Taxonomical and functional microbial community dynamics in an Anammox-ASBR system under different Fe (III) supplementation

In the present study, we explored the metabolic versatility of anaerobic ammonium oxidation (anammox) bacteria in a variety of Fe (III) concentrations. Specifically, we investigated the impacts of Fe (III) on anammox growth rates, on nitrogen removal performance, and on microbial community dynamics. The results from our short-term experiments revealed that Fe (III) concentrations (0.04–0.10 mM) significantly promote the specific anammox growth rate from 0.1343 to 0.1709 d^?1. In the long-term experiments, the Anammox-anaerobic sequencing batch reactor (ASBR) was operated over 120 days and achieved maximum NH₄ ⁺-N, NO₂ ^?-N, and TN efficiencies of 90.98 ± 0.35, 93.78 ± 0.29, and 83.66 ± 0.46 %, respectively. Pearson’s correlation coefficients between anammox-(narG + napA), anammox-nrfA, and anammox-FeRB all exceeded r = 0.820 (p < 0.05), confirming an interaction and ecological association among the nitrogen and iron-cycling-related microbial communities. Illumina MiSeq sequencing indicated that Chloroflexi (34.39–39.31 %) was the most abundant phylum in an Anammox-ASBR system, followed by Planctomycetes (30.73–35.31 %), Proteobacteria (15.40–18.61 %), and Chlorobi (4.78–6.58 %). Furthermore, we found that higher Fe (III) supplementation (>0.06 mM) could result in the community succession of anammox species, in which Candidatus Brocadia and Candidatus Kuenenia were the dominant anammox bacteria species. Combined analyses indicated that the coupling of anammox, dissimilatory nitrogen reduction to ammonium, and iron reduction accounted for nitrogen loss in the Anammox-ASBR system. Overall, the knowledge gained in this study provides novel insights into the microbial community dynamics and metabolic potential of anammox bacteria under Fe (III) supplementation.     

Short-term effects of crop rotation,residue management,and soil water on carbon mineralization in a tropical cropping system

The purpose of this study was to investigate the short-term effects of maize (Zea mays)-fallow rotation, residue management, and soil water on carbon mineralization in a tropical cropping system in Ghana. After 15 months of the trial, maize–legume rotation treatments had significantly (P?C ₀ (μg CO₂–C g^?1) than maize–elephant grass (Pennisetum purpureum) rotations. The C ₀ for maize–grass rotation treatments was significantly related to the biomass input (r?=?0.95; P?=?0.05), but that for the maize–legume rotation was not. The soil carbon mineralization rate constant, k (per day), was also significantly related to the rotation treatments (P?k values for maize–grass and maize–legume rotation treatments were 0.025 and 0.036 day^?1 respectively. The initial carbon mineralization rate, m ₀ (μg CO₂–C g^?1 day ^?1), was significantly (P?θ. The m ₀ ranged from 3.88 to 18.67 and from 2.30 to 15.35 μg CO₂–C g^?1 day^?1 for maize–legume and maize–grass rotation treatments, respectively, when the soil water varied from 28% to 95% field capacity (FC). A simple soil water content (θ)-based factor, f _w, formulated as: \(f_{\text{w}} = \left[ {\frac{{\theta - \theta _{\text{d}} }}{{\theta _{{\text{FC}}} - \theta _{\text{d}} }}} \right]\), where θ _d and θ _FC were the air-dry and field capacity soil water content, respectively, adequately described the variation of the m ₀ with respect to soil water (R ²?=?0.91; RMSE?=?1.6). Such a simple relationship could be useful for SOC modeling under variable soil water conditions.     

