In vitro water stress bioassays with the nematophagous fungus Pochonia chlamydosporia: Effects on growth and parasitism

The biocontrol potential of Pochonia chlamydosporia, a fungus with parasitic activity against economically important plant-parasitic nematodes, can be influenced by abiotic factors such as water availability. The objective of this study was to evaluate the effects of different water stress regimes on in vitro growth, sporulation, germination and parasitism of P. chlamydosporia isolates. The osmotic water potential of 1.7% corn meal agar (CMA) was modified by addition of potassium chloride (KCl) or glycerol, and the matric water potential was modified using polyethylene glycol (PEG 8000). The fungus was able to grow over a range of potentials but radial growth rates decreased with the increase of osmotic and matric stress. No growth was observed at −10 MPa on 1.7% CMA amended with glycerol and at −7.1 MPa on medium with PEG 8000 but all isolates were able to resume growth when transferred onto unmodified 1.7% CMA. The production of chlamydospores was repressed in both osmotic and matric modified media. Although the production of conidia increased in medium modified with KCl, the germination rate was lower. Spores/hyphal fragments remained viable in all isolates that were previously inoculated onto media with growth-limiting water potential (−10 MPa on 1.7% CMA amended with glycerol and −10 MPa on medium with PEG 8000). The percentage of viable conidia produced on 1.7% CMA, after inoculation under osmotic or matric stress conditions for 25 days, was over 74.5% in all isolates (osmotic stress) and ranged from 1% (Pc1) to 65.8% (Pc280) (matric stress). The in vitro infection of potato cyst nematodes, Globodera rostochiensis eggs by P. chlamydosporia isolates, grown under these limiting conditions, was studied using a standard bioassay. The percentage of parasitized eggs was significantly higher under osmotic stress except for isolates Pc2 and Pc3. P. chlamydosporia spores/hyphal fragments can remain viable at water potentials limiting for growth, for prolonged periods of time, suggesting that the osmoregulation mechanisms, used to compensate water stress, affect in vitro sporulation and increased pathogenicity. Knowledge on water requirements of P. chlamydosporia enables a better understanding of its survival and growth strategies in the soil environment and could aid the development of effective strategies to increase the production and quality of inoculum, thus contributing to the implementation of biosafe, sustainable management strategies against plant-parasitic nematodes.     

Additive and synergistic effects of Bacillus spp. isolates and essential oils on the control of phytopathogenic and saprophytic fungi from medicinal plants and marigold seeds

Contamination of plants and seeds with microorganisms is one of the main problems in the production and distribution of various agricultural products, as well as raw herbal material for the preparation of herbal remedies. In targeting microbial contamination, among other bacteria, Bacillus species showed a significant capacity for biocontrol. The antifungal activity of 14 isolates of Bacillus spp. against 15 fungal isolates from medicinal plants was examined utilizing a dual plate assay. The strongest and broadest antagonistic activity against all fungi tested was exhibited by isolates SS-12.6 and SS-13.1 (from a 43% to 74% reduction in fungal growth), while isolates SS-39.1 and SS-39.3 were effective against the fewest fungus species and also had the weakest antifungal activity. The effect of a crude lipopeptide extract (CLE) of Bacillus sp. SS-12.6 was similar to that achieved by a dual culture with isolate SS-12.6, confirming that the antagonism was the result of the antifungal activities of lipopeptides. In addition, essential oils of thyme (0.55 mg/mL) and savory (0.32 mg/mL) in various combinations with the CLE of SS-12.6 were tested for antifungal activity, and additive and synergistic effects for some of the fungi were obtained. When testing the effect of CLE, oils (0.40 mg/mL for thyme oil and 0.21 mg/mL for savory oil) and combinations in situ on marigold seeds, a reduction of total fungal infection without an adverse effect on germination was accomplished by 6-h treatments with CLE of SS-12.6 (85% reduction of fungal infection and 63% germination), supernatant from liquid culture of SS-12.6 (more than 90% reduction of fungal infection with 69% seed germination) and combinations of CLE and savory oil (77% reduction of fungal infection and 62% seed germination) and CLE with thyme and savory oils (about 75% reduction of fungal infection with 69% seed germination).     

Bacillus spp. and Pseudomonas putida as inhibitors of the Colletotrichum acutatum group and potential to control Glomerella leaf spot

The control of Glomerella leaf spot (GLS) in Brazil is solely based on fungicide sprays and new alternatives are needed. In apple, few biological control methods have been evaluated, and most have focused on post-harvest pathogens. Therefore, the objectives of this work were to study the mode of action of three bacterial strains and the commercial product Serenade® (Bacillus subtilis) against the Colletotrichum acutatum group, the causal agents of GLS, and to evaluate the influence of bacterial isolates and Serenade® on the development of the first cycle of infection disease under controlled conditions. To assess the mode of action of the bacterial isolates against strains of the C. acutatum group, in vitro tests were performed. It was tested the effect of the bacteria on conidial germination and mycelial growth, using three methodologies, (i) fungal-bacterial co-cultivation, (ii) bacterial thermostable metabolites and (iii) bacterial volatile compounds. The influence of the bacterial isolates on the GLS development was assessed using apple seedlings. The seedlings were first sprayed weekly with bacterial suspension for 5 weeks, and were then inoculated with conidia suspensions (10⁴ conidia mL⁻¹) of C. acutatum group isolates. Seedlings were maintained in chambers (CONVIRON) at 25 °C and a 12-h light regime. Disease severity of GLS was evaluated daily by counting typical lesions caused by C. acutatum group on all leaves during 12 consecutive days. The disease progress curve was fitted to nonlinear models for incidence and severity data. The treatments were compared by contrasting epidemiological parameters. Bacillus sp. isolated from the apple phylloplane inhibited more than 60% of the C. acutatum group conidial germination. The mode of action of Bacillus sp. and Bacillus alcalophilus on the C. acutatum group was through the production of fixed and volatile compounds, which inhibited mycelial growth. The primary mode of action of Serenade® on the C. acutatum group was the production of thermostable metabolites capable of completely inhibiting mycelial growth. In the GLS disease cycle, it was possible to adjust the monomolecular model for incidence and the number of lesions. There were significant differences between the epidemiological parameters of GLS in seedlings treated with apple phylloplane bacteria or with Serenade® as compared to the controls, indicating a potential for the use of biological control to manage GLS in apple orchards.     

Diversity of culturable sulfidogenic bacteria in two oil–water separation tanks in the north-eastern oil fields of India

Sulfidogenic communities in the production waters of onshore oil fields in north-eastern India were examined using a culturing approach. Production water samples were inoculated into medium selective for Sulfate reducing bacteria (SRB) and Thiosulfate Reducing Bacteria (TRB). The total number of viable sulfidogenic microorganisms in the samples obtained from the two production water tanks was approximately 10⁵ MPN ml^?1 (most probable number per ml). Most of the isolates were thermo-tolerant and could be grown between 40 and 45 °C. Hydrogen sulfide production by TRB was significantly higher than by SRB. Based on 16S rRNA gene sequencing, the isolates were grouped in nine different phylotypes. Phylogenetic analysis indicated that most of the SRB were affiliated with the phylum Proteobacteria, encompassing Gram-negative bacteria, belonging to the genera Desulfovibrio, Desulfomicrobium, and Desulfobulbus. However, five isolates grouped with the genus Desulfotomaculum were found to be gram-positive SRB. Most of the thiosulfate reducing isolates was affiliated with the phylum Firmicutes, including Clostridium and Fusibacter and also with the phylum Proteobacteria, including the genera Enterobacter and Citrobacter. Phylotypes related to Clostridium (69%) and Desulfovibrio (53%) dominated the community in the production water samples. This study demonstrates the diversity of the TRB and SRB that play a critical role in the souring mediated corrosion of the oil–water separation tanks in the north-eastern India oil fields.     

