipitation and solid-phase extraction (SPE). Furthermore, the antimicrobial potential of the bacteriocin was evaluated by the agar well diffusion assay and quantified using the tube dilution method. The purified peptide demonstrated broad-spectrum antimicrobial activity, particularly against the test Gram-positive bacteria Bacillus cereus and retained its bioactivity across a wide pH range (3-9) and high thermal conditions (up to 100 {degrees}C). Furthermore, it had high sensitivity to proteolytic enzymes (Proteinase K and Trypsin). Notably, the peptide was thermostable and retained up to 90% of its initial activity after thermal treatment and maintained consistent inhibitory performance after extended storage. These findings highlight the potential application of Nisin 2A as a natural biopreservative in food systems.

Trace elements, such as iron, nickel, and cobalt are known to regulate methanogenic activity in anaerobic digestors used for waste valorisation, but the potential role of rare earth elements remains poorly understood. This study investigated the effects of lanthanum (La) supplementation on biogas production, methane generation, volatile fatty acid (VFA) formation, and carbohydrate utilisation in anaerobic digestion (AD). Biomethane potential (BMP) assays conducted under mesophilic conditions (37C) using methanogenic sludge granules, and glucose as substrate, were supplemented with 0.1, 1, 10, and 100 mg/L lanthanum chloride (LaCl3). Biogas production and composition was monitored over a 96-h incubation, while sacrificial, batch bioreactors were used to evaluate temporal VFA and carbohydrate profiles. La supplementation significantly enhanced biogas and methane production in a concentration-dependent manner. The highest cumulative biogas yield (478.9 mL, corresponding to 179.5 mL biogas/g COD) and methane production (285.7 mL, corresponding to 107.1 mL CH4/g COD) were observed with 100 mg/L LaCl3, corresponding to increases of 88.7% and 186%, respectively, compared with La-free controls. CO2 production also increased with La concentration, whereas hydrogen production remained comparatively low. Acetic and butyric acids represented the dominant fermentation products (80-88% of total VFAs), but profiles of accumulated VFA in the bioreactors diversified with La addition, including showing caproate production, indicating changed biodegradation dynamics in the methanogenic microbiome. These findings demonstrate that lanthanum can stimulate anaerobic digestion performance and methane generation, highlighting the potential as a novel trace element additive to enhance biogas production. Research is now required to elucidate the underlying microbial and biochemical mechanisms, and establish optimal dosing strategies for large-scale applications.

Given the current global environmental challenges, waste biomass is an attractive renewable resource for circular economies. Gasification of biomass yields syngas (CO, CO2, and H2) that is a suitable feedstock for gas fermentation in biomanufacturing of fuels and chemicals using acetogen microbes. While it is generally known that syngas composition influences both acetogen growth and process performance, we are lacking a consistent dataset quantifying these effects under controlled fermentation conditions. Here, we mapped the metabolic response of the model-acetogen Clostridium autoethanogenum to seven synthetic syngas mixtures during exponential batch growth in bioreactor fermentations. Notably, distinct gas compositions resulted in different fermentation profiles, affecting both growth and metabolite production. Maximum specific growth rates ranged within 0.05 0.13 h-1, with slower growth for low-CO mixtures. While acetate and ethanol production yields varied between 20-133 and 76-353 mmol per gram dry cell weight, respectively, minor production of 2,3-butanediol was detected. All syngas mixtures supported co-utilization of CO and H2, though gas uptake stoichiometry only moderately correlated with syngas content. Importantly, gas uptake stoichiometry strongly influenced carbon partitioning, with higher relative H2 uptake reducing CO2 loss or even realizing CO2 fixation together with increasing carbon flow towards metabolites. Interestingly, higher syngas H2 content favored ethanol and 2,3-butanediol production, while higher H2:CO uptake ratios increased total flux through the Wood-Ljungdahl pathway rather than selectively favoring reduced by-products. Our results are valuable for a better understanding of syngas composition effects on the acetogen biocatalyst and for process engineering towards optimizing gas fermentation performance. HighlightsO_LISyngas composition affects acetogen growth, gas uptake, and carbon distribution C_LIO_LIHigher H2:CO uptake ratios increase carbon flow through the Wood-Ljungdahl pathway C_LIO_LIHigher relative H2 uptake reduces CO2 loss and increases metabolite production C_LI