Occurrence and distribution of multiple antibiotic-resistant <Emphasis Type="Italic">Enterococcus</Emphasis> and <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. from Indian poultry: in vivo transferability of their erythromycin,tetracycline and vancomycin resistance

The objective of this study was to determine the occurrence and distribution of antibiotic resistant (AR) lactic acid bacteria (LAB) in Indian poultry. LAB from poultry farm feces (n = 21) and samples from slaughter houses comprising chicken intestine (n = 46), raw meat (n = 23), and sanitary water (n = 4) were evaluated and compared with those from organic chicken (OC) collected from nearby villages. Screening studies showed 5–7 log units higher erythromycin (ER), tetracycline (TC) and vancomycin (VAN) resistant LAB from conventional poultry chicken (CC) compared to OC. Molecular characterization of isolated cultures (n = 32) with repetitive-PCR profiling and 16S rRNA gene sequencing revealed their taxonomical status as Enterococcus faecium (n = 16), Enterococcus durans (n = 2), Lactobacillus plantarum (n = 10), Lactobacillus pentosus (n = 1) and Lactobacillus salivarius (n = 3). The isolates were found to harbor erm(B), msr(C), msr(A/B), tet(M), tet(L) and tet(K) genes associated with Tn916 and Tn917 family transposons. Expression studies through real-time PCR revealed antibiotic-induced expression of the identified AR genes. In vitro and in vivo conjugational studies revealed transfer of ER and TC resistant (ER^R and TC^R) genes with transfer frequencies of 10^?7 and 10^?4 transconjugants recipient^?1, respectively. Although no known VAN resistance (VAN^R) genes were detected, high phenotypic resistance was observed and was transferable to the recipient. From a public health point of view, this study reports Indian poultry as a major source of high levels of AR bacteria contaminating the food chain and the environment. Thus, urgent and determined strategies are needed to control the spread of multiple AR bacteria.     

Optimization of continuous-flow solid-phase denitrification via coupling carriers in enhancing simultaneous removal of nitrogen and organics for agricultural runoff purification

Coupling of biodegradable corncob and plastic carrier was optimized in continuous-flow solid-phase denitrification systems for enhancing simultaneously removal of nitrogen and organics in agricultural runoff. In compared with preposition of plastic carriers and mixed distribution method, it was demonstrated that the preposition of corncobs simultaneously enhanced nitrate (6.64 ± 1.35 mg L^?1 day ^?1) and organics removal (6.33 ± 1.44 mg L^?1 day^?1) at a hydraulic retention time (HRT) of 6 h. The operation performance could be further enhanced with extension of HRT to 12 h. The dominant genera found in corncob were denitrifiers for nitrate reduction (Bosea, Simplicispira, Desulfovibrio, Klebsiella, etc.) and fermentative bacteria (Pleomorphomonas, Actinotalea, Opitutus, Cellulomonas, Bacteroides, etc.) responsible for corncob degrading to simple organics for other denitrifiers. However, much lower and different denitrifiers abundances (Bradyrhizobium, Acinetobacter, Bacillus, etc.) exhibited on plastic filler than those of corncob. It well explained that the biofilm on plastic carrier was mainly related with organics removal while the biofilm on corncobs inclined to effectively remove nitrate, and simultaneous removal of nitrogen and organics could be achieved in coupling carriers system with preposition of biodegradable corncob.     

Different responses in the expression of alginases,alginate polymerase and acetylation genes during alginate production by <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> under oxygen-controlled conditions

Alginate production and gene expression of genes involved in alginate biosynthesis were evaluated in continuous cultures under dissolved oxygen tension (DOT) controlled conditions. Chemostat at 8% DOT showed an increase in the specific oxygen uptake rate \((q_{{{\text{O}}_{ 2} }} )\) from 10.9 to 45.3 mmol g^?1 h^?1 by changes in the dilution rate (D) from 0.06 to 0.10 h^?1, whereas under 1% DOT the \(q_{{{\text{O}}_{ 2} }}\) was not affected. Alginate molecular weight was not affected by DOT. However, chemostat at 1% DOT showed a downregulation up to 20-fold in genes encoding both the alginate polymerase (alg8, alg44), alginate acetylases (algV, algI) and alginate lyase AlgL. alyA1 and algE7 lyases gene expressions presented an opposite behavior by changing the DOT, suggesting that A. vinelandii can use specific depolymerases depending on the oxygen level. Overall, the DOT level have a differential effect on genes involved in alginate synthesis, thus a gene expression equilibrium determines the production of alginates of similar molecular weight under DOT controlled.     

