Benzene, toluene, and o-xylene degradation by free and immobilized P. putida F1 of postconsumer agave-fiber/polymer foamed composites

Benzene, toluene, and o-xylene (BTX) degradation by immobilized Pseudomonas putida F1 of postconsumer agave-fiber/polymer foamed-composites (AFPFC) and suspended cultures was studied under controlled conditions. Analyses using FTIR-ATR and SEM showed that P. putida F1 adhered onto the composite surface and developed a biofilm. In this sense, the AFPFC were successfully used as a support for bacterial immobilization. Both systems, immobilized and suspended cells of P. putida F1, were able to completely degrade benzene and toluene from initial concentrations of 15, 30, 60, and 90 mg l⁻¹. An inhibitory effect of the intermediary catechol from benzene degradation was observed in suspended cultures but it was not presented in the immobilized system. The degradation of o-xylene was partially accomplished in both systems. The Monod equation was used to model the experimental data obtained from the biodegradation kinetics, and they were adequately described with this model.     

Low molecular weight dextran: Immobilization of cells of Leuconostoc mesenteroides KIBGE HA1 on calcium alginate beads

Dextran is a long chain polymer of d-glucose produced by different bacterial strains including Leuconostoc, Streptococcus and Acetobacter. The bacterial cells from Leuconostoc mesenteroides KIBGE HA1 were immobilized on calcium alginate for dextran production. It was observed that dextran production increases as the temperature increases and after reaching maxima (30 °C) production started to decline. It was also observed that at 50 °C free cells stopped producing dextran, while immobilized cells continued to produce dextran even after 60 °C and still not exhausted. It was found that when 10 g% substrate (sucrose) was used, maximum dextran production was observed. Immobilized cells produced dextran upto 12 days while free cells stopped producing dextran only after 03 days. Molecular mass distribution of dextran produced by immobilized cells is low as compared to free cells.     

Metabolic characterization of a strain (BM90) of <Emphasis Type="Italic">Delftia tsuruhatensis</Emphasis> showing highly diversified capacity to degrade low molecular weight phenols

A novel bacterium, strain BM90, previously isolated from Tyrrhenian Sea, was metabolically characterized testing its ability to use 95 different carbon sources by the Biolog system. The bacterium showed a broad capacity to use fatty-, organic- and amino-acids; on the contrary, its ability to use carbohydrates was extremely scarce. Strain BM90 was identified and affiliated to Delftia tsuruhatensis by molecular techniques based on 16S rRNA gene sequencing. D. tsuruhatensis BM90, cultivated in shaken cultures, was able to grow on various phenolic compounds and to remove them from its cultural broth. The phenols used, chosen for their presence in industrial or agro-industrial effluents, were grouped on the base of their chemical characteristics. These included benzoic acid derivatives, cinnamic acid derivatives, phenolic aldehyde derivatives, acetic acid derivatives and other phenolic compounds such as catechol and p-hydroxyphenylpropionic acid. When all the compounds (24) were gathered in the same medium (total concentration: 500 mg/l), BM90 caused the complete depletion of 18 phenols and the partial removal of two others. Only four phenolic compounds were not removed. Flow cytometry studies were carried out to understand the physiological state of BM90 cells in presence of the above phenols in various conditions. At the concentrations tested, a certain toxic effect was exerted only by the four compounds that were not metabolized by the bacterium.     

Effect of biosurfactant and fertilizer on biodegradation of crude oil by marine isolates of Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa

This study was conducted to investigate the effects of fertilizers and biosurfactants on biodegradation of crude oil by three marine bacterial isolates; Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa. Five sets of experiments were carried out in shake flask and microcosm conditions with crude oil as follows: Set 1-only bacterial cells added (no fertilizer and biosurfactant), Set 2-with additional fertilizer only, Set 3-with additional biosurfactant only, Set 4-with added biosurfactant + fertilizer, Set 5-with no bacterial cells added (control), all the above experimental sets were incubated for 168 h. The biosurfactant + fertilizer added Set 4, resulted in maximum crude oil degradation within shake flask and microcosm conditions. Among the three bacterial isolates, P. aeruginosa and biosurfactant produced by this strain resulted in maximum crude oil degradation compared to the other two bacterial strains investigated. Interestingly, when biosurfactant and bacterial cells were used (Set 3), significant oil biodegradation activity occurred and the difference between this treatment and that in Set 4 with added fertilizer + biosurfactant were only 4-5% higher degradation level in shake flask and 3.2-7% in microcosm experiments for all three bacterial strains used. It is concluded that, biosurfactants alone capable of promoting biodegradation to a large extent without added fertilizers, which will reduce the cost of bioremediation process and minimizes the dilution or wash away problems encountered when water soluble fertilizers used during bioremediation of aquatic environments.     