Metabolic engineering of Bacillus sp. for diacetyl production

Diacetyl, a highly valuable product that is extensively used as an ingredient of food, tobacco, and daily chemicals such as perfumes, can be produced from the nonenzymatic oxidative decarboxylation of α-acetolactate during bacterial fermentation and converted to acetoin and 2,3-butanediol by 2,3-butanediol dehydrogenase. In the present study, Bacillus sp. DL01, which gives high acetoin production, was metabolically engineered to improve diacetyl production. After the deletion of α-acetolactate decarboxylase (ALDC)-encoding gene (alsD) by homologous recombination, the engineered strain, named Bacillus sp. DL01-ΔalsD, lost ALDC activity and produced 1.53 g/L diacetyl without acetoin and 2,3-butanediol accumulation. The channeling of carbon flux into diacetyl biosynthetic pathway was amplified by an overexpressed α-acetolactate synthase (ALS)-encoding gene (alsS) in Bacillus sp. DL01-ΔalsD-alsS, which produced 4.02 g/L α-acetolactate and 1.94 g/L diacetyl, and the conversion from α-acetolactate to diacetyl was increased by 1-fold after 20 mM Fe³⁺ was added to the fermentation medium. A titer of 8.69 g/L diacetyl, the highest reported diacetyl production, was achieved by fed-batch fermentation in optimal conditions using the metabolically engineered strain of Bacillus sp. DL01-ΔalsD-alsS. These results are of great importance as a new method for the efficient production of diacetyl by food-safe bacteria.     

Evaluation of fluorescent pseudomonads for plant growth promotion,antifungal activity against Rhizoctonia solani on common bean,and biocontrol potential

Fluorescent pseudomonads are ubiquitous bacteria that are common inhabitants of the rhizosphere and are the most studied group within the genus Pseudomonas. Bacterial isolates (n = 103) from the rhizosphere of wheat and common bean were assessed as potential biocontrol agents in this study. Fungal inhibition tests were performed by a plate assay in which each isolate was tested directly for the production of hydrogen cyanide, protease, siderophore and cellulase. Production of DAPG was verified by using an analytical high performance liquid chromatography assay (HPLC). Plant growth promotion was assessed in phytochamber trials and biocontrol activity was evaluated in greenhouse trials. In all, 52 bacterial isolates with antifungal activity against Rhizoctonia solani were found. Of the 52 isolates, 41 were selected according to their high efficiency in in vitro antagonism, which was shown as inhibition zones in the dual-culture assay. Six of the 41 rhizobacteria, including isolates UTPF7, UTPF13, UTPF18, UTPF22, UTPF27 and strain CHA0 produced HCN. Production of protease enzyme was detected for all isolates excluding UTPF30 isolate. Although some stains appeared not to produce any compound with affinity for ferric iron, other isolates produced prolific amounts, creating a large zone of orange (up to 160 mm², i.e., UTPF16). Seventeen of 41 isolates of fluorescent pseudomonads including strain CHAO produced different amounts of DAPG ranging from 0.6 to 11.4 ng/10⁸ cfu. A total of 39 isolates induced statistically significant effects on plant growth compared with the non-treated control for at least one parameter. The predominant influence observed was increased root length. No bacteria could completely protect the plant against R. solani, although all isolates significantly increased fresh weight as compared to the infested control in greenhouse trials. Pseudomonas fluorescens isolates UTPF16 and UTPF26 significantly (P < 0.05) decreased the number of seedlings with damping-off symptoms in the means of the experiments.     

A Ca-independent α-amylase that is active and stable at low pH from the Bacillus sp. KR-8104

Bacillus sp. KR-8104 was selected from a set of 18 bacteria strains isolated from soil samples and screened for production of amylase. The maximum productivity obtained at pH 5–6 and 60–65 h after cultivation in production medium. New extracellular Ca-independent α-amylase was highly purified using ion exchange and hydrophobic interaction chromatography, which showed a single band with an apparent molecular weight of 59 kDa by SDS-PAGE. This enzyme is active in a wide pH range with its maximum activity at low pH values (4.0–6.0) and has the 90% of its maximum activity at pH 3.5. The α-amylase is optimally active at 75–80 °C. The presence or absence of Ca²⁺ and EDTA did not affect enzyme activity and thermal stability.     

Statistical optimization of antifungal iturin A production from Bacillus amyloliquefaciens RHNK22 using agro-industrial wastes

Biosurfactants are secondary metabolites with surface active properties and have wide application in agriculture, industrial and therapeutic products. The present study was aimed to screen bacteria for the production of biosurfactant, its characterization and development of a cost effective media formulation for iturin A production. A total of 100 bacterial isolates were isolated from different rhizosphere soil samples by enrichment culture method and screened for biosurfactant activity. Twenty isolates were selected for further studies based on their biosurfactant activity [emulsification index (EI%), emulsification assay (EA), surface tension (ST) reduction] and antagonistic activity. Among them one potential isolate Bacillus sp. RHNK22 showed good EI% and EA with different hydrocarbons tested in this study. Using biochemical methods and 16S rRNA gene sequence, it was identified as Bacillus amyloliquefaciens. Presence of iturin A in RHNK22 was identified by gene specific primers and confirmed as iturin A by FTIR and HPLC. B. amyloliquefaciens RHNK22 exhibited good surface active properties and antifungal activity against Sclerotium rolfsii and Macrophomina phaseolina. For cost-effective production of iturin A, 16 different agro-industrial wastes were screened as substrates, and Sunflower oil cake (SOC) was favouring high iturin A production. Further, using response surface methodology (RSM) model, there was a 3-fold increase in iturin A production (using SOC 4%, inoculum size 1%, at pH 6.0 and 37 °C temperature in 48 h). This is the first report on using SOC as a substrate for iturin A production.     

Does soil nutrient availability influence night-time water flux of aspen saplings?

Previous investigations have been demonstrated that night-time water flux may increase or remain unchanged at low mineral nutrient availability. At the same time, it is a well-known fact that night-time water flux is relatively high in fast-growing species, which typically grow in fertile soil. To test the impact of soil nutrient deficiency on night-time water flux and fine-root acclimation in saplings of hybrid aspen (Populus tremula L. × Populus tremuloides Michx.), a growth chamber experiment was carried out. We set up a hypothesis that night-time sap flux density (F) and night-time water use percentage from daytime water use (NWU) are more intense in fertile conditions, in order to enhance or sustain the high intrinsic growth potential of hybrid aspen. The main limiting element in the low nutrient availability treatment (low-n) was nitrogen. The nitrogen concentrations of leaves and fine-roots exhibited the strongest (R² = 0.95; P < 0.001) positive relationship with NWU and foliar biomass. Both the night-time F and NWU were several times higher in the case of fertilized soil (high-n treatment) compared to low-n treatment (P < 0.01). The differences in nocturnal (and in diurnal) F disappeared at the end of the period of sap flow measurements, when the foliage area of trees was almost full-grown. Endogenous increase in water flux during predawn hours was observable only in the high-n treatment. Significantly greater NWU (P < 0.01) and specific fine-root length (P < 0.05), but smaller fine-root biomass (P < 0.05) in saplings of the high-n treatment potentially allow plants to use mass flow in soil more efficiently for transportation nutrients towards roots and to decrease construction costs for fine-root biomass production. Our results suggest that decreased night-time water flux as a result of strong nutrient (especially nitrogen) deficit could be characteristic to fast-growing tree species, which are adapted to grow in fertile soil.     

Diversity and heavy metal tolerance of endophytic fungi from six dominant plant species in a Pb–Zn mine wasteland in China

The diversity and metal tolerance of endophytic fungi from six dominant plant species in a Pb–Zn mine wasteland in Yunnan, China were investigated. Four hundred and ninety-five endophytic fungi were isolated from 690 tissue segments. The endophytic fungal colonization extent and isolation extent ranged from 59 % to 75 %, and 0.42–0.93, respectively, and a positive correlation was detected between them. Stems harboured more endophytic fungi than leaves in each plant species, and the average colonization extent of stems was 82 %, being significantly higher than that of leaves (47 %) (P ≤ 0.001, chi-square test). The fungi were identified to 20 taxa in which Phoma, Alternaria and Peyronellaea were the dominant genera and the relative frequencies of them were 39.6 %, 19.0 % and 20.4 %, respectively. Metal tolerance test showed that 3.6 mM Pb²⁺ or 11.5 mM Zn²⁺ exhibited the greatest toxicity to some isolates and they did not grow on the metal-amended media. In contrast, some isolates were growth stimulated in the presence of tested metals. The isolates of Phoma were more sensitive to Zn²⁺ than the isolates of Alternaria and Peyronellaea. However, the sensitivity of isolates to Pb²⁺ was not significantly different among Phoma, Alternaria, Peyronellaea and other taxa (P > 0.05, chi-square test). Our results suggested that fungal endophyte colonization in Pb–Zn polluted plants is moderately abundant and some isolates have a marked adaptation to Pb²⁺ and Zn²⁺ metals, which has a potential application in phytoremediation in this area.     

Isolation and molecular characterization of Bacillus thuringiensis found in soils of the Cerrado region of Brazil,and their toxicity to Aedes aegypti larvae

This study investigated the potential of Bacillus thuringiensis isolates obtained in the Cerrado region of the Brazilian state of Maranhão for the biological control of Aedes aegypti larvae. The isolates were obtained from soil samples and the identification of the B. thuringiensis colonies was based on morphological characteristics. Bioassays were run to assess the pathogenicity and toxicity of the different strains of the B. thuringiensis against third-instar larvae of A. aegypti. Protein profiles were obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polymerase chain reaction assays were used to detect the toxin genes found in the bacterial isolates. Overall, 12 (4.0%) of the 300 isolates obtained from 45 soil samples were found to present larvicidal activity, with the BtMA-104, BtMA-401 and BtMA-560 isolates causing 100% of mortality. The BtMA-401 isolate was the most virulent, with the lowest median lethal concentration (LC₅₀) (0.004 × 10⁷ spores/mL), followed by the Bacillus thuringiensis var. israelensis standard (0.32 × 10⁷ spores/mL). The protein profiles of BtMA-25 and BtMA-401 isolates indicated the presence of molecular mass consistent with the presence of the proteins Cry4Aa, Cry11Aa and Cyt1, similar to the profile of Bacillus thuringiensis var. israelensis IPS-82. Surprisingly, however, none of the cry and cyt genes analyzed were amplified in the isolate BtMA-401. The results of the present study revealed the larvicidal potential of B. thuringiensis isolates found in the soils of the Cerrado region from Maranhão, although further research will be necessary to better elucidate and describe other genes associated with the production of insecticidal toxins in these isolates.     

Isolation and identification of gastrointestinal microbiota from the short-nosed fruit bat Cynopterus brachyotis brachyotis

Studies on the microbial ecology of gut microbiota in bats are limited and such information is necessary in determining the ecological significance of these hosts. Short-nosed fruit bats (Cynopterus brachyotis brachyotis) are good candidates for microbiota studies given their close association with humans in urban areas. Thus, this study explores the gut microbiota of this species from Peninsular Malaysia by means of biochemical tests and 16S rRNA gene sequences analysis. The estimation of viable bacteria present in the stomach and intestine of C. b. brachyotis ranged from 3.06 × 10¹⁰ to 1.36 × 10¹⁵ CFU/ml for stomach fluid and 1.92 × 10¹⁰ to 6.10 × 10¹⁵ CFU/ml for intestinal fluid. A total of 34 isolates from the stomach and intestine of seven C. b. brachyotis were retrieved. A total of 16 species of bacteria from eight genera (Bacillus, Enterobacter, Enterococcus, Escherichia, Klebsiella, Pantoea, Pseudomonas and Serratia) were identified, Enterobacteriaceae being the most prevalent, contributing 12 out of 16 species isolated. Most isolates from the Family Enterobacteriaceae have been reported as pathogens to humans and wildlife. With the possibility of human wildlife transmission, the findings of this study focus on the importance of bats as reservoirs of potential bacterial pathogens.     

Cold-active extracellular α-amylase production from novel bacteria Microbacterium foliorum GA2 and Bacillus cereus GA6 isolated from Gangotri glacier,Western Himalaya

Microbial cold-active α-amylases offer various economical and ecological benefits through energy savings by overcoming the heating requirements and also provide large biotechnological potentials. The objective of present study was to isolate new cold-adapted bacterial strains for production of cold-active α-amylases and their production optimization. Out of 30 cold-active α-amylase producing bacteria, isolated from soil of Gangotri glacier, Western Himalaya, India, two potential isolates, designated as GA2 and GA6, were selected for enzyme production. The α-amylase production was found maximum at 20 °C and pH 9 after 120 h incubation for GA2; and 20 °C and pH 10 after 96 h incubation for GA6. Among the carbon sources, lactose and glycerol was most suitable for GA2 and GA6, respectively. However, yeast extract and ammonium acetate was found best as nitrogen source by GA2 and GA6, respectively. Out of two potential isolates, maximum enzyme production (5870 units) was achieved with GA2 followed by GA6 (4746 units). GA2 was resistant to penicillin (10 μg) among tested antibiotics and as per plasmid curing results, amylase production was a plasmid mediated characteristic. The phylogenetic analysis revealed that GA2 and GA6 have highest homology with Microbacterium foliorum (99%) and Bacillus cereus (98%), respectively. This was the first report on cold-active α-amylase production by M. foliorum strain GA2 and B. cereus strain GA6, also their 16S rRNA sequences assigned an accession number HQ832574 and HQ832575, respectively from NCBI.     

Wool-associated proteolytic bacteria,isolated from Portuguese Merino breed

The main purpose of this study was to isolate and briefly characterize proteolytic bacteria from a poorly known habitat – raw wool. Fleece samples were accordingly collected from Merino raw wool – a Portuguese ewe breed, at three distinct areas of their body, from animals exhibiting no symptoms or signs of abnormalities; they were then subjected to enumeration and isolation of a total of 158 bacterial strains. Said isolates were screened for protease activity, using the spot technique, on Calcium Caseinate Agar containing 1% (w/v) skim milk. The 36 isolates displaying the highest protease activity underwent a more refined assessment of enzymatic performance – by examining their cell-free supernatant extracts, using casein as substrate. Two Bacillus isolates were eventually chosen owing to their highest proteolytic activities (24.6 and 15.9 U/mL), and identified using molecular biology tools.     

The involvement of acetic acid in programmed cell death for the elimination of Bacillus sp. used in bioremediation

A bacterium capable of tolerating some textile effluent conditions such as salinity and high temperature was used to decolorize methyl red (MR). Bacillus sp. isolated from Dead Sea salt was identified by 16S rRNA phylogenetic identification as Bacillus ST (accession number: HQ013327). The bacterium showed 98% color removal under microaerophilic conditions in 24 h at 30 °C and 78% at 80 °C in the presence of mannitol and NaCl. After decolorization an induced programmed cell death (PCD) was proposed to eliminate all living Bacillus ST cells. Acetic acid, which was linked to programmed cell death in yeast, was used in this study to examine its ability to induce PCD in bacteria as well. The acetic acid concentration used was enough to shift the culture pH from alkalinity to neutral. The formation of cytosolic caspase-3 suggested that the programmed cell death occurs through an extrinsic pathway by which mitochondria are by passed and caspases are directly activated. Comet assay which proved death upon the addition of 80 mM acetic acid in 120 min as compared to gamma radiation killed cells. Transmission electron microscopy (TEM) showed cell shrinking, chromatin condensation and membrane blebbing which are all signs of PCD. The mode by which this bacterium follows in programmed cell death is very similar to those in mammalian and yeast cells. The process is proposed to be safe, efficient and cheap for getting rid of cells after bioremediation of colored effluent.     

RAPD-PCR and 16S rDNA phylogenetic analysis of alkaline protease producing bacteria isolated from soil of India: Identification and detection of genetic variability

178 bacterial strains were isolated from the soil samples collected from different regions of India out of which, 20 bacterial isolates were selected for alkaline protease production. The alkaline protease production efficiency of organisms was monitored at regular intervals (24 h) upto 7 days at 37 °C, pH 10. The 16S rDNA sequencing and RAPD-PCR based technique were used to identify the genetic variability among the 20 isolates of alkaline protease producing bacteria. The phylogenetic analysis indicated that the isolates can be separated into two clusters which could be further subdivided into five groups. Group 1 and 5 represented the family Bacillaceae, Groups 2 represented the Micrococcaceae family while Group 3 included the Arthrobacter bacterial group (family Micrococcaceae) from different geographical locations, respectively. Group 4 was identified as Pseudomonadaceae which was gram (−) bacteria. 21 different oligonucleotide primers were used to amplify approximately 261 fragments from each DNA sample. The bands were scored on the basis of their presence and absence and similarity between DNA samples was checked using Jaccard’s coefficient. Isolates were distinguished into distinct groups based on RAPD profiles from different geographical locations, morphological features and enzyme production efficiency. For cluster analysis the dendrogram was constructed using the unweighted pair group method with arithmetic averages (UPGMA). The results indicated that 16S rDNA and RAPD-PCR are suitable methods for rapid identification and differentiation of alkaline protease producing bacteria.     

Purification and characterization of cellulase from a marine Bacillus sp. H1666: A potential agent for single step saccharification of seaweed biomass

In this study, 115 marine bacterial isolates were screened for cellulase enzymatic activity and enzyme with a molecular mass of 40 kDa was purified from culture supernatant of the marine bacterium Bacillus sp. H1666 using ion exchange and size exclusion chromatography method. Growth of bacterial strain H1666 with efficient cellulase enzyme production was observed on untreated wheat straw and rice bran. The biochemical properties of the extracted cellulase were studied and enzyme was found active over a range of pH 3–9. The optimum cellulase activity was observed at pH 7 and temperature 50 °C. The enzyme was also shown to be slightly thermo-stable with 40% residual activity at 60 °C for 4 h. The potential applicability of enzyme was tested on dried green seaweed (Ulva lactuca) and 450 mg/g increase in glucose yield was obtained after saccharification. MALDI TOF–TOF analysis of cellulase peptide fingerprint showed similarity to the sequence of the glycoside hydrolase family protein.     

Trametes versicolor: Potential for atrazine bioremediation in calcareous clay soil,under low water availability conditions

This study examined the feasibility of Trametes versicolor to actively degrade atrazine (0.5 μg g^?1) in non-sterile calcareous clay soil (Algarve, Portugal) microcosms for up to 24 weeks (20 °C), under low water availability (soil water potentials of ?0.7 and ?2.8 MPa). Soil respiration, laccase activity, and atrazine quantification by high-performance liquid chromatography (HPLC) were assessed. Respiration was significantly (p < 0.05) enhanced in soil containing the inoculant, particularly in the presence of atrazine, indicating that it remained metabolically active throughout the study. Furthermore, up to 98% and 85% (at ?0.7 and ?2.8 MPa, respectively) of atrazine was degraded in soil containing both the atrazine and the inoculant, compared to 96% and 50% in soil containing atrazine only. The contribution of T. versicolor to atrazine degradation was only significant (p < 0.005) under the driest soil treatment conditions. The strategies used for enhancing colonisation and biodegradation capabilities of the inoculant, as well as the selection of sawdust as carrier, were thus effective. However, there were no differences (p > 0.05) in quantified laccase activity in soil containing the inoculant and the control. Overall, this study demonstrated that T. versicolor was a strong candidate for atrazine bioremediation in soil with low moisture and organic matter contents, such as that found in semi-arid and Mediterranean-like ecosystems.     

The first crenarchaeon capable of growth by anaerobic carbon monoxide oxidation coupled with H2 production

The ability to grow by anaerobic CO oxidation with production of H₂ from water is known for some thermophilic bacteria, most of which belong to Firmicutes, as well as for a few hyperthermophilic Euryarchaeota isolated from deep-sea hydrothermal habitats. A hyperthermophilic, neutrophilic, anaerobic filamentous archaeon strain 1505 = VKM B-3180 = KCTC 15798 was isolated from a terrestrial hot spring in Kamchatka (Russia) in the presence of 30% CO in the gas phase. Strain 1505 could grow lithotrophically using carbon monoxide as the energy source with the production of hydrogen according to the equation CO + H₂O → CO₂ + H₂; mixotrophically on CO plus glucose; and organotrophically on peptone, yeast extract, glucose, sucrose, or Avicel. The genome of strain 1505 was sequenced and assembled into a single chromosome. Based on 16S rRNA gene sequence analysis and in silico genome-genome hybridization, this organism was shown to be closely related to the Thermofilum adornatum species. In the genome of Thermofilum sp. strain 1505, a gene cluster (TCARB_0867-TCARB_0879) was found that included genes of anaerobic (Ni,Fe-containing) carbon monoxide dehydrogenase and genes of energy-converting hydrogenase ([Ni,Fe]-CODH–ECH gene cluster). Compared to the [Ni,Fe]-CODH–ECH gene clusters occurring in the sequenced genomes of other H₂-producing carboxydotrophs, the [Ni,Fe]-CODH–ECH gene cluster of Thermofilum sp. strain 1505 presented a novel type of gene organization. The results of the study provided the first evidence of anaerobic CO oxidation coupled with H₂ production performed by a crenarchaeon, as well as the first documented case of lithotrophic growth of a Thermofilaceae representative.     

Antibiotic resistance profiling and phenotyping of Aeromonas species isolated from aquatic sources

This study aimed to investigate antibiotics resistance pattern and phenotyping of Aeromonas species isolated from different aquatic sources in Melaka, Malaysia. A total of 53 Aeromonas species were isolated from the following sources: sediment (n = 13), bivalve (n = 10), sea cucumber (n = 16) and sea water (n = 14) and resistance to 12 antibiotics – Tetracycline (30 μg), Kanamycin (30 μg), Oxytetracycline (30 μg), Ampicillin (10 μg), Streptomycin (10 μg), Gentamicin (10 μg), Sulphamethoxazole (25 μg), Nalixidic acid (30 μg), Trimethoprim (1.25 μg), Novobiocin (5 μg), Penicilin (10 μg) and Chloramphenicol (10 μg) was tested. The results obtained from this study reveal multi drug resistance pattern among the isolates. All the isolates were completely resistant to Ampicillin, Novobiocin, Sulphamethoxazole and Trimethoprim, respectively but susceptible to Tetracycline (100%), Kanamycin (5.7%), Gentamicin (5.7%) and Oxytetracycline (24.5%). Antibiotics phenotyping of the bacteria revealed 21 different phenotypes among the isolates.