Response of performance and bacterial community to oligotrophic stress in biofilm systems for raw water pretreatment

Understanding the dynamics of performance and bacterial community of biofilm under oligotrophic stress is necessary for the process optimization and risk management in biofilm systems for raw water pretreatment. In this study, biofilm obtained from a pilot-scale biofilm reactor was inoculated into a pilot-scale experimental tank for the treatment of oligotrophic raw water. Results showed that the removal of NH₄ ⁺–N was impaired in biofilm systems when influent NH₄ ⁺–N was less than 0.35 mg L^?1 or NH₄ ⁺–N loading rate of less than 7.51 mg L^?1 day^?1. The dominant bacteria detected in biofilm of different carrier were obvious distinct from phylum to genus level under oligotrophic stress. The dominant bacteria in elastic stereo media carrier changed from Proteobacteria (51.1%) to Firmicutes (32.7%), while Proteobacteria was always dominant in suspended ball carrier after long-term operation under oligotrophic conditions. Oligotrophic stress largely decreased the functional bacteria for the removal of nitrogen and organics including many genera in Proteobacteria and Nitrospirae, but increased several genera with spore forming organisms or potential bacterial pathogens in ESM carrier mainly including Bacillus, Mycobacterium, Pseudomonas, etc.     

Changes in Immunity,Expression of some Immune-Related Genes of Shabot Fish, <Emphasis Type="Italic">Tor grypus</Emphasis>, Following Experimental Infection with <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis>: Effects of Autochthonous Probiotics

In this study, the effects of orally administrated two native probiotics (Lactobacillus plantarum and Lactobacillus delbrueckii ssp. bulguricus), isolated from the intestine of Shabot fish, Tor grypus, on some immune response parameters and immune-related genes expression against Aeromonas hydrophila in T. grypus were evaluated. Four hundred and eighty juveniles weighing 45?±?10 g were randomly divided into four groups (with three replications) and fed with the experimental diet containing 5?×?10⁷ cfu g^?1 of L. plantarum (G1), Lactobacillus bulgaricus (G2), Lactobacillus casei (G3), and a control diet (without probiotics) for 60 continuous days. At the end of the dietary treatments, fish were challenged with a lethal concentration of A. hydrophila (5?×?10⁸ CFU ml^?1) via intra peritoneal (i.p) injection. Blood and head kidney samples were taken from six fish in each treatment before challenging and 6, 12, 24, and 48 h and also 7 days after injection. The results showed that lysozyme, complement, bactericidal, and NBT activity of probiotic-treated groups were significantly elevated (P?<?0.05). The IL-8, IL-1β, and TNF-α gene expressions were significantly higher in all probiotic-treated groups (P?<?0.05). Meanwhile, a high direct correlation was observed between serum immune parameters and expression of immune-related genes (P?<?0.0001); furthermore, the highest correlation (R ²?=?0.634, P?<?0.0001) was recorded between IL-1β expression and NBT activity. It can be concluded that not only two native probiotics strains stimulate serum immune responses parameters and immune-related gene expression in T. grypus, but also a high correlation was seen among these indices. The study suggests that gastrointestinal colonization is preferred for host specificity as the strain previously derived from shabot fish displayed better colonization than the non-indigenous bacteria strain such as L. casei. Therefore, these native probiotics bacteria can be accounted as suitable candidates to immune stimulation in fish.     

Dynamics of the composition of a microbial consortium during start-up of a single-stage constant flow laboratory nitritation/anammox setup

Dynamics of the composition of the microbial community was studied during start-up of a single-stage completely mixed constant flow laboratory setup for ammonium removal by the nitritation/anammox process from the filtrate of digested sludge of the Kuryanovo wastewater treatment plant (KWTP), Moscow. To decrease the period of the start-up, the setup was initially inoculated with two types of activated sludge (nitrifying sludge from a KWTP aeration tank and sludge from a sequencing batch reactor enriched with anammox bacteria). The start-up and adjustment stage was therefore decreased to 35–40 days, and nitrogen removal efficiency reached 80% after 120 days of the setup operation. Taxonomic analysis of the composition of the microbial community was carried out by pyrosequencing of the 16S rRNA fragments obtained using the universal and planctomycetes-specific primers. In the course of adaptation of activated sludge to increasing nitrogen load, microbial community of the setup became less diverse and more specialized. The contribution of anammox bacteria of the family Brocadiaceae, closely related to Candidatus “Brocadia caroliniensis,” increased gradually. Members of the order Nitrosomonadales were involved in ammonium oxidation to nitrite. While nitrite-oxidizing bacteria of the genus Nitrospira were also detected, their share decreased with accumulation of the activated sludge. The contribution of other bacteria varied as well: the shares of the phyla Ignavibacteria, Chloroflexi, and Acidobacteria increased significantly (up to 13, 12, and 10%, respectively of the total number of reads), while relative abundance of the Proteobacteria, Bacteroidetes, Actinobacteria, Firmicutes, Synergistetes, Aminicenantes, Thermotogae, and Cloacimonetes decreased. Thus, application of pyrosequencing made it possible to monitor succession of the bacterial community involved in nitrogen removal by nitritation/anammox process.     

Analysis of the effect of plant growth regulators and organic elicitors on antibacterial activity of <Emphasis Type="Italic">Eucomis autumnalis</Emphasis> and <Emphasis Type="Italic">Drimia robusta</Emphasis> ex vitro-grown biomass

The effects of plant growth regulators (PGRs) and organic elicitors (OEs) on in vitro propagation of Eucomis autumnalis was established. Three-year-old ex vitro grown plants from organogenesis of E. autumnalis and somatic embryogenesis (previously reported protocol) of Drimia robusta were investigated for antibacterial activity. In vitro propagation from leaf explants of E. autumnalis was established using different PGRs and OE treatments for mass propagation, biomass production and bioactivity analysis to supplement the use of wild plant material. Prolific shoots (16.0?±?0.94 shoots per explant) were obtained with MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium containing 100 mg l^?1 haemoglobin (HB), 10 µM benzyladenine (BA) and 2 µM naphthaleneacetic acid (NAA). The shoots were rooted effectively with a combination of 2.5 µM indole-3-acetic acid and 5.0 µM indole-3-butyric acid. The plantlets were successfully acclimatized in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Three-year-old ex vitro-grown E. autumnalis and D. robusta plants derived via organogenesis and somatic embryogenesis respectively exhibited antibacterial activity and varied with PGR and OE treatments, plant parts and bacteria. The leaves of E. autumnalis ex vitro-derived from a combination of HB, BA and NAA followed by the individual treatments of BA and HB gave the best antibacterial activities (<?1 mg ml^?1: minimum inhibitory concentration from 0.098 to 0.78 mg ml^?1) against all tested pathogenic bacteria (Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa). The bulbs of D. robusta ex vitro-derived from solid culture with 10 µM picloram, 1 µM thidiazuron and 20 µM glutamine exhibited good antibacterial activity against E. faecalis, M. luteus and S. aureus when compared with other treatments and mother plants. The ex vitro-grown E. autumnalis and D. robusta biomass produced with PGRs along with OE treatments confirmed a good potent bioresource and can be used as antibacterial agents. The in vitro plant regeneration of E. autumnalis and D. robusta protocols and ex vitro plants could be used for conservation strategies, bioactivity and traditional medicinal use.     

Interspecific variation in extracellular polysaccharide content and colony formation of <Emphasis Type="Italic">Microcystis</Emphasis> spp. cultured under different light intensities and temperatures

Single cells of five different Microcystis species (M. ichthyoblabe, M. viridis, M. flos-aquae, M. wesenbergii, and M. aeruginosa) were batch-cultured at different temperatures and light intensities: (a) 25 °C and 50 μmol photons m^?2 s^?1 (control culture); (b) 25 °C and 10 μmol photons m^?2 s^?1; and (c) 15 °C and 50 μmol photons m^?2 s^?1. The extracellular polysaccharide content was significantly higher in treatments b and c than in the control treatment. All Microcystis species existed as single cells under the control treatment but formed colonies in treatments b and c. All of the colonies were irregular with indistinct margins. M. ichthyoblabe, M. viridis, M. flos-aquae, and M. wesenbergii formed colonies with similar morphologies and their cells were loosely aggregated. In contrast, M. aeruginosa formed denser colonies with no distinct holes. The colony morphologies differed from the classic morphology of M. ichthyoblabe field-grown colonies but resembled that of small colonies found in Lake Taihu (Yangtze Delta Plain, China) during early spring. This indicates that field- and laboratory-grown colonies are governed by similar formation processes. We suggest that in laboratory and field environments, M. ichthyoblabe (or M. flos-aquae) colonies are representative of small colonies formed from single Microcystis cells, whereas the morphology of older colonies evolves to resemble M. wesenbergii and M. aeruginosa colonies.     

Oxygen transfer as a tool for fine-tuning recombinant protein production by <Emphasis Type="Italic">Pichia pastoris</Emphasis> under glyceraldehyde-3-phosphate dehydrogenase promoter

Effects of oxygen transfer on recombinant protein production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter were investigated. Recombinant glucose isomerase was chosen as the model protein. Two groups of oxygen transfer strategies were applied, one of which was based on constant oxygen transfer rate where aeration rate was Q _O/V = 3 and 10 vvm, and agitation rate was N = 900 min^?1; while the other one was based on constant dissolved oxygen concentrations, C _DO = 5, 10, 15, 20 and 40 % in the fermentation broth, by using predetermined exponential glucose feeding with μ _o = 0.15 h^?1. The highest cell concentration was obtained as 44 g L^?1 at t = 9 h of the glucose fed-batch phase at C _DO = 20 % operation while the highest volumetric and specific enzyme activities were obtained as 4440 U L^?1 and 126 U g^?1 cell, respectively at C _DO = 15 % operation. Investigation of specific enzyme activities revealed that keeping C _DO at 15 % was more advantageous with an expense of relatively higher by-product formation and lower specific cell growth rate. For this strategy, the highest oxygen transfer coefficient and oxygen uptake rate were K _L a = 0.045 s^?1 and OUR = 8.91 mmol m^?3 s^?1, respectively.     

Survey of Philippine coffee beans for the presence of ochratoxigenic fungi

In 2012 to 2014, Philippine green coffee beans from Coffea arabica in Benguet and Ifugao; Coffea canephora var. Robusta in Abra, Cavite, and Ifugao; and Coffea liberica and Coffea excelsea from Cavite were collected and assessed for the distribution of fungi with the potential to produce ochratoxin A (OTA). The presence of fungal species was evaluated both before and after surface sterilization. There were remarkable ecological and varietal differences in the population of OTA-producing species from the five provinces. Aspergillus ochraceus, A. westerdijkiae, and Penicillium verruculosum were detected from Arabica in Benguet and Ifugao while Aspergillus carbonarius, Aspergillus niger, and Aspergillus japonicus were isolated in Excelsa, Liberica, and Robusta varieties from Abra, Cavite, and Davao. Contamination by Aspergillus and Penicillium species was found on 59 and 19 %, respectively, of the 57 samples from five provinces. After disinfection with 1 % sodium hypochlorite, the levels of infection by Aspergillus and Penicillium fell to 40 and 17 %, respectively. A total of 1184 fungal isolates were identified to species level comprising Aspergillus sections Circumdati (four species), Clavati (one), Flavi (one), Fumigati (one), Nigri (three), and Terrie (one). Within section Circumdati, 70 % of A. ochraceus produced OTA as high as 16238 ng g^?1 while 40 % of A. westerdijkiae produced maximum OTA of 36561 ng g^?1 in solid agar. Within section Nigri, 16.76 % of A. niger produced OTA at the highest 18439 ng g^?1, 10 % of A. japonicus at maximum level of 174 ng g^?1, and 21.21 % of A. carbonarius yielded maximum OTA of 1900 ng g^?1. Of the 12 species of Penicillium isolated, P. verruculosum was ochratoxigenic, with a maximum OTA production of 12 ng g^?1.     

Inheritance pattern and genetic correlations among growth and wood quality traits in Para rubber tree (<Emphasis Type="Italic">Hevea brasiliensis</Emphasis>) and implications for breeding

Inheritance pattern of wood traits viz. specific gravity, fibre dimensions and fibre-derived biometrical indices and their interactions among themselves and with that of growth are reported in Hevea brasiliensis. Girth (h² =???0.02?±?0.44 to h² =?0.35?±?0.24) showed moderate genetic control. Among wood traits, specific gravity (h²?=?0.15?±?0.31 to h² =?0.33?±?0.28) was found to be under moderate genetic control. Fibre traits viz., fibre length (h² =???0.26?±?0.30 to h² =?0.50?±?0.34), fibre diameter (h² =?0.19?±?0.49 to h² =?0.70?±?0.11), fibre lumen diameter (h² =???0.18?±?0.35 to h² =?0.56?±?0.47) and fibre wall thickness (h² =???5.17?±?5.26 to h² =?0.50?±?0.50) were under moderate to strong genetic control. Among fibre-derived indices, flexibility coefficient (h² =?0.48?±?0.21 to h² =?0.89?±?0.29) showed moderate to very strong genetic control. The Runkel ratio (h² =???0.40?±?0.27 to h² =?0.42?±?0.29) and slenderness ratio (h² =???0.36?±?0.29 to h² =?0.43?±?0.28) showed moderate genetic control. Girth showed very strong positive genetic correlation with fibre wall thickness and strong positive correlation with fibre width indicating scope of indirect selection potential for these traits. Wood specific gravity was not correlated with either girth or fibre traits. Hence, it would be possible to concomitantly improve growth and fibre traits without adversely affecting wood specific gravity. Moderate to very high estimates of heritability for fibre traits, girth and specific gravity indicated that considerable genetic gain can be realised for these traits. Implications of the above findings in genetic improvement of wood in Hevea are discussed.     

Perchlorate reduction by hydrogen autotrophic bacteria and microbial community analysis using high-throughput sequencing

Hydrogen autotrophic reduction of perchlorate have advantages of high removal efficiency and harmless to drinking water. But so far the reported information about the microbial community structure was comparatively limited, changes in the biodiversity and the dominant bacteria during acclimation process required detailed study. In this study, perchlorate-reducing hydrogen autotrophic bacteria were acclimated by hydrogen aeration from activated sludge. For the first time, high-throughput sequencing was applied to analyze changes in biodiversity and the dominant bacteria during acclimation process. The Michaelis–Menten model described the perchlorate reduction kinetics well. Model parameters q _max and K _s were 2.521–3.245 (mg ClO₄ ^?/gVSS h) and 5.44–8.23 (mg/l), respectively. Microbial perchlorate reduction occurred across at pH range 5.0–11.0; removal was highest at pH 9.0. The enriched mixed bacteria could use perchlorate, nitrate and sulfate as electron accepter, and the sequence of preference was: NO₃ ^? > ClO₄ ^? > SO₄ ^2?. Compared to the feed culture, biodiversity decreased greatly during acclimation process, the microbial community structure gradually stabilized after 9 acclimation cycles. The Thauera genus related to Rhodocyclales was the dominated perchlorate reducing bacteria (PRB) in the mixed culture.     

Earthworm Communities in the Bamboo Plantations of West Tripura (India)

Present study revealed the presence of 16 earthworm species belonging to 11 genera and four families viz. Megascolecidae (Amynthus alexandri, Metaphire houlleti, Lampito mauritii, Kanchuria sp1, Perionyx excavatus), Octochaetidae (Eutyphoeus gigas, Eutyphoeus comillahnus, Eutyphoeus orientalis, Octochaetona beatrix, Dichogaster bolaui, Lennogaster chittagongensis, Lennogaster yeicus), Moniligastridae (Drawida papillifer papillifer, Drawida assamensis, Drawida nepalensis) and Glossoscolecidae (Pontoscolex corethrurus) in the soils of five bamboo species [Bambusa balcooa (Sil Barak), Melocanna baccifera (Muli), Bambusa polumorpha (Bari), Bambus cacharensis (Bom) and Bambus bambus (Katabarak)] of West-Tripura. While four earthworm species viz. Metaphire houlleti, Drawida assamensis, Drawida papillifer papillifer and Pontoscolex corethrurus were common to all species of bamboo plantations, the rest showed restricted distribution. Among the earthworm species 4 were exotic (Amynthus alexandri, Metaphire houlleti, Dichogaster bolaui and Pontoscolex corethrurus) and the others were native to the Indian sub-continent. In general, earthworms under the bamboo plantations occurred within temperature range of 21.6 °C–28.0 °C, pH 4.0–7.0, organic matter 0.56–5.99 %, moisture 9.6–31.7 %, water holding capacity 14.6–43.9 % and bulk density 0.7–1.8 g cm^?3. The average density and biomass of the earthworms in the studied places were 108 ind m^?2 and 44 g m^?2 respectively. Earthworm diversity, dominance and evenness indices showed the values 1.00, 0.47 and 0.70 respectively. Earthworm density and biomass showed a negative correlation with temperature whereas those had a strong positive correlation with pH, moisture and organic matter of the soils.     

The nitrite reductase activity of horse heart carboxymethylated-cytochrome <Emphasis Type="Italic">c</Emphasis> is modulated by cardiolipin

Horse heart carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous CM-cytc [CM-cytc-Fe(II)], in the presence of sodium dithionite, is reported between pH 5.5 and 7.6, at 20.0 °C. Cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO₂ ^?-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO [k _on = (7.3 ± 0.7) × 10^?2 M^?1 s^?1; at pH 7.4], whereas the value of k _on for NO₂ ^? reduction by CM-cytc-Fe(II) is 1.1 ± 0.2 M^?1 s^?1 (at pH 7.4). CL facilitates the NO₂ ^?-mediated nitrosylation of CM-cytc-Fe(II) in a dose-dependent manner, the value of k _on for the NO₂ ^?-mediated conversion of CL–CM-cytc-Fe(II) to CL–CM-cytc-Fe(II)-NO (5.6 ± 0.6 M^?1 s^?1; at pH 7.4) being slightly higher than that for the NO₂ ^?-mediated conversion of CL–cytc-Fe(II) to CL–cytc-Fe(II)-NO (2.6 ± 0.3 M^?1 s^?1; at pH 7.4). The apparent affinity of CL for CM-cytc-Fe(II) is essentially pH independent, the average value of B being (1.3 ± 0.3) × 10^?6 M. In the absence and presence of CL liposomes, the nitrite reductase activity of CM-cytc-Fe(II) increases linearly on lowering pH and the values of the slope of the linear fittings of Log k _on versus pH are ?1.05 ± 0.07 and ?1.03 ± 0.03, respectively, reflecting the involvement of one proton for the formation of the transient ferric form, NO, and OH^?. These results indicate that Met80 carboxymethylation and CL binding cooperate in the stabilization of the highly reactive heme-Fe atom of CL–CM-cytc.     

A calcium-stimulated serine peptidase from a true-branching cyanobacterium, <Emphasis Type="Italic">Westiellopsis ramosa</Emphasis> sp. nov.

Unbranched heterocytous cyanobacteria produce a number of serine peptidases. We have characterized several peptidases in the cell-free extracts of a true-branched N₂-fixing cyanobacterium, Westiellopsis ramosa sp. nov. Upon substrate-gel zymography of intact filaments and heterocytes, five peptidase bands were resolved, whereas in vegetative cells, a single band was discernible. No band was detected in \({\text{NO}}_{3}^{ - } /{\text{NH}}_{4}^{ + }\)-grown cultures suggesting that the peptidases were present under diazotrophic conditions with much of them confined to heterocytes. Using salt precipitation and chromatography, a caseinolytic peptidase, called Wrp49, was purified which also demonstrated fibrinolytic activity. In SDS-PAGE, the purified peptidase was resolved into 17 and 27 kDa fragments. The enzyme in its native state exhibited M_r ≈ 49 kDa, and digested gelatin in a substrate gel at a corresponding position. The enzyme showed amidolytic activity on a plasmin specific substrate, D-Val-Leu-Lys p-nitroanilide. Moreover, a trypsin specific substrate, N-benzoyl-DL-Arg p-nitroanilide was hydrolyzed at an apparent K_m = 0.195 mM and V_max = 5 × 10^?7 M s^?1. The enzyme was stable in a wide pH and temperature range. While Ca²⁺ stimulated the activity; phenylmethane sulfonyl fluoride, leupeptin, EDTA and chelants were inhibitory. The activity of the EDTA-inactivated enzyme was completely restored upon adding Ca²⁺, suggesting that both compounds competed with each other in modulating the enzyme activity. The enzyme showed similarities with a Ca²⁺ stimulated subtilisin-like serine peptidase of Anabaena variabilis ATCC 29413, but also presented several unique features of metallopeptidases, such as the chelant’s response. Moreover, the N-terminal sequence (MTVENLARTGVGPGWR) did not match with any of the known peptidases.     

Single point mutations enhance activity of <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase

Objectives

To enhance activity of cis-epoxysuccinate hydrolase from Klebsiella sp. BK-58 for converting cis-epoxysuccinate to tartrate.

Results

By semi-saturation mutagenesis, all the mutants of the six important conserved residues almost completely lost activity. Then random mutation by error-prone PCR and high throughput screening were further performed to screen higher activity enzyme. We obtained a positive mutant F10D after screening 6000 mutations. Saturation mutagenesis on residues Phe10 showed that most of mutants exhibited higher activity than the wild-type, and the highest mutant was F10Q with activity of 812 U mg^?1 (k _cat /K _m , 9.8 ± 0.1 mM^?1 s^?1), which was 230 % higher than that of wild-type enzyme 355 U mg^?1 (k _cat /K _m , 5.3 ± 0.1 mM^?1 s^?1). However, the thermostability of the mutant F10Q slightly decreased.

Conclusions

The catalytic activity of a cis-epoxysuccinate hydrolase was efficient improved by a single mutation F10Q and Phe10 might play an important role in the catalysis.

     

Prokaryotic diversity in a Tunisian hypersaline lake,Chott El Jerid

Prokaryotic diversity was investigated in a Tunisian salt lake, Chott El Jerid, by quantitative real-time PCR, denaturing gradient gel electrophoresis (DGGE) fingerprinting methods targeting the 16S rRNA gene and culture-dependent methods. Two different samples S1-10 and S2-10 were taken from under the salt crust of Chott El Jerid in the dry season. DGGE analysis revealed that bacterial sequences were related to Firmicutes, Proteobacteria, unclassified bacteria, and Deinococcus-Thermus phyla. Anaerobic fermentative and sulfate-reducing bacteria were also detected in this ecosystem. Within the domain archaea, all sequences were affiliated to Euryarchaeota phylum. Quantitative real-time PCR showed that 16S rRNA gene copy numbers of bacteria was 5 × 10⁶ DNA copies g^?1 whereas archaea varied between 5 × 10⁵ and 10⁶ DNA copies g^?1 in these samples. Eight anaerobic halophilic fermentative bacterial strains were isolated and affiliated with the species Halanaerobium alcaliphilum, Halanaerobium saccharolyticum, and Sporohalobacter salinus. These data showed an abundant and diverse microbial community detected in the hypersaline thalassohaline environment of Chott El Jerid.     

Growth and phosphorus removal by <Emphasis Type="Italic">Synechococcus elongatus</Emphasis> co-immobilized in alginate beads with <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>

This study examined the co-immobilization of the cyanobacterium Synechococcus elongatus with the plant growth-promoting bacterium Azospirillum brasilense in alginate beads and its potential application for the removal of phosphorus from aquaculture wastewater. Co-immobilization of both microorganisms significantly increased the cell density of S. elongatus (2852.5?×?10⁴ cells mL^?1) compared with that of immobilization of cyanobacteria alone (1325.2?×?10⁴ cells mL^?1). Chlorophyll a content was similar in co-immobilized (11.1?±?3.5 pg cell^?1) and immobilized S. elongatus (14.5?±?4.9 pg cell^?1). Azospirillum brasilense showed continuous growth until day 2, after which its cell concentration declined until the end of the assay. Co-immobilized S. elongatus removed more phosphorus (44.8 %) than immobilized cyanobacteria cells alone (32.0 %). In conclusion, phosphate removal was greater with free cells of S. elongatus but overlapped with the values that were obtained with the treatment of co-immobilization of cells. Our results demonstrate that A. brasilense enhances the growth of S. elongatus and improves its removal of phosphorus when they are co-immobilized in alginate beads compared with only immobilization of cyanobacteria cells alone.