Augmentation of physiology and productivity, and reduction of lead accumulation in lettuce grown in lead contaminated soil by rhizobacteria-assisted rhizoengineeing.

Microbial-assisted rhizoengineering is a promising biotechnology for improving crop productivity. In this study, lettuce roots were bacterized with two lead (Pb) tolerant rhizobacteria including Pseudomonas azotoformans ESR4 and P. poae ESR6, and a consortium consisted of ESR4 and ESR6 to increase productivity, physiology and antioxidants, and reduce Pb accumulation grown in Pb-contaminated soil i.e., 80 (Pb in native soil), 400 and 800 mg kg-1 Pb. In vitro studies showed that these strains and the consortium produced biofilms, synthesized indole-3-acetic acid and NH3, and solubilized phosphate challenging to 0, 100, 200 and 400 mg L-1 of Pb. In static conditions and 400 mg L-1 Pb, ESR4, ESR6 and the consortium adsorbed 317.0, 339.5 and 357.4 mg L-1 Pb, respectively, while 384.7, 380.7 and 373.2 mg L-1 Pb, respectively, in shaking conditions. Fourier transform infrared spectroscopy results revealed that several functional groups [Pb-S, M - O, O-M-O (M = metal ions), S-S, PO, CO, -NH, -NH2, C-C-O, and C-H] were involved in Pb adsorption. ESR4, ESR6 and the consortium-assisted rhizoengineering (i) increased leaf numbers and biomass production, (ii) reduced H2O2 production, malondialdehyde, electrolyte leakages, and transpiration rate, (iii) augmented photosynthetic pigments, photosynthetic rate, water use efficiency, total antioxidant capacity, total flavonoid content, total phenolic content, and minerals like Ca2+ and Mg2+ in comparison to non-rhizoengineering plants grown in Pb-contaminated soil. Principal component analysis revealed that higher pigment production and photosynthetic rate, improved water use efficiency and increased uptake of Ca2+ were interlinked to increased productivity by bacterial rhizoengineering of lettuce grown in different levels of Pb exposures. Surprisingly, Pb accumulation in lettuce roots and shoots was remarkably decreased by rhizoengineering than in non-rhizoengineering. Thus, these bacterial strains and this consortium could be utilized to improve productivity and reduce Pb accumulation in lettuce.

Decolorization, degradation and detoxification of mutagenic dye Methyl orange by novel biofilm producing plant growth-promoting rhizobacteria.

Discharge of untreated dyeing wastewater nearby water-bodies is one of major causes of water pollution. Generally, bacterial strains isolated from industrial effluents and/or contaminated soils are used for the bioremediation of Methyl orange (MO), a mutagenic recalcitrant mono-azo dye, used in textiles and biomedical. However, MO degradation by biofilm producing plant growth-promoting rhizobacteria (BPPGPR) was not studied yet. In this study, 19 out of 21 BPPGPR strains decolorized 96.3-99.9% and 89.5-96.3% MO under microaerophilic and aerobic conditions, respectively from Luria-Bertani broth (LBB) followed by yeast-extract peptone and salt-optimized broth plus glycerol media within 120 h of incubation at 28 °C. Only selected BPPGPR including Pseudomonas fluorescens ESR7, P. veronii ESR13, Stenotrophomonas maltophilia ESR20, Staphylococcus saprophyticus ESD8, and P. parafulva ESB18 were examined for process optimization of MO decolorization using a single factor optimization method. This study showed that under optimal conditions (e.g., LBB, 100 mg L-1 MO, pH 7, incubation of 96 h, 28 °C), these strains could remove 99.1-99.8% and 97.6-99.5% MO under microaerophilic and aerobic conditions, respectively. Total azoreductase and laccase activities responsible for biodegradation were also remarkably activated in the biodegraded samples under optimal conditions, while these activities were repressed under unfavorable conditions (e.g., 40 °C and 7.5% NaCl). This study confirmed that MO was degraded and detoxified by these bacterial strains through breakage of azo bond. So far, this is the first report on bioremediation of MO by the BPPGPR strains. These BPPGPR strains are highly promising to be utilized for the bioremediation of dyeing wastewater in future.

The first report on a new Tor species, Tor barakae Arunkumar & Basudha 2003, from Bangladesh using DNA barcoding technique.

Mahseer are large-scale fish of the Cyprinidae family that inhabit South and Southeast Asian mountainous streams, rivers, and reservoirs. Tor tor and Tor putitora, two species of the Tor genus, were reportedly found in Bangladesh. This study aimed to confirm the species level of specimens collected from the Sangu River. The collected samples were identified using the DNA barcoding technique, followed by amplifying 645 bp of the cytochrome oxidase c subunit 1 gene (COI) using the FishF1/FishR1 universal primer. The sequence similarity was conducted using BOLD and NCBI databases which showed 99.85-100 % similarity to the reference genome. The genetic divergence between T. putitora vs. SRI, BT, and ST was found to be 0.0239, 0.0239, and 0.0238, respectively. The genetic divergence between T. tor vs. SRI, BT, and ST was 0.0272, 0.0272, and 0.0270, respectively. In the phylogenetic tree, two clusters were formed where collected specimens (SRI, BT, and ST) formed a subcluster with the reference genome (NC_056296.1 T. barakae) with 100 % bootstrap support. This study's findings revealed the presence of a new Tor species named Tor barakae in the Sangu River basin in Bangladesh.

Biofilm-mediated decolorization, degradation and detoxification of synthetic effluent by novel biofilm-producing bacteria isolated from textile dyeing effluent.

Biofilm-mediated bioremediation of xenobiotic pollutants is an environmental friendly biological technique. In this study, 36 out of 55 bacterial isolates developed biofilms in glass test tubes containing salt-optimized broth plus 2% glycerol (SOBG). Scanning electron microscopy, Fourier transform infrared (FTIR) spectroscopy, and Congo red- and Calcofluor binding results showed biofilm matrices contain proteins, curli, nanocellulose-rich polysaccharides, nucleic acids, lipids, and peptidoglycans. Several functional groups including -OH, N-H, C-H, CO, COO-, -NH2, PO, C-O, and C-C were also predicted. By sequencing, ten novel biofilm-producing bacteria (BPB) were identified, including Exiguobacterium indicum ES31G, Kurthia gibsonii ES43G, Kluyvera cryocrescens ES45G, Cedecea lapagei ES48G, Enterobacter wuhouensis ES49G, Aeromonas caviae ES50G, Lysinibacillus sphaericus ES51G, Acinetobacter haemolyticus ES52G, Enterobacter soli ES53G, and Comamonas aquatica ES54G. The Direct Red (DR) 28 (a carcinogenic and mutagenic dye used in dyeing and biomedical processes) decolorization process was optimized in selected bacterial isolates. Under optimum conditions (SOBG medium, 75 mg L-1 dye, pH 7, 28 °C, microaerophilic condition and within 72 h of incubation), five of the bacteria tested could decolorize 97.8% ± 0.56-99.7% ± 0.45 of DR 28 dye. Azoreductase and laccase enzymes responsible for biodegradation were produced under the optimum condition. UV-Vis spectral analysis revealed that the azo (-NN-) bond peak at 476 nm had almost disappeared in all of the decolorized samples. FTIR data revealed that the foremost characteristic peaks had either partly or entirely vanished or were malformed or stretched. The chemical oxygen demand decreased by 83.3-91.3% in the decolorized samples, while plant probiotic bacterial growth was indistinguishable in the biodegraded metabolites and the original dye. Furthermore, seed germination (%) was higher in the biodegraded metabolites than the parent dye. Thus, examined BPB could provide potential solutions for the bioremediation of industrial dyes in wastewater.

Halotolerant biofilm-producing rhizobacteria mitigate seawater-induced salt stress and promote growth of tomato.