Enzymatic conversion of sunflower oil to biodiesel in a solvent-free system: Process optimization and the immobilized system stability

The feasibility of using the commercial immobilized lipase from Candida antarctica (Novozyme 435) to synthesize biodiesel from sunflower oil in a solvent-free system has been proved. Using methanol as an acyl acceptor and the response surface methodology as an optimization technique, the optimal conditions for the transesterification has been found to be: 45 ^oC, 3% of enzyme based on oil weight, 3:1 methanol to oil molar ratio and with no added water in the system. Under these conditions, >99% of oil conversion to fatty acid methyl ester (FAME) has been achieved after 50 h of reaction, but the activity of the immobilized lipase decreased markedly over the course of repeated runs. In order to improve the enzyme stability, several alternative acyl acceptors have been tested for biodiesel production under solvent-free conditions. The use of methyl acetate seems to be of great interest, resulting in high FAME yield (95.65%) and increasing the half-life of the immobilized lipase by about 20.1 times as compared to methanol. The reaction has also been verified in the industrially feasible reaction system including both a batch stirred tank reactor and a packed bed reactor. Although satisfactory performance in the batch stirred tank reactor has been achieved, the kinetics in a packed bed reactor system seems to have a slightly better profile (93.6 ± 3.75% FAME yield after 8–10 h), corresponding to the volumetric productivity of 48.5 g/(dm³ h). The packed bed reactor has operated for up to 72 h with almost no loss in productivity, implying that the proposed process and the immobilized system could provide a promising solution for the biodiesel synthesis at the industrial scale.     

Biological treatment of olive mill wastewater by non-conventional yeasts

The ability of lipolytic yeasts to grow on olive mill wastewater (OMW)-based medium and to produce high-value compounds while degrading this waste, was tested. OMW collected from three-phase olive mills from the North region of Portugal were characterized and used. OMW with COD ranging from 100 g L⁻¹ to 200 g L⁻¹ were supplemented with yeast extract and ammonium chloride. Studies of OMW consumption were carried out in batch cultures of Candida rugosa, Candida cylindracea and Yarrowia lipolytica. All strains were able to grow in the OMW-based media, without dilution, to consume reducing sugars and to reduce COD. C. cylindracea was the best strain concerning the lipase production and the reduction of phenolic compounds and COD. For all strains, the phenols degradation was quite difficult, mostly when more easily degradable carbon source is still present in the medium. Among the phenolic compounds tested catechol is the most inhibitory to the cells.     

Evaluation of colour removal in synthetic saline wastewater containing azo dyes using an immobilized halotolerant cell system

Studies were carried out to evaluate the colour removal capacity of a moderately halotolerant bacterium, Bacillus firmus, in synthetic saline wastewater medium (SSWM) under static condition. The bacterial strain effectively decolourized Polar red B (an azo dye) in a wide range of sodium chloride (1-6%, w/v), dye (5-100 mg/L) and SDS (0.1-5.0 mg/L) concentrations and at pH range of 6-10 after 24 h of incubation. Cell immobilization studies indicated that colour removal was significantly higher (p < 0.05) in immobilized halotolerant cell systems than with free cells of B. firmus especially at salt concentrations higher than 4%. Results suggest the potential of using the immobilized halotolerant cell system for effective treatment of dye-contaminated saline wastewaters.     

Bacteriophage-based biosorbents coupled with bioluminescent ATP assay for rapid concentration and detection of Escherichia coli

Wild type T4 bacteriophage and recombinant T4 bacteriophages displaying biotin binding peptide (BCCP) and cellulose binding module (CBM) on their heads were immobilized on nano-aluminum fiber-based filter (Disruptor™), streptavidin magnetic beads and microcrystalline cellulose, respectively. Infectivity of the immobilized phages was investigated by monitoring the phage-mediated growth inhibition of bioluminescent E. coli B and cell lysis using bioluminescent ATP assay. The results showed that phage immobilization resulted in a partial loss of infectivity as compared with the free phage. Nevertheless, the use of a biosorbent based on T4 bacteriophage immobilized on Disruptor™ filter coupled with a bioluminescent ATP assay allowed simultaneous concentration and detection of as low as 6 × 10³ cfu/mL of E. coli in the sample within 2 h with high accuracy (CV = 1-5% in log scale). Excess of interfering microflora at levels 60-fold greater than the target organism did not affect the results when bacteriophage was immobilized on the filter prior to concentration of bacterial cells.     

Colonization of Aspergillus japonicus on synthetic materials and application to the production of fructooligosaccharides

The ability of Aspergillus japonicus ATCC 20236 to colonize different synthetic materials (polyurethane foam, stainless steel sponge, vegetal fiber, pumice stones, zeolites, and foam glass) and to produce fructooligosaccharides (FOS) from sucrose (165 g/L) is described. Cells were immobilized in situ by absorption, through direct contact with the carrier particles at the beginning of fermentation. Vegetal fiber was the best immobilization carrier as A. japonicus grew well on it (1.25 g/g carrier), producing 116.3 g/L FOS (56.3 g/L 1-kestose, 46.9 g/L 1-nystose, and 13.1 g/L 1-β-fructofuranosyl nystose) with 69% yield (78% based only in the consumed sucrose amount), giving also elevated activity of the β-fructofuranosidase enzyme (42.9 U/mL). In addition, no loss of material integrity, over a 2 day-period, was found. The fungus also immobilized well on stainless steel sponge (1.13 g/g carrier), but in lesser extents on polyurethane foam, zeolites, and pumice stones (0.48, 0.19, and 0.13 g/g carrier, respectively), while on foam glass no cell adhesion was observed. When compared with the FOS and β-fructofuranosidase production by free A. japonicus, the results achieved using cells immobilized on vegetal fiber were closely similar. It was thus concluded that A. japonicus immobilized on vegetal fiber is a potential alternative for high production of FOS at industrial scale.     

Biochemical degradation pathway of reactive blue 13 by Candida rugopelliculosa HXL-2

A strain HXL-2 from a lab-scale sequence batch reactor (SBR) was identified as Candida rugopelliculosa based on its physiological, biochemical characteristics, and 26S rDNA D1/D2 gene phylogenetic analysis. About 90% of the 50 mg/L Reactive blue 13(RB13) was degraded in 48 h after inoculation with strain HXL-2. The optimum efficiency of pH on decolorization was obtained at pH 5.The optimum efficiency temperature of C. rugopelliculosa HXL-2 decolorization RB13 was obtained at 28 °C. The color removal efficiency was obtained at 80.3% when the feed concentration reached 2000 mg/L. We first detected naphthalene-like compound is produced as degradation intermediate after the cleavage of RB13 azo bond, detected 1-chloro-3-aniline- 2,4,6-triazine. We proposed degradation pathway of Reactive blue 13 by Candida sp and proved RB13 degradation pathway by Candida sp. has some difference from RB13 degradation pathway by Pseudomonas sp.     

Microbial tolerance in secondary peritonitis is dose dependent

Local microbial tolerance was investigated in a murine model of peritonitis. Peritoneal bacterial burden and inflammatory cytokine concentrations were determined at different times, within 48 h after infection. Peritoneal macrophages were harvested from naïve mice or from mice 48 h after infection and underwent ex vivo stimulation with different concentrations of Klebsiella. Cytokine secretion was determined in the supernatants. Peritoneal bacteria concentrations, remained relatively steady between 24 h (median: 5.04 log CFU) and 48 h (median: 5.19 log CFU) after infection. Peritoneal cytokine concentrations peaked early but were already diminished at 48 h after infection, despite persistent high bacteria levels. Macrophages, harvested from naïve mice responded vigorously to ex vivo stimulation with 10⁵ CFU and 2 × 10⁸ CFU Klebsiella. Cells harvested from animals 48 h after infection, were unresponsive to an ex vivo stimulation with 10⁵ CFU Klebsiella, but fully responded to 10⁸ CFU. Persistent intraabdominal bacterial infection induced dose dependent microbial tolerance in peritoneal macrophages.     

Leishmania (Viannia) peruviana (MHOM/PE/LCA08): Comparison of THP-1 cell and murine macrophage susceptibility to axenic amastigotes for the screening of leishmanicidal compounds

This study, undertaken to compare the susceptibility of THP-1 cells and murine peritoneal macrophages to Leishmania peruviana amastigotes, obtained THP-1 infection with 10 parasites/cell compared to 2 parasites/murine macrophage. The parasite burden was maximal at 72 h post-infection (h.p.i.) for THP-1 cells, while it was still increasing at 120 h.p.i. for murine macrophages. Since in both cases the infection with L. peruviana affected cell viability, we recommend evaluating any leishmanicidal activity at 72 h.p.i. Amphotericin B reduced Leishmania infection by 50% at concentrations of 0.1 μM in THP-1 and murine macrophages at 72 h.p.i.Our results demonstrate that amastigotes of L. peruviana can infect THP-1 cells and murine macrophages and indicate the suitability of this model to screen compounds for leishmanicidal activity.     

One-step process for debromination and aerobic mineralization of tetrabromobisphenol-A by a novel Ochrobactrum sp. T isolated from an e-waste recycling site

A novel bacterium, Ochrobactrum sp. T, capable of simultaneous debromination and aerobic mineralization of tetrabromobisphenol-A (TBBPA), was isolated from a sludge sample collected from an electronic-waste recycling site. The bacterium exhibited maximal debrominase activity at pH 6.5, 35 °C, and 200 rpm in Luria-Bertani culture medium. Initial TBBPA concentration and pH had more significant effects on degradation efficiency than those of temperature and inoculum size. Degradation and debromination efficiencies of 91.8% and 86.7%, respectively, were achieved within 72 h under optimized conditions of 35 °C, pH 7.0, inoculum volume of 25 mL, and TBBPA concentration of 3 mg L⁻¹. In addition, a 35.6% decrease in total organic carbon was observed after the degradation of 5 mg L⁻¹ TBBPA for 120 h. Eight metabolic intermediates were identified during the biodegradation of TBBPA. This study is the first report to propose a one-step process for TBBPA debromination and mineralