Simple stain-free screening method for pectinolytic microorganisms under alkalophilic conditions.

OBJECTIVES: To develop a simple pectin-degrading microorganism screening method. RESULTS: We developed a method utilizing the phenomenon whereby cooling an alkaline agar medium containing pectin causes the agar to become cloudy. This highly simplified method involves culturing the microorganisms on pectin-containing agar medium until colony formation is observed, and subsequent overnight cooling of the agar medium to 4 °C. Using this simple procedure, we successfully identified pectin-degrading microorganisms by observing colonies with halos on the clouded agar medium. We used alkaline pectinase and Bacillus halodurans, which is known to secrete alkaline pectinase, to establish the screening method. We demonstrated the screening of pectin-degrading microorganisms using the developed method and successfully isolated pectin-degrading microorganisms (Paenibacillus sp., Bacillus clausii, and Bacillus halodurans) from a soil sample. CONCLUSIONS: The developed method is useful for identifying pectin-degrading microorganisms.

Evidencia molecular de la ocurrencia de bacterias celulolíticas del género Cohnella en especies neotropicales de Nasutitermitinae colectadas en el NEA / Molecular evidence that cellulolytic bacterial genus Cohnella is widespread among Neotropical Nasutitermitinae from NE Argentina

Cohnella is a highly cellulolytic bacterial genus, which can be found in a variety of habitats. The aim of this study was to assess its presence in the digestive tract of termite species collected in North-eastern Argentina: Nasutitermes aquilinus, N. corniger and Cortaritermes fulviceps. Gut homogenates were incubated with cellulosic substrate for bacterial growth. Bacterial 16S rDNA was partially amplified using new primers for Cohnella spp. and cloned. Sequences obtained showed highest similarity (97.2-99.9%) with those of Cohnella spp. previously reported from diverse environments. Phylogenetic analysis tended to group the clones according to their host species and sampling sites. These results indicate the association of Cohnella-related intestinal symbionts with three common Neotropical termites. Their potential industrial application encourages further research.

Cohnella es un género de bacterias celulolíticas que puede ser encontrado en una variedad de hábitats. El propósito de este estudio fue registrar su presencia en el tracto digestivo de termitas (Nasutitermes aquilinus, N. corniger y Cortaritermes fulviceps) colectadas en el noreste argentino (NEA). Se incubaron homogenados de intestinos en sustrato celulósico para multiplicar las bacterias. Utilizando nuevos cebadores para Cohnella spp., se amplificó una porción del ADN ribosomal 16S bacteriano, el cual fue posteriormente clonado. Las secuencias obtenidas mostraron su mayor porcentaje de similitud (97,2-99,9%) con Cohnella spp., previamente reportadas en diversos ambientes. El análisis filogenético tendió a agrupar a los clones de acuerdo a la especie hospedante y al sitio de muestreo. Estos resultados indican que especies de termitas frecuentes en el NEA albergan simbiontes intestinales relacionados con el género Cohnella. Las potenciales aplicaciones industriales de estos microorganismos animan a profundizar los estudios.

Consistent associations with beneficial bacteria in the seed endosphere of barley (Hordeum vulgare L.).

The importance of the plant microbiome for host fitness has led to the concept of the "plant holobiont". Seeds are reservoirs and vectors for beneficial microbes, which are very intimate partners of higher plants with the potential to connect plant generations. In this study, the endophytic seed microbiota of numerous barley samples, representing different cultivars, geographical sites and harvest years, was investigated. Cultivation-dependent and -independent analyses, microscopy, functional plate assays, greenhouse assays and functional prediction were used, with the aim of assessing the composition, stability and function of the barley seed endophytic bacterial microbiota. Associations were consistently detected in the seed endosphere with Paenibacillus, Pantoea and Pseudomonas spp., which were able to colonize the root with a notable rhizocompetence after seed germination. In greenhouse assays, enrichment with these bacteria promoted barley growth, improved mineral nutrition and induced resistance against the fungal pathogen Blumeria graminis. We demonstrated here that barley, an important crop plant, was consistently associated with beneficial bacteria inside the seeds. The results have relevant implications for plant microbiome ecology and for the holobiont concept, as well as opening up new possibilities for research and application of seed endophytes as bioinoculants in sustainable agriculture.

Regulated ploidy of Bacillus subtilis and three new isolates of Bacillus and Paenibacillus.

Bacteria were long assumed to be monoploid, maintaining one copy of a circular chromosome. In recent years it became obvious that the majority of species in several phylogenetic groups of prokaryotes are oligoploid or polyploid. The present study aimed at investigating the ploidy in Gram-positive aerobic endospore-forming bacteria. First, the numbers of origins and termini of the widely used laboratory strain Bacillus subtilis 168 were quantified. The strain was found to be mero-oligoploid in exponential phase (5.9 origins, 1.2 termini) and to down-regulate the number of origins in stationary phase. After inoculation of fresh medium with stationary-phase cells the onset of replication preceded the onset of mass increase. For the analysis of the ploidy in fresh isolates, three strains were isolated from soil, which were found to belong to the genera of Bacillus and Paenibacillus. All three strains were found to be mero-oligoploid in exponential phase and exhibit a growth phase-dependent down-regulation of the ploidy level in stationary phase. Taken together, these results indicate that mero-oligoploidy as well as growth phase-dependent copy number regulation might be widespread in and typical for Bacillus and related genera.

An extractive membrane biofilm reactor as alternative technology for the treatment of methyl tert-butyl ether contaminated water.

Among the strategies developed for contaminated groundwater bioremediation, those based on the use of bacteria adhering to inert supports and establishing biofilms have gained great importance in this field. Extractive membrane biofilm reactor (EMBFR) technology offers productive solutions for the removal of volatile and semi-volatile compounds. EMBFR technology is based on the use of extractive semipermeable membranes through which contaminants migrate to the biological compartment in which microorganisms with pollutant biotransformation and/or mineralization capacities can grow, forming an active biofilm on the membrane surface. The objective of this study was to assess the use of three bacterial strains (Paenibacillus sp. SH7 CECT 8558, Agrobacterium sp. MS2 CECT 8557, and Rhodococcus ruber EE6 CECT 8612), as inoculum in a lab-scale EMBFR running for 28 days under aerobic conditions to eliminate methyl tert-butyl ether (MTBE) from water samples. Three different hydraulic retention times (1, 6, and 12 h) were employed. MTBE degradation values were determined daily by a gas GC-MS technique, as well as suspended bacterial growth. The biofilm established by the bacterial strains on the semipermeable membrane was detected by Field-Emission Scanning Electron Microscopy (FESEM) at the end of each experiment. The acute toxicity of the treated effluents and biomedium was determined by Microtox© assay (EC50 ).The results achieved from the MTBE degradation, biofilm formation, and toxicity analysis indicated that bacterial strains MS2 and EE6 were the best options as selective inoculum, although further research is needed, particularly with regard to their possible use as a mixed culture. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1238-1245, 2016.

Iron deficiency resistance mechanisms enlightened by gene expression analysis in Paenibacillus riograndensis SBR5.

Despite its importance in growth and cell division, iron metabolism is still poorly understood in microorganisms, especially in Gram-positive bacteria. In this work, we used RNA sequencing technology to elucidate global mechanisms involved in iron starvation resistance in Paenibacillus riograndensis SBR5, a potential plant growth-promoting bacterium. Iron deficiency caused several changes in gene expression, and 150 differentially expressed genes were found: 71 genes were overexpressed and 79 genes were underexpressed. Eight genes for which expression was at least twice as high or twice as low in iron-limited condition compared with iron-sufficient condition were chosen for RT-qPCR analysis to validate the RNA seq data. In general, most genes exhibited the same pattern of expression after 24 h of P. riograndensis growth under iron-limiting condition. Our results suggest that, during iron deficiency, bacteria express several genes related to nutrient uptake when they start to grow to obtain all of the molecules necessary for maintaining major cellular processes. However, once iron becomes highly limiting and is no longer able to sustain exponential growth, bacteria begin to express genes related to several processes, like sporulation and DNA protection, as a way of resisting this stress.

Screening and characterization of endophytic Bacillus and Paenibacillus strains from medicinal plant Lonicera japonica for use as potential plant growth promoters

Abstract A total of 48 endophytic bacteria were isolated from surface-sterilized tissues of the medicinal plant Lonicera japonica, which is grown in eastern China; six strains were selected for further study based on their potential ability to promote plant growth in vitro (siderophore and indoleacetic acid production). The bacteria were characterized by phylogenetically analyzing their 16S rRNA gene similarity, by examining their effect on the mycelial development of pathogenic fungi, by testing their potential plant growth-promoting characteristics, and by measuring wheat growth parameters after inoculation. Results showed that the number of endophytic bacteria in L. japonica varied among different tissues, but it remained relatively stable in the same tissues from four different plantation locations. Among the three endophytic strains, strains 122 and 124 both had high siderophore production, with the latter showing the highest phosphate solubilization activity (45.6 mg/L) and aminocyclopropane-1-carboxylic acid deaminase activity (47.3 nmol/mg/h). Strain 170 had the highest indoleacetic acid (IAA) production (49.2 mg/L) and cellulase and pectinase activities. After inoculation, most of the six selected isolates showed a strong capacity to promote wheat growth. Compared with the controls, the increase in the shoot length, root length, fresh weight, dry weight, and chlorophyll content was most remarkable in wheat seedlings inoculated with strain 130. The positive correlation between enzyme (cellulose and pectinase) activity and inhibition rate on Fusarium oxysporum, the IAA production, and the root length of wheat seedlings inoculated with each tested endophytic strain was significant in regression analysis. Deformity of pathogenic fungal mycelia was observed under a microscope after the interaction with the endophytic isolates. Such deformity may be directly related to the production of hydrolytic bacterial enzymes (cellulose and pectinase). The six endophytic bacterial strains were identified to be Paenibacillus and Bacillus strains based on the results of 16S rRNA gene sequencing analysis and their physiological and biochemical characteristics. Results indicate the promising application of endophytic bacteria to the biological control of pathogenic fungi and the improvement of wheat crop growth.

Initial pH of medium affects organic acids production but do not affect phosphate solubilization

The pH of the culture medium directly influences the growth of microorganisms and the chemical processes that they perform. The aim of this study was to assess the influence of the initial pH of the culture medium on the production of 11 low-molecular-weight organic acids and on the solubilization of calcium phosphate by bacteria in growth medium (NBRIP). The following strains isolated from cowpea nodules were studied: UFLA03-08 (Rhizobium tropici), UFLA03-09 (Acinetobacter sp.), UFLA03-10 (Paenibacillus kribbensis), UFLA03-106 (Paenibacillus kribbensis) and UFLA03-116 (Paenibacillus sp.). The strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 solubilized Ca3(PO4)2 in liquid medium regardless of the initial pH, although without a significant difference between the treatments. The production of organic acids by these strains was assessed for all of the initial pH values investigated, and differences between the treatments were observed. Strains UFLA03-09 and UFLA03-10 produced the same acids at different initial pH values in the culture medium. There was no correlation between phosphorus solubilized from Ca3(PO4)2 in NBRIP liquid medium and the concentration of total organic acids at the different initial pH values. Therefore, the initial pH of the culture medium influences the production of organic acids by the strains UFLA03-08, UFLA03-09, UFLA03-10 and UFLA03-106 but it does not affect calcium phosphate solubilization.

Paenibacillus yonginensis DCY84(T) induces changes in Arabidopsis thaliana gene expression against aluminum, drought, and salt stress.

Current agricultural production methods, for example the improper use of chemical fertilizers and pesticides, create many health and environmental problems. Use of plant growth-promoting bacteria (PGPB) for agricultural benefits is increasing worldwide and also appears to be a trend for the future. There is possibility to develop microbial inoculants for use in agricultural biotechnology, based on these beneficial plant-microbe interactions. For this study, ten bacterial strains were isolated from Yongin forest soil for which in vitro plant-growth promoting trait screenings, such as indole acetic acid (IAA) production, a phosphate solubilization test, and a siderophore production test were used to select two PGPB candidates. Arabidopsis thaliana plants were inoculated with Paenibacillus yonginensis DCY84(T) and Micrococcus yunnanensis PGPB7. Salt stress, drought stress and heavy metal (aluminum) stress challenges indicated that P. yonginensis DCY84(T)-inoculated plants were more resistant than control plants. AtRSA1, AtVQ9 and AtWRKY8 were used as the salinity responsive genes. The AtERD15, AtRAB18, and AtLT178 were selected to check A. thaliana responses to drought stress. Aluminum stress response was checked using AtAIP, AtALS3 and AtALMT1. The qRT-PCR results indicated that P. yonginensis DCY84(T) can promote plant tolerance against salt, drought, and aluminum stress. P. yonginensis DCY84(T) also showed positive results during in vitro compatibility testing and virulence assay against X. oryzae pv. oryzae Philippine race 6 (PXO99). Better germination rates and growth parameters were also recorded for the P. yonginensis DCY84(T) Chuchung cultivar rice seed which was grown on coastal soil collected from Suncheon. Based on these results, P. yonginensis DCY84(T) can be used as a promising PGPB isolate for crop improvement.

Biocontrol of Fusarium wilt disease in tomato by Paenibacillus ehimensis KWN38.

This study was conducted to investigate biocontrol potential of Paenibacillus ehimensis KWN38 against Fusarium oxysporum f.sp. lycopersici causing Fusarium wilt disease in tomato. Our result showed that P. ehimensis KWN38 produced extracellular organic compounds and crude enzyme to inhibit F. oxysporum f.sp. lycopersici conidial germination in in vitro assays. Tomato seedlings were treated with water (W), grass medium (G), G with P. ehimensis KWN38 inoculation (GP) and G along with synthetic fungicide (GSf). Disease symptoms were was first observed in G and W at 12 days after infection (DAI) while symptoms were noticeable in the GP and GSf treatments at 20 and 24 DAI, respectively. Tomato plants treated with P. ehimensis KWN38 or fungicide significantly reduced Fusarium wilt disease incidence and severity as compared to control tomato plants treated with water and grass medium. The similar results were also found in the root mortality of tomato plants. At 25 DAI, most plants in control treatments (W and G) wilted and the brown vascular systems of infected plants was clearly differentiable from normal green vascular system of healthy plants from GP and GSf. Plants in the GP showed higher fresh and dry weights of both root and shoots than those in W and G treatments. Leaf peroxidase and polyphenol oxidase activities of tomato plants in G and W were higher than those in GP and GSf. Root enzyme activities showed a similar pattern but the values were higher than leaf enzyme. The results clearly demonstrated that P. ehimensis KWN38 may be considered as biocontrol agent of Fusarium wilt disease in tomato.

Investigation of Paenibacillus alvei ARN63 ability for biodemulsifier production: medium optimization to break heavy crude oil emulsion.

The demulsifying performance of Paenibacillus alvei ARN63 (P. alvei), as a biodemulsifier-producing bacterium, for breaking water-in-heavy crude oil emulsion has been investigated. The produced lipopeptide biodemulsifier showed the potential to be used in the petroleum industry as an environmentally friendly and non-toxic material. To optimize the biodemulsifier production, the impacts of parameters such as temperature, pH, carbon source and carbon concentration at a constant agitation speed of 180 rpm and with ammonium sulfate as the sole nitrogen source (1.0 g/l) were studied in detail. Several normal paraffin compounds, vegetable oils and motor oil revealed the ability to be used as the carbon source for synthesis of biodemulsifier. The best biodemulsifier production was obtained employing motor oil as the carbon source with a concentration of 42.5 g/l at 37°C and pH 7.0 after 72 h of incubation. Under these conditions, the surface tension of the medium reduced from 58 mN/m to 24.7 mN/m and the biodemulsifier yield reached a value of 2.1 g/l. The demulsification ratio approached 77% and the produced biodemulsifier by P. alvei strain effectively broke water-in-heavy crude oil emulsion. According to biodemulsifier production and growth time course profiles, the biosynthesis was growth associated. Besides, the produced biodemulsifier had good stability during exposure to salinities up to 20%, temperatures up to 80°C and a wide pH range of 2-12.

Detection of secreted antimicrobial peptides isolated from cell-free culture supernatant of Paenibacillus alvei AN5.

An antimicrobial substance produced by the Paenibacillus alvei strain AN5 was detected in fermentation broth. Subsequently, cell-free culture supernatant (CFCS) was obtained by medium centrifugation and filtration, and its antimicrobial activity was tested. This showed a broad inhibitory spectrum against both Gram-positive and -negative bacterial strains. The CFCS was then purified and subjected to SDS-PAGE and infrared spectroscopy, which indicated the proteinaceous nature of the antimicrobial compound. Some de novo sequencing using an automatic Q-TOF premier system determined the amino acid sequence of the purified antimicrobial peptide as Y-S-K-S-L-P-L-S-V-L-N-P (1,316 Da). The novel peptide was designated as peptide AN5-1. Its mode of action was bactericidal, inducing cell lysis in E. coli ATCC 29522 and S. aureus, and non-cell lysis in both S. marcescens and B. cereus ATCC 14579. Peptide AN5-1 displayed stability at a wide range of pH values (2-12) and remained active after exposure to high temperatures (100 °C). It also maintained its antimicrobial activity after incubation with chemicals such as SDS, urea and EDTA.

Interactive optimization of biosurfactant production by Paenibacillus alvei ARN63 isolated from an Iranian oil well.

The potential of an indigenous bacterial strain isolated from an Iranian oil field for the production of biosurfactant was investigated in this study. After isolation, the bacterium was characterized to be Paenibacillus alvei by biochemical tests and 16S ribotyping. The biosurfactant, which was produced by this bacterium, was able to lower the surface tension of media to 35 mN/m. Accordingly, thin layer chromatography (TLC) and FT-IR has been carried out to determine compositional analysis of the produced biosurfactant. After all the tests related to characterization of the biosurfactant produced by the isolated bacterium, it was characterized as lipopeptide derivative. The combination of central composite rotatable design (CCRD) and response surface methodology (RSM) was exploited to optimize biosurfactant production. Therefore, variations of four impressive parameters, pH, temperature, glucose and salinity concentrations were selected for optimization of growth conditions. The empirical model developed through RSM in terms of effective operational factors mentioned above was found to be adequate to describe the biosurfactant production. A maximum reduction in surface tension was obtained under the optimal conditions of 13.03 g/l glucose concentration, 34.76 °C, 51.39 g/l total salt concentration and medium pH 6.89.

Growth, Fe3+ reductase activity, and siderophore production by Paenibacillus polymyxa SQR-21 under differential iron conditions.

An experiment was planned to evaluate the behavior of Paenibacillus polymyxa SQR-21 under differential iron availability. P. polymyxa was grown under three differential iron conditions (0, 2, 20 µM). Iron starvation slowed bacterial growth and at all iron levels, pH of liquid culture was decreased, but maximum decrease was observed at highest iron level. Cell surface ferrireductase activity decreased as culture aged, while extracellular Fe(3+)-reducing activity constantly increased. Hydroxamates type siderophores production was increased with the decrease in iron levels. Numerous cellular proteins were expressed by P. polymyxa in the range of 5-140 kDa and several of them showed conspicuous differential iron regulation. P. polymyxa seems to have more than one type of iron acquisition mechanism including gradual release of organic acids, cell surface ferrireductases, extracellular reductants, and secretion of low molecular weight hydroxamates chelators. This article is the first to report the kinetic study of P. polymyxa under differential iron availability. The information provided here gives initial information about the iron uptake mechanism of P. polymyxa.