Cloning and Characterization of a Novel Alginate Lyase from Paenibacillus sp. LJ-23

As a low molecular weight alginate, alginate oligosaccharides (AOS) exhibit improved water solubility, better bioavailability, and comprehensive health benefits. In addition, their biocompatibility, biodegradability, non-toxicity, non-immunogenicity, and gelling capability make them an excellent biomaterial with a dual curative effect when applied in a drug delivery system. In this paper, a novel alginate lyase, Algpt, was cloned and characterized from a marine bacterium, Paenibacillus sp. LJ-23. The purified enzyme was composed of 387 amino acid residues, and had a molecular weight of 42.8 kDa. The optimal pH of Algpt was 7.0 and the optimal temperature was 45 °C. The analysis of the conserved domain and the prediction of the three-dimensional structure indicated that Algpt was a novel alginate lyase. The dominant degradation products of Algpt on alginate were AOS dimer to octamer, depending on the incubation time, which demonstrated that Algpt degraded alginate in an endolytic manner. In addition, Algpt was a salt-independent and thermo-tolerant alginate lyase. Its high stability and wide adaptability endow Algpt with great application potential for the efficient preparation of AOS with different sizes and AOS-based products.

Plant growth‐promoting activity of endophytic bacteria from sweet sorghum (Sorghum bicolor (L.) Moench)

Application of high levels of chemical fertilizers for optimal growth of sweet sorghum causes environmental degradation. Plant growth‐promoting bacteria have biotechnological importance because they can improve the growth and health of important agronomic plants. This study aimed to isolate, characterize, and identify endophytic bacteria associated with sweet sorghum (cv. KCS105), and also to study the inoculation effects of selected isolates on sorghum growth. In this study, 35 isolates were evaluated for their ability to support plant growth. The results showed that seven isolates were diazotrophic, six were capable of dissolving phosphate, six produced IAA and could detect ACC‐deaminase activity, and three inhibited the growth of pathogenic fungi. Nine isolates exhibiting mechanisms for promoting plant growth from the Alphaproteobacteria (Devosia), Firmicutes (Bacillus, Paenibacillus, Staphylococcus), and Actinobacteria (Microbacterium, Brachybacterium) phyla were identified. In addition, the Paenibacillus sp. BB7, Bacillus sp. PIB1B, and Bacillus sp. PLB1B isolates showed increasing effects on plant growth in greenhouse tests. Endophytic bacterial isolates which display plant growth‐promoting features can potentially be employed as biofertilizer agents. They may also address environmental damage problems resulting from the use of chemical fertilizers and pesticides.

Comparison of Synergistic Effect of Nisin and Monolaurin on the Inactivation of Three Heat Resistant Spores Studied by Design of Experiments in Milk

Spore forming bacteria are special problems for the dairy industry. Heat treatments are insufficient to kill the spores. This is a continuously increasing problem for the industry, but we should be able to control it. In this context, we investigated the combined effect of nisin, monolaurin, and pH values on three heat resistant spores in UHT milk and distilled water and to select an optimal combination for the maximum spore inactivation. The inhibitory effect of nisin (between 50 and 200 IU/ml), monolaurin (ranging from 150 to 300 µg/ml), and pH (between 5 and 8) was investigated using a central composite plan. Results were analyzed using the response surface methodology (RSM). The obtained data showed that the inactivation of Bacillus spores by the combined effect of nisin-monolaurin varies with spore species, acidity, and nature of the medium in which the bacterial spores are suspended. In fact, Terribacillus aidingensis spores were more resistant, to this treatment, than Paenibacillus sp. and Bacillus sporothermodurans ones. The optimum process parameters for a maximum reduction of bacterial spores (∼3log) were obtained at a concentration of nisin >150 IU/ml and of monolaurin >200 µg/ml. The current study highlighted the presence of a synergistic effect between nisin and monolaurin against heat bacterial spores. So, such treatment could be applied by the dairy industry to decontaminate UHT milk and other dairy products from bacterial spores.

ANTAGONISTIC ACTIVITY AND PLANT GROWTH PROMOTING RHIZOBACTERIA ISOLATED FROM FOREST PLANT RHIZOSPHERE AGAINST FUSARIUM SOLANI ON THUJA SEEDLINGS

Root diseases are one of the main forest nursery problems that have a significant impact on forest production which are caused by Fusarium solani. Rhizobacteria from healthy forest soils were isolated and screened in streak method to select antagonistic strains against F. solani. Two isolates showed high antagonistic activity and molecularly identified as Paenibacillus sp. and Pseudomonas sp. The capability of the Paenibacillus sp. and Pseudomonas sp. were tested in greenhouse plastic containers experiments against F. solani. Soil bacterization with Paenibacillus sp. and Pseudomonas sp. significantly protected thuja seedlings from F. solani compared to the untreated control seedlings. The containers added by Paenibacillus sp. and pseudomonas sp. are also showed plant growth promotion including shoot length, root length, dry and wet weights of the seedlings as well as the chlorophyll contents of the thuja seedlings compared to the untreated control plants. In this research it has been showing that the rhizobacterial treatments have potential to decrease the effect of fungal disease severity, promoting the plant growth and also helps plants to maintain a good health.

Diferentes estratégias para aumentar a produção de biossurfactante de um isolado de Paenibacillus sp. (BR13834)

Os biossurfactantes são metabólitos produzidos por diversos microorganismos, e nos últimos anos estão atraindo um interesse na comunidade científica devido suas vantagens em relação aos surfactantes sintéticos. O presente estudo teve como objetivo incrementar a produção de biossurfactante por Paenibacillus sp., otimizando os fatores de crescimento fonte de carbono, pH, temperatura e tempo de cultivo. Inicialmente foram avaliadas as fontes de carbono glicose, lactose, azeite de oliva, óleo de soja, glicerol e querosene. Em seguida, foram avaliados os fatores pH, Temperatura e Tempo, utilizado um delineamento fatorial para a identificação dos fatores que influenciam o processo de produção de biossurfactante. Por fim, os fatores ambientais temperatura e tempo de cultivo foram avaliados usando delineamento central composto rotacional (DCCR). Em todos os ensaios foi utilizado o isolado BR13834 pertencente ao gênero Paenibacillus. O modelo de previsão empírica desenvolvido foi considerado adequado para descrever a produção de biossurfactante em relação à tensão superficial (R2 = 0,755). O valor mínimo para a tensão superficial foi de 34,6 mN/m, obtido nas condições ideais de 30 °C e 24 horas de cultivo. Os resultados demonstraram que o DCCR foi adequado para identificar as melhores condições de produção de biossurfactante produzido por Paenibacillus sp.

Potential Antagonistic Bacteria against Verticillium dahliae Isolated from Artificially Infested Nursery

As an ecofriendly biocontrol agent, antagonistic bacteria are a crucial class of highly efficient fungicides in the field against Verticillium dahliae, the most virulent pathogen for cotton and other crops. Toward identifying urgently needed bacterial candidates, we screened bacteria isolated from the cotton rhizosphere soil for antagonisitic activity against V. dahliae in an artificially infested nursery. In preliminary tests of antagonistic candidates to characterize the mechanism of action of on culture medium, 88 strains that mainly belonged to Bacillus strongly inhibited the colony diameter of V. dahliae, with inhibiting efficacy up to 50% in 9 strains. Among the most-effective bacterial strains, Bacillus sp. ABLF-18, and ABLF-50 and Paenibacillus sp. ABLF-90 significantly reduced the disease index and fungal biomass of cotton to 40–70% that of the control. In further tests to elucidate the biocontrol mechanism (s), the strains secreted extracellular enzymes cellulase, glucanase, and protease, which can degrade the mycelium, and antimicrobial lipopeptides such as surfactin and iturin homologues. The expression of PAL, MAPK and PR10, genes related to disease resistance, was also elicited in cotton plants. Our results clearly show that three candidate bacterial strains can enhance cotton defense responses against V. dahliae; the secretion of fungal cell-wall-degrading enzymes, synthesis of nonribosomal antimicrobial peptides and induction of systemic resistance shows that the strains have great potential as biocontrol fungicides.

Priming of Resistance-Related Phenolics: A Study of Plant-Associated Bacteria and Hymenoscyphus fraxineus

European ash (Fraxinus excelsior) is highly affected by the pathogenic fungus Hymenoscyphus fraxineus in all of Europe. Increases in plant’s secondary metabolite (SM) production is often linked tol enhanced resistance to stress, both biotic and abiotic. Moreover, plant-associated bacteria have been shown to enhance SM production in inoculated plants. Thus, our hypothesis is that bacteria may boost ash SM production, hence priming the tree’s metabolism and facilitating higher levels of resilience to H. fraxineus. We tested three different ash genotypes and used Paenibacillus sp. and Pseudomonas sp. for inoculation in vitro. Total phenol (TPC), total flavonoid (TFC) and carotenoid contents were measured, as well as the chlorophyll a/b ratio and morphometric growth parameters, in a two-stage trial, whereby seedlings were inoculated with the bacteria during the first stage and with H. fraxineus during the second stage. While the tested bacteria did not positively affect the morphometric growth parameters of ash seedlings, they had a statistically significant effect on TPC, TFC, the chlorophyll a/b ratio and carotenoid content in both stages, thus confirming our hypothesis. Specifically, in ash genotype 64, both bacteria elicited an increase in carotenoid content, TPC and TFC during both stages. Additionally, Pseudomonas sp. inoculated seedlings demonstrated an increase in phenolics after infection with the fungus in both genotypes 64 and 87. Our results indicate that next to genetic selection of the most resilient planting material for ash reforestation, plant-associated bacteria could also be used to boost ash SM production.

Cadmium Resistance, Microbial Biosorptive Performance and Mechanisms of a Novel Biocontrol Bacterium Paenibacillus sp. LYX-1

In order to explore whether the newly discovered biocontrol strain Paenibacillus sp., LYX-1 having antagonistic effect on peach brown rot was resistant to Cd2+, a series of growth of strain LYX-1 under different Cd concentration and biosorption experiments were conducted to living and dead strain LYX-1. Meanwhile, the Cd2+ resistance and biosorption mechanisms were further identified by Cd-resistant genes, TEM, SEM-EDS, FTIR and XPS analysis. The results showed that strain LYX-1 could resist 50 mg/L Cd2+ and the adsorption process of both living and dead strain LYX-1 all satisfied the pseudo-second kinetic equation. Under pH 8.0 and at a dose of 1.0 g/L strain, the removal capacities of living and dead cells were as high as 90.39% and 75.67% at 20 mg/L Cd2+, respectively. For the adsorption isotherm test, results revealed that both Langmuir (R2=0.9704) and Freundlich (R2=0.9915) model could describe the Cd2+ biosorption well for living strain LYX-1. The maximum equilibrium biosorption capacities of living and dead biomass were 30.6790 and 24.3752 mg/g, respectively. The adsorption mechanism was controlled by chemisorption with -OH, -NH, -C=O, O=C-O, C-N, S2− and phosphate functional groups on the cell surface of strain LYX-1, which were further identified by XPS. The insignificant biosorption difference of living and dead biomass was caused by CzcD gene in strain LYX-1 detoxifying cadmium through the heavy metal efflux system. The above results indicated that strain LYX-1 had higher tolerance and fixed capacity to Cd2+.

Polyhydroxyalkanoate Recovery From Newly Screened Bacillus Sp. LPPI-81 Using Various Methods of Extraction From Loktak Lake Sediment Sample

Nowadays the conventional plastic wastes are very challenging to environments and its production cost also creates an economic crisis due to petrochemical-based plastic. In order to solve this problem, the current studies were aimed at screening and characterizing these PHA producing isolates and evaluating the suitability of some carbon source for newly screened PHA producing isolates. Some carbon sources such as D-fructose, glucose, molasses, D-ribose and sucrose were evaluated for PHA production. Data were analyzed using SPSS version 20. The 16SrRNA gene sequence of these isolates was performed. This newly isolated taxa was related to Bacillus species. It was designed as Bacillus sp. LPPI-18 and affiliated Bacillus cereus ATCC 14577T (AE01687) (99.10%). Paenibacillus sp. 172 (AF273740.1) was used as an out-group. Bacillus sp. LPPI-18 is a gram-positive, rod-shaped, endospore former, and citrate test positive. This isolate showed positive for amylase, catalase, pectinase, and protease test. They produced intracellular PHA granules when this isolate was stained with Sudan Black B (SBB) and Nile Blue A (NBA) preliminary and specific staining dyes, respectively. Both Temperature and pH used to affect PHA productivity. Bacteria are able to reserve PHA in the form of granules during stress conditions. This isolate produces only when supplied with carbon sources. More PHA contents (PCs) were obtained from glucose, molasses, and D-fructose. In this regard, the maximum mean value of PC was obtained from glucose (40.55±0.7%) and the minimum was obtained from D-Ribose (12.4±1.4%). Great variations (p≤0.05) of PCs were observed among glucose & sucrose, molasses & sucrose and D-fructose & sucrose carbon sources for PHA productivity (PP) of Cell Dry Weight (CDW) g/L. After extraction, PHA film was produced for this typical isolate using glucose as a sole carbon source. Fourier transform infrared spectrum was performed for this isolate and showed the feature of polyester at 1719.64 to 1721.16 wavelength for these extracted samples. The peak of fingerprinting (band of carboxylic acid group) at this wave-length is a characteristic feature of PHB and corresponds to the ester functional group (C=O).