Biofilm-producing rhizobacteria (BPR) enhance productivity and mitigate abiotic stresses in plants. This study showed that 21 out of 65 halotolerant rhizobacteria could build biofilms. The components of the biofilm matrices i.e., extracellular polymeric substances (EPS) are proteins, curli, nanocelloluse, nucleic acids, lipids, and peptidoglycans. Various functional groups including carbonyl, carboxyl, amino, hydroxyl, and phosphate were identified. Positions of these groups were shifted by application of 5% NaCl, suggesting Na+ biosorption. By sequencing, Glutamicibacter arilaitensis (ESK1, ESM4 and ESM7), G. nicotianae (ESK19, ESM8 and ESM16), Enterobacter ludwigii (ESK15, ESK17, ESM2 and ESM17), E. cloacae (ESM5 and ESM12), Exiguobacterium acetylicum (ESM24 and ESM25), Staphylococcus saprophyticus ESK6, Leclercia adecarboxylata ESK12, Pseudomonas poae ESK16, Bacillus subtilis ESM14, and P. putida ESM17 were identified. These rhizobacteria exhibited numerous plant growth-promoting (PGP) activities including producing IAA, ACC deaminase, and siderophores, and solubilizing phosphate. Under non-stress, bacterized plants increased biomass accumulation (8-23.2% roots and 23-49.4% shoots), while under seawater-induced salt stress only ESK12, ESM4, ESM12, and ESM14 enhanced biomass production (5.8-52.9% roots and 8.8-33.4% shoots). Bacterized plants induced antioxidant defense system (19.5-142% catalase and 12.3-24.2% DPPH radical scavenging activity), retained a greater relative water content (17-124%), showed lesser membrane injuries (19.9-26.5%), and a reduced Na+ (6-24% in roots) and increased K+/Na+ ratio (78.8 and 103% in roots by ESK12 and ESM24, respectively) than the non-bacterized plants in saline conditions. Thus, native halotolerant BPR can be utilized as ameliorators of salt stress.

Biofilm Formation, Production of Matrix Compounds and Biosorption of Copper, Nickel and Lead by Different Bacterial Strains.

Bacterial biofilms play a key role in metal biosorption from wastewater. Recently, Enterobacter asburiae ENSD102, Enterobacter ludwigii ENSH201, Vitreoscilla sp. ENSG301, Acinetobacter lwoffii ENSG302, and Bacillus thuringiensis ENSW401 were shown to form air-liquid (AL) and solid-air-liquid (SAL) biofilms in a static condition at 28 and 37°C, respectively. However, how environmental and nutritional conditions affect biofilm formation; production of curli and cellulose; and biosorption of copper (Cu), nickel (Ni), and lead (Pb) by these bacteria have not been studied yet. In this study, E. asburiae ENSD102, E. ludwigii ENSH201, and B. thuringiensis ENSW401 developed the SAL biofilms at pH 8, while E. asburiae ENSD102 and Vitreoscilla sp. ENSG301 constructed the SAL biofilms at pH 4. However, all these strains produced AL biofilms at pH 7. In high osmolarity and ½-strength media, all these bacteria built fragile AL biofilms, while none of these strains generated the biofilms in anaerobic conditions. Congo red binding results showed that both environmental cues and bacterial strains played a vital role in curli and cellulose production. Calcofluor binding and spectrophotometric results revealed that all these bacterial strains produced significantly lesser amounts of cellulose at 37°C, pH 8, and in high osmotic conditions as compared to the regular media, at 28°C, and pH 7. Metal biosorption was drastically reduced in these bacteria at 37°C than at 28°C. Only Vitreoscilla sp. ENSG301 and B. thuringiensis ENSW401 completely removed (100%) Cu and Ni at an initial concentration of 12.5 mg l-1, while all these bacteria totally removed (100%) Pb at concentrations of 12.5 and 25 mg l-1 at pH 7 and 28°C. At an initial concentration of 100 mg l-1, the removal of Cu (92.5 to 97.8%) and Pb (89.3 to 98.3%) was the highest at pH 6, while it was higher (84.7 to 93.9%) for Ni at pH 7. Fourier transform infrared spectroscopy results showed metal-unloaded biomass biofilms contained amino, hydroxyl, carboxyl, carbonyl, and phosphate groups. The peak positions of these groups were shifted responding to Cu, Ni, and Pb, suggesting biosorption of metals. Thus, these bacterial strains could be utilized to remove Cu, Ni, and Pb from aquatic environment.

Decolorization, degradation and detoxification of carcinogenic sulfonated azo dye methyl orange by newly developed biofilm consortia.

Metabolites of azo dyes are often carcinogenic, teratogenic, mutagenic and recalcitrant in nature. In this study, four biofilm consortia such as C1 (Vitreoscilla sp. ENSG301, Acinetobacter lwoffii ENSG302, Klebsiella pneumoniae ENSG303 and Pseudomonas fluorescens ENSG304), C2 (Escherichia coli ENSD101, Enterobacter asburiae ENSD102 and E. ludwigii ENSH201), C3 (E. asburiae ENSD102, Vitreoscilla sp. ENSG301 and Bacillus thuringiensis ENSW401), and C4 (E. coli ENSD101, E. ludwigii ENSH201 and B. thuringiensis ENSW401) were applied to degrade and detoxify methyl orange (MO), a carcinogenic, sulfonated mono azo dye, used in textile dyeing industry worldwide. The consortia of C1, C2, C3 and C4 showed 97.30, 98.75, 99.51 and 99.29% decolorization, respectively in yeast extract peptone (YEP) broth containing 200 mg L-1 MO within 60 h of incubation in static condition. The optimum pH and temperature for decolorization was 7.0 and 28 °C, respectively. Some divalent metal ions including Mg2+, Ca2+, Zn2+ and Mn2+ could stimulate MO decolorization. UV-Vis spectral analysis showed that the absorption peak at 465 nm originated from the azo (N[bond, double bond]N) bond was completely disappeared within 60 h of incubation. Fourier transform infrared spectroscopy (FTIR) results also revealed that several major peaks including azo bond peak at 1602.6 cm-1 are completely or partly vanished, deformed or shifted. Activities of azoreductase, NADH-DCIP reductase and laccase were significantly increased in the bacterial cells within 60 h of incubation in comparison to that of control (0 h). The chemical oxygen demand was incredibly reduced by 85.37 to 91.44% by these consortia. Accordingly, plant (wheat seed germination) and microbial (growth of the plant probiotic bacteria such as Pseudomonas cedrina ESR12 and Bacillus cereus ESD3 on biodegraded products) toxicity studies showed that biodegraded products of MO are non-toxic. Thus, all these consortia can be utilized in bioremediation of MO from wastewater for safe disposal into environment. To our knowledge, this is the first report on degradation and detoxification of MO from wastewater by bacterial biofilm consortia.

Novel bacterial biofilm consortia that degrade and detoxify the carcinogenic diazo dye Congo red.

Free-living planktonic single bacterial strain can decolorize Congo red (CR) but often produces the carcinogenic, mutagenic and genotoxic aromatic amines. Planktonic single and bacterial consortia are more susceptible to toxic pollutants than their biofilm counterparts. In the present study, four biofilm consortia (C1 = Vitreoscilla sp. ENSG301, Acinetobacter lwoffii ENSG302, Klebsiella pneumoniae ENSG303 and Pseudomonas fluorescens ENSG304, C2 = Escherichia coli ENSD101, Enterobacter asburiae ENSD102 and E. ludwigii ENSH201, C3 = E. asburiae ENSD102, Vitreoscilla sp. ENSG301 and Bacillus thuringiensis ENSW401, and C4 = E. coli ENSD101, E. ludwigii ENSH201 and B. thuringiensis ENSW401) were prepared and assessed for bioremediation of CR. All these biofilm consortia remarkably decolorized (96.9 to 99.5%) the CR (100 mg/L) in static condition within 72 h incubation at 28 °C. These consortia also synthesized significantly more intracellular azoreductase and laccase enzyme than extracellular of these enzymes. UV-Vis spectral analysis revealed that the major peak at 478 nm wavelength of CR was completely disappeared. FTIR analysis showed several major peaks along with azo bonds are completely or partly disappeared, deformed or widened. Chemical oxygen demand was reduced by 86.4, 85.5, 87.0 and 86.2% by C1, C2, C3 and C4, respectively. Accordingly, biodegraded metabolites of CR by different biofilm consortia did not inhibit the germination of wheat seeds and bacterial growth. Thus, these biofilm consortia can be applied in bioremediation of wastewater containing CR for safe disposal into the environment. To our knowledge, this is the first report on degradation and detoxification of aqueous solution containing CR by bacterial biofilm consortia.

Biofilm Producing Rhizobacteria With Multiple Plant Growth-Promoting Traits Promote Growth of Tomato Under Water-Deficit Stress.

Plant growth-promoting rhizobacteria (PGPR) not only enhance plant growth but also control phytopathogens and mitigate abiotic stresses, including water-deficit stress. In this study, 21 (26.9%) rhizobacterial strains isolated from drought-prone ecosystems of Bangladesh were able to form air-liquid (AL) biofilms in the glass test tubes containing salt-optimized broth plus glycerol (SOBG) medium. Based on 16S rRNA gene sequencing, Pseudomonas chlororaphis (ESR3 and ESR15), P. azotoformans ESR4, P. poae ESR6, P. fluorescens (ESR7 and ESR25), P. gessardii ESR9, P. cedrina (ESR12, ESR16, and ESR23), P. veronii (ESR13 and ESR21), P. parafulva ESB18, Stenotrophomonas maltophilia ESR20, Bacillus cereus (ESD3, ESD21, and ESB22), B. horikoshii ESD16, B. aryabhattai ESB6, B. megaterium ESB9, and Staphylococcus saprophyticus ESD8 were identified. Fourier transform infrared spectroscopy studies showed that the biofilm matrices contain proteins, polysaccharides, nucleic acids, and lipids. Congo red binding results indicated that these bacteria produced curli fimbriae and nanocellulose-rich polysaccharides. Expression of nanocellulose was also confirmed by Calcofluor binding assays and scanning electron microscopy. In vitro studies revealed that all these rhizobacterial strains expressed multiple plant growth-promoting traits including N2 fixation, production of indole-3-acetic acid, solubilization of nutrients (P, K, and Zn), and production of ammonia, siderophores, ACC deaminase, catalases, lipases, cellulases, and proteases. Several bacteria were also tolerant to multifarious stresses such as drought, high temperature, extreme pH, and salinity. Among these rhizobacteria, P. cedrina ESR12, P. chlororaphis ESR15, and B. cereus ESD3 impeded the growth of Xanthomonas campestris pv. campestris ATCC 33913, while P. chlororaphis ESR15 and B. cereus ESD21 prevented the progression of Ralstonia solanacearum ATCC® 11696TM. In a pot experiment, tomato plants inoculated with P. azotoformans ESR4, P. poae ESR6, P. gessardii ESR9, P. cedrina ESR12, P. chlororaphis ESR15, S. maltophilia ESR20, P. veronii ESR21, and B. aryabhattai ESB6 exhibited an increased plant growth compared to the non-inoculated plants under water deficit-stressed conditions. Accordingly, the bacterial-treated plants showed a higher antioxidant defense system and a fewer tissue damages than non-inoculated plants under water-limiting conditions. Therefore, biofilm-producing PGPR can be utilized as plant growth promoters, suppressors of plant pathogens, and alleviators of water-deficit stress.

The first record of two trichodinid ectoparasites, Trichodina pseudoheterodentata Tang et al. 2017 and Trichodina hafizuddini Asmat, 2005 (Ciliophora: Peritricha) from the freshwater fishes in the Baikka Beel of Moulvibazar district in Sylhet division, Bangladesh

A parasitological investigation on trichodinid ciliates from several freshwater fishes in the Baikka Beel of Moulvibazar district of Sylhet was carried out between January and December 2015. During the investigation period, two parasitic trichodinid ciliates (Ciliophora: Peritrichia), Trichodina pseudoheterodentata Tang et al. 2017 and Trichodina hafizuddini Asmat, 2005 were morphologically studied and described with using the silver nitrate impregnation technique. Trichodina pseudoheterodentata was isolated from the gills of Mystus bleekeri, while Trichodina hafizuddini from the gills of Amblypharyngodon mola. This investigation has been revealed that these two Trichodina species fall within the range of morphometry and agree closely in the overall appearance of the adhesive disc with the original populations. Geographical variation was also observed by considering the body size and number of the denticles. Depending on country's available taxonomical survey on trichodinid ciliates, this study is the first formal report on these trichodinids from any part of Bangladesh.

Trichodina amblypharyngodoni sp. n. and Trichodina hoffmani Wellborn, 1967 (Ciliophora: Trichodinidae) from the freshwater fishes in the Baikka Beel of Moulvibazar district in Sylhet division, Bangladesh

During a survey on species diversity of trichodinid ciliates from freshwater fishes in the Baikka Beel of Moulvibazar district in Sylhet division from January to December 2015, two species of ciliates were identified, of which one is new to science. Trichodina hoffmani Wellborn, 1967 is collected from the gills of Mystus tengara, and Trichodina amblypharyngodoni sp. n. is described from Amblypharyngodon mola using the silver nitrate impregnation method. T. amblypharyngodoni sp. n. is characterized by having a large, darkly stained central area; rectangularly-rounded butterspoon-shaped blade with very narrow interblade space and blunt tangent point; indistinct anterior and posterior blade apophysis, but a rounded apex near the base of the central part of denticle; slender, sometimes slightly triangular central part with rounded tip; and filamentous, curved, anteriorly directed rays with inflated tip and indistinct central groove. Based on these characters and the unique shape and absence of variability of the denticles among the silver impregnated specimens of the present species, it may be said that to a lesser extent, it resembles Trichodina prowazeki Grupcheva and Lom, 1980.