Acetate is a promising carbon source for microbial biotechnology as it can be produced sustainably from lignocellulosic biomass or C1 gases. Since acetate is directly activated to acetyl-CoA, it is especially suitable for producing acetyl-CoA-derived products, showcased here with the production of 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs). P. putida KT2440 can natively metabolize acetate, but the weak acid has also inhibitory effects on microbial growth. We present an in-depth study on the physiology of P. putida KT2440 using acetate as carbon and energy source and evaluate acetate as feedstock for the biosynthesis of HAAs. Initially, a rational engineering approach to overexpress acetyl-CoA synthetase for acetate activation resulted in an improved growth rate of 16% and reduced lag phase by six hours. To further increase the performance of P. putida KT2440 on acetate, adaptive laboratory evolution was performed. This resulted in an improvement in the growth rate from 0.4 h-1 to 0.6 h-1 and enabled growth on up to 12.5 g L-1 acetate with a shortened lag phase compared to the wild type. Whole-genome sequencing revealed mutations in proteins involved in gene expression regulation and signal transduction. This evolutionary engineering approach informed the deletions of gacS and crc, which resulted in a reduction in the lag phase from seven hours to one hour and an improvement of the growth rate by 25 %, matching the growth properties of the evolved clones. Using the evolved strains for the production of HAAs resulted in faster biomass and product formation with product titers reaching up to 94 % of that of the wild type. In conclusion, we identified mechanisms in the acetate metabolism of P. putida KT2440 and improved the growth performance of the strain by rational and evolutionary engineering, demonstrating the potential of the promising, but challenging 3rd generation feedstock acetate.

The increasing world population necessitates new sustainable nutrient sources, making microalgae like Chlorella sorokiniana interesting due to its rich nutrient profile and sustainable cultivation methods. With genetic optimization tools like CRISPR/Cas9, microalgae as a nutrient source can be improved even further. However, degradation of the rigid cell wall of microalgae, and thereby developing protoplasts, is often necessary prior to transformation, but monitoring protoplast development in spherical, single-celled organisms like C. sorokiniana is challenging using bright-field microscopy. Carbotrace 480 and 630 were tested as fluorescent markers of the cell wall of a C. sorokiniana mutant for protoplast detection, and Carbotrace 480 was successfully used to distinguish protoplast from normal cells in a cell suspension. The enzymes Driselase, Glucanex, Snailase, and Saczyme were tested in different combinations to degrade the cell wall of the mutant, with Snailase as the most effective yielding ~60 % protoplasts. This study provides a quick and easy tool for monitoring protoplast development in the microalgae C. sorokiniana, the first step to improve C. sorokiniana as a sustainable nutrient source using genetic optimization tools like CRISPR/Cas9.

Polycyclic aromatic hydrocarbons (PAHs) are hazardous organic contaminants for which microbial bioaugmentation is a promising remediation strategy, but poor persistence of introduced microorganisms can limit efficacy. Encapsulation may improve persistence, yet the influence of capsule design, microbial species, and environmental conditions on performance remains poorly understood. We evaluated alginate encapsulation of the PAH-degrading bacteria Pseudomonas putida and Novosphingobium aromaticivorans across nutrient conditions and capsule formulations. Encapsulation effects varied by species and medium, influencing growth rate, maximum cell density, overall growth, and lag time; notably, encapsulation shortened lag time of N. aromaticivorans in sRB15 medium (36.9 h to 3.9-5.3 h). Enumeration methods also affected apparent cell recovery. After 8 weeks, encapsulation had no significant effect on P. putida but resulted in increased concentrations of N. aromaticivorans relative to planktonic cultures (1.22 x 10; vs. 2.05 x 10; CFU/mL). Capsule composition further influenced cell retention: increasing alginate approximately doubled capsule-associated cell concentrations, while chitosan coatings reduced cell concentrations within capsules without affecting external concentrations. These findings demonstrate that the benefits of encapsulation are species- and environment-dependent and that capsule formulation can be tuned to influence bacterial persistence and release, informing the design of encapsulated inoculants for bioaugmentation applications.

Nisin is a promising antimicrobial peptide widely used in food preservation due to its efficacy against Gram-positive spoilage and pathogenic bacteria. Although Nisin is increasingly applied in the food sector, the biopeptide suffers from instability within food matrixes and can rapidly lose its antimicrobial potential following interaction with food biomolecules. Thus, it is necessary to investigate approaches that can be employed to extend the stability and activity of Nisin. Hence, the aim of this study was to develop and characterise a chitosan-alginate polyelectrolyte microencapsulation system capable of enhancing Nisin stability while retaining antimicrobial activity. The microencapsulation of Nisin was achieved by pre-gelation of alginate using calcium chloride and subsequent direct electrostatic interaction between cationic Nisin and chitosan with pre-gelled anionic alginate at pH 5.0. Following microcapsule formation, physicochemical and structural characterisation was performed using Zeta potential determination and measurement of the polydispersity index (PDI) via dynamic light scattering. SEM micrographs were used to confirm morphology, while Fourier-transform infrared (FTIR) spectroscopy and high-performance liquid chromatography (HPLC) were utilised to assess chemical integrity and functional group preservation of encapsulated Nisin. Following this, stable microcapsules with diameters ranging from 150-200 nm and smooth surface morphology were obtained. Microcapsule formation was strongly influenced by formulation parameters, particularly pH, calcium ion concentration, and chitosan content, with deviations from optimal acidic conditions (< pH 5.0) resulting in aggregation, increased polydispersity, and reduced encapsulation efficiency. The microcapsules were monodispersed (PDI {approx} 0.30) and electrostatically stable, exhibiting a Zeta potential of approximately +36 mV. These microcapsules remained stable over a prolonged storage period of 21 days under refrigerated conditions while retaining antimicrobial activity against Bacillus cereus. Encapsulation efficiency reached approximately 65%, confirming effective retention of Nisin within the polymer matrix. Overall, the findings demonstrate that chitosan-alginate ionic gelation is a non-denaturing and effective encapsulation strategy for extending the functional stability of Nisin. These microcapsules show strong potential as natural antimicrobial delivery systems for food and beverage applications, particularly in acidic food matrices, with implications for improved food safety and shelf-life extension.

The Bacillus cereus group comprises bacteria of biotechnological interest, but also raises health concerns. Some bacteria in this group are opportunistic human pathogens, mainly causing foodborne gastrointestinal infections. As of today, the presence, sequence variability, or expression of genes encoding toxins or other virulence factors are insufficient to predict the potential of a given isolate to cause the diarrheal form of the disease. To address this limitation, we developed a sandwich ELISA to quantify the NheA and Sphingomyelinase (SMase) proteins in culture supernatants to test them as markers of pathogenic potential. Application of the assay to a collection of B. cereus group isolates revealed that strains associated with food poisoning outbreaks produce significantly more NheA and SMase than those isolated from the environment or from commercial products. Statistical analyses show that the combined quantification of NheA and SMase provides robust discrimination between pathogenic and non-pathogenic (environmental and commercial) profiles. These results demonstrate that the quantitative assessment of both NheA and SMase production can serve as a reliable biomarker for distinguishing diarrheic food poisoning isolates from harmless strains.

Compact Cas nucleases offer advantages over the widely used SpCas9 due to their smaller size, which enables more efficient delivery for in vivo applications. Among these, the phage-encoded Cas{Phi}2 (Cas12j2) is highly promising due to its relaxed PAM requirement (5-TTN-3) and compact size (757 aa); however, its translational potential is limited by low editing activity. To enhance the efficacy of Cas{Phi}2, we optimized the previously reported EPICA system, developing EPICA.2, a eukaryotic directed evolution platform to improve nucleases with nearly undetectable activity. EPICA.2 integrates additional yeast evolution rounds to enrich for active variants along with a low background mammalian reporter system that improves detection and selection of enhanced variants. Finally, we set up a long-read sequencing protocol which uses unique molecular identifiers (UMIs) to reduce sequencing errors, enabling accurate identification of the mutation combinations in each evolved variant. Among the most frequent variants, we obtained evoCas{Phi}2, which contains six activity-boosting mutations with a synergistic effect not predictable by rational engineering. Overall, evoCas{Phi}2 showed up to 70-fold increased activity in human cells compared to wild-type and outperformed variants generated through rational approaches, highlighting the potential of EPICA.2 as a powerful strategy to evolve genome editing tools with low native activity.

Romanian bors, a traditional fermented wheat bran beverage, is produced through spontaneous fermentation and represents a complex microbial ecosystem. Despite its cultural importance and presumed health benefits, its microbial ecology and functional potential remain poorly characterized. The present study aimed to elucidate the microbial community structure of bors and link it to functional traits relevant to fermentation performance and food functionality by integrating culture-independent sequencing with culture-dependent isolation and functional characterization. A total of 32 bors samples (12 commercial and 20 homemade) were analyzed. Amplicon-based sequencing revealed a microbiome dominated by lactic acid bacteria (LAB), with lactobacilli accounting for the majority of the bacterial communities and Lactobacillus amylolyticus being identified as the most prevalent and abundant species. The yeast communities were mainly composed of fermentative taxa, including Pichia kudriavzevii and Kluyveromyces marxianus. Lactobacillus amylolyticus and P. kudriavzevii were also the most frequently isolated species among bacteria and yeasts, respectively. These results highlighted a strong adaptation of the microbial isolates to starch-rich cereal substrates and underscored the central role of these microorganisms in wheat bran fermentation for bors production. Whereas the sequencing-based analyses showed no significant differences in overall diversity between the commercial and homemade bors samples, the cultivation-based results indicated a higher bacterial richness in the commercial products. Notably, the culture-dependent method captured substantially fewer taxa, highlighting the complementary nature of the two approaches. Of a total of 101 bacterial strains (88 LAB and 13 acetic acid bacteria) isolated, many exhibited rapid growth and strong acidification capacity, reaching pH values below 4.5 within 12 h. A functional screening revealed that 21 % of these strains displayed -amylase activity, 65 % phytase activity, and 50 % {beta}-glucosidase activity, highlighting their capacity to metabolize cereal substrates and enhance the nutrient availability of bors. All strains showed antibacterial activity against at least one indicator bacterium tested, with a universal inhibition of Listeria monocytogenes. Overall, Romanian bors harbored a lactic acid bacteria-dominated core microbiome with a significant functional diversity. These findings underscored its potential as a rich source of functional and technologically important strains for application in starter and protective culture development.

Cyclic oligosaccharides possess industrial advantages, including molecular encapsulation capability and high physicochemical stability, owing to the absence of a reducing end. Recently, a novel cyclic tetrasaccharide, cycloisomaltotetraose (CI4), consisting of four -1,6-linked glucose units, and the enzymes responsible for its synthesis, cycloisomaltotetraose glucanotransferases (CI4Tases), were discovered. Unlike known cycloisomaltooligosaccharide glucanotransferases (CITases) that yield a wide distribution of cyclic products with a degree of polymerization (DP) of 7 or higher, CI4Tases strictly produce CI4. To elucidate the molecular mechanism underlying this strict DP4 specificity, we determined the crystal structures of CI4Tase from Agreia sp. D1110, in its ligand-free form, as well as in complex with the linear hydrolysis product isomaltotetraose (IG4) and with CI4. Structural comparisons revealed that a loop (M247 to R251) blocks the region corresponding to the -5 subsite of typical CITases, narrowing the substrate-binding pocket. This "molecular ruler" mechanism ensures that only a glycan chain of exactly four glucose units is accommodated for cyclization. Among mutants of the residue positioned at the center of bound CI4, the formation of by-products other than CI4 was significantly suppressed in F245L, F245A, and F245W. While the cyclization activity of all F245 mutants decreased, the CI4 hydrolysis activity of these three mutants was also significantly reduced, resulting in an increased specificity for cyclic sugar production. These findings elucidate the strict size-control mechanism of CI4Tase and provide a structural foundation for engineering cycloisomaltooligosaccharide-producing enzymes with optimized transglycosylation efficiency and specificity for industrial applications.

Microbial fuel cells (MFCs) have been widely investigated as decentralized bioelectrochemical systems capable of converting organic substrates into electricity. However, their long-term autonomous operation is constrained by substrate depletion in the anode compartment, leading to metabolic starvation of electroactive biofilms and a decline in power output. Conventional MFC design treats substrate crossover through the membrane separator as a parasitic loss that reduces coulombic efficiency. In this work, we propose a conceptual inversion of this paradigm by considering controlled cathodic-to-anodic substrate crossover as a passive mechanism to sustain basal microbial metabolism during periods of substrate scarcity. A transport-reaction framework is developed to quantify the balance between membrane-mediated substrate flux and microbial maintenance demand within the anode biofilm. Based on this balance, a dimensionless maintenance crossover Damkohler number (Dam) is introduced to define three operational regimes: starvation-dominated (Dam >> 1), balanced autonomous (Dam {approx} 1), and crossover-dominated (Dam << 1). The framework integrates membrane transport theory with biofilm kinetics to evaluate the effects of separator properties, substrate gradients, and current-dependent electro-osmotic transport on system stability. Order-of-magnitude analysis indicates that achievable crossover fluxes span several orders of magnitude depending on separator characteristics, suggesting that membrane properties critically influence system behavior. This perspective reframes substrate crossover from a loss mechanism to a potential design variable, offering a conceptual tool for enhancing resilience and guiding separator selection in MFCs intended for long-duration, and low-maintenance operation. HighlightsO_LIControlled crossover can sustain microbial metabolism in MFCs C_LIO_LIIntroduces maintenance crossover Damkohler number (Dam) C_LIO_LIIdentifies regimes for autonomous and starvation operation C_LIO_LILinks membrane properties to long-term system stability C_LIO_LIReframes crossover as a design variable, not only a loss C_LI

New maritime regulations restricting high-sulfur fuels have led to the transition to new low sulfur fuel oils (LFSOs). We do not know how LSFOs will behave in marine environments and how they will respond to available remediation strategies, presenting an environmental risk. The risk will be even higher in the remote high Arctic, especially along the Northwest Passage (NWP), for which an increase in shipping traffic is expected by the end of the century. In this study, we evaluated the long-term (one year) biodegradation potential of the native microbial community of NWP beach sediment using in situ mesocosm experiments with two different types of LSFOs: a marine gas oil (Marine diesel) and an ultra-low sulfur fuel oil (ULSFO). We observed that the lighter Marine diesel was biodegraded better (72.0%) than the heavier ULSFO (32.5%). We described composition of the microbial community of the mesocosms using 16S rRNA gene amplicon sequencing and observed a decrease in microbial diversity for the fuel-treated samples compared to the untreated controls. Despite the decrease in overall diversity, we observed significantly higher abundances of known hydrocarbon degrading microbes (e.g., Oleispira, Altererythrobacter, Gilvibacter, Pseudohongiella) in the fuel mesocosms. Our study showed the potential to implement biodegradation as a remediation strategy under the cold and oligotrophic environmental conditions present throughout the NWP. However, we also observed that microbes on their own cannot degrade the entirety of the removed fuel and other types of remediation will need to be considered to complement the natural biodegradation observed here.

The coprophilous agaricomycete Coprinopsis cinerea is a model organism for antagonistic fungal-bacterial interactions. Previous studies showed that C. cinerea responds to antagonistic bacteria with strong induction of a set of genes encoding secreted antibacterial molecules. However, little is known about the elicitors of this response. Key open questions in this respect include whether individual antibacterial defence genes are induced by different bacteria and/or by specific bacterial soluble molecules. Here, we present a new C. cinerea reporter system to monitor antibacterial defence induction and address related outstanding issues with minimal hands-on time. In this system, the promoter of the endogenous bacterial-induced gene cclys1 drives the expression of cnluc, which encodes a secreted variant of the deep-sea shrimp luciferase Nluc. We show that cNluc allows to detect and quantify cclys1 induction by measuring luminescence directly in the culture medium of reporter strain colonies. Building on these features, we successfully leveraged the inducible cNluc reporter strain for the development of a novel 96-well plate assay that allows the high-throughput screening of antibacterial defence elicitors. As cNluc can be subject to degradation by secreted proteases of fungal or bacterial origin in the culture medium, we coupled this assay to confirmatory qRT-PCR. Testing this set-up by confronting the reporter strain with several different bacteria revealed that cclys1 induction occurs independently of the bacterial ecological niche. Based on these results, we also recommend qRT-PCR exclusively for validation of negative results. We conclude that cNluc offers significant advantages over cytoplasmic reporter proteins, especially for preliminary rapid screening of multiple conditions.

Protein engineering and precision fermentation provide an opportunity to increase the value of food proteins by improving their solubility, stability, functionality, or nutritional composition. Here, we use {beta}-lactoglobulin ({beta}LG) as a model protein to investigate how state-of-the-art computational protein design approaches affect these properties. First, the deep learning-based design tool ProteinMPNN was used to alter up to 20% of {beta}LG residues for increased stability. Second, the physics-based modeling platform PyRosetta was used to find positions in {beta}LG accommodating increased branched-chain amino acid (BCAA) content and up to 10 residues were simultaneously exchanged. Experimental characterisation of ProteinMPNN and stabilised BCAA-enriched variants showed similar secondary structure and oligomeric state as native {beta}LG. ProteinMPNN variants gave increased titers and increased thermal stability up to 15 {degrees}C, and this correlated with changes in the rate of surface pressure in droplet tensiometry. Stabilized BCAA-enriched mutants had altered acid solubility. Correlations between computationally derived biophysical metrics and experimental properties are presented and suggest some predictive power for surface hydrophobicity on protein yield.

Poly(ethylene terephthalate) (PET) is one of the most widely produced plastics, and enzymatic depolymerization offers a promising route to closed-loop recycling under mild conditions. However, most known bacterial PET hydrolases belong to a conserved canonical-fold cutinase family, leaving much of alpha/beta-hydrolase diversity unexplored. Here, we mapped bacterial cutinase sequence space by combining bioinformatics-guided sequence selection with high-throughput secretion screening in Bacillus subtilis. A library of 1,120 genes encoding 954 unique bacterial cutinases, spanning canonical- and minimal-fold families, was screened for activity on Impranil DLN and semicrystalline PET. We identified 156 secreted cutinases with polyester activity, broadly distributed across sequence space, but only ten showed detectable PET hydrolysis, all from the canonical-fold family. These PET hydrolases were active at 40-50{degrees}C, preferred alkaline pH, and showed moderate thermostability. Our results demonstrate that PET activity is rare among bacterial cutinases and provide a scalable workflow for discovering diverse enzyme starting points.

"Lactic acid bacteria (LAB) are one of the dominant groups of bacteria in the palm sap (Arenga pinnata) microbiome. Previous research in the sago palm sap production centers of Tomohon City (Kayawu, Pinaras, and Lahendong) has successfully identified various LAB species, including Lactobacillus casei, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus buchneri, Leuconostoc mesenteroides, and Leuconostoc sp. This study aims to identify LAB species in sago palm sap from a new location, namely the Wawo Plantation in Tomohon, and to evaluate their potential as natural antibacterial agents. Through 16S rDNA gene sequencing analysis, the isolates obtained were identified as belonging to the newly described genera Lacticaseibacillus and Lactiplantibacillus. Four promising isolates Lactiplantibacillus fabifermentans A1.4, Lacticaseibacillus casei B1.5, Lacticaseibacillus paracasei B1.6, and Lacticaseibacillus paracasei B3.5 were tested for their inhibitory activity against the enteric pathogens Salmonella sp. and Escherichia coli using the well diffusion method. The results showed that all isolates exhibited a strong spectrum of pathogen inhibition. The highest inhibitory activity against Salmonella sp. was demonstrated by the L. paracasei B1.6 isolate, with an inhibition zone of 21.25 mm, while optimal inhibition against E. coli was achieved by L. casei B1.5 at 11.0 mm. These findings confirm that the local BAL strain from Tomohon palm sap has great potential for large-scale development as a biopreservative in the food industry and as a functional probiotic agent"

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and associated nuclease gene (Cas), originating from the bacteria acquired immune system, have revolutionized gene editing technology. In this regard, type II (Cas9) been extensively studied and widely applied CRISPR system so far. The mechanism for precise manipulation of genomic sequences is guided by small RNA called CRISPR RNA (crRNA). In this study we devised and optimized CRISPR-Cas9 screening system based on Cas9 gene detection, targeting a conserved part of recognition domain (REC) consisting of arginine rich bridge helix (BH). We used hemi-nested PCR approach for screening sensitivity and reproducibility. The recombinant E. coli DH5 alpha containing the pRGEB32 vector (DH5 alpha/pRGEB32) with the Cas9 gene was used for system optimization. Subsequently, the screening system was applied and validated on different environmental bacterial strains including Alcaligenes faecalis and Pseudomonas stutzeri, isolated from sewerage samples. The optimized hemi-nested PCR resulted in amplification of targeted region in environmental bacterial strains and results were reproduced successfully. Furthermore, nucleotides and amino acid sequence, motif and domain analysis of PCR products, confirmed the targeted Cas9 REC-BH domain. Presently, no rapid and cost effective CRISPR-Cas screening system is available except expensive whole genome sequencing approach. Our investigation aimed to device rapid and cost effective screening system for identification of new variants of Cas9 proteins in environmental bacterial species. In this context, the developed Cas9 gene-based CRISPR-Cas screening system (C9CSS) may be a potential rapid screening tool to identify new Cas9 orthologs in different bacterial genomes with improved functions.

The pulp and paper industry produces large volumes of condensed kraft lignin, which is challenging to convert to single chemical products. For this purpose, tandem chemical depolymerization and bioconversion to a single atom-efficient product is a potentially promising strategy. In this study, we conducted copper-catalyzed oxidative depolymerization using pine-derived kraft lignin to generate multiple bioavailable aromatic monomers at a yield of 4.5 weight% (wt%; g monomers per g lignin) from both C--O and C--C bond cleavage, followed by counter-current extraction with a 52 wt% monomer recovery. This resulted in an oxidized lignin product containing vanillin, vanillate, 4-hydroxybenzaldehyde, 4-hydroxybenzoate, 5-formylvanillin, 5-carboxyvanillin, 5-carboxyvanillate, acetovanillone, and vanillyl glyoxylate. Based on this stream composition, we engineered the industrially relevant soil bacterium Pseudomonas putida KT2440 to catabolize the latter five compounds via overexpression of ten heterologous genes (acvABCDEFSYK-6, vceABSYK-6, ligW2SYK-6, and mdlCPP). We combined these engineered pathways with previously reported strategies for muconate production from G- and H-type monomers to generate P. putida KMM428, which utilized 93.6 {+/-} 0.2 mol% of the quantified aromatic monomers in a depolymerized kraft lignin mixture, and produced muconate at a yield of 99 {+/-} 3 mol%, on a quantified monomer basis. Together, this work increases the theoretical carbon conversion efficiency of this process by 37.6 {+/-} 0.1 mol% through incorporation of three {beta}-5 cleavage products, in addition to traditional G-type monomers.

The marine bacterium Vibrio alginolyticus and the food poisoning bacterium V. parahaemolyticus possess two types of flagella in one cell: proton-driven lateral flagella (Laf) extending from the periphery of the cell body, and sodium ion-driven polar flagella (Pof) extending from a cell pole. For swimming in seawater they use polar flagella, suppressing the expression of lateral flagella. When they attach to the body surface of fish or intestinal tract, lateral flagella are induced, allowing it to crawl along the surface or through mucus. The dynamometer hypothesis, which proposes that polar flagella sense rotation and control the expression of lateral flagellar genes, has been widely accepted. However, how rotation is sensed and how expression is controlled remains unclear. Although swarming has recently been analyzed by physical, biological, or biochemical perspectives, it remains unclear how this motility is controlled, or which substances and conditions are necessary for swarming ability. In this study, we discovered that adding gelatin to agar medium promotes swarming on the agar surface by the lateral flagella of Vibrio. Our data suggested that surfactants or viscous polysaccharides secreted extracellularly are important for promoting swarming on the agar surface and we identified that swarming is likely to be driven by S (social)-motility, in which bacteria move by interacting with each other, and A (adventure)-motility, in which bacteria move by interacting with the agar surface. Our study provides clues that help clarify the mechanism of bacterial swarming IMPORTANCEWe discovered that adding gelatin to hard agar medium promoted swarming on agar surfaces by the lateral flagella of Vibrio cells. The surfactants or viscous polysaccharides secreted extracellularly seem to be important for swarming ability on agar surfaces. We proposed that the swarming is thought to occur through S(social)-motility, where cells move by interacting with cell bodies each other, and A(adventure)-motility, where cells move by interacting with the agar surface and cell body. The present study should provide the clues to clarify the mechanism of bacterial swarming and how to move in a viscous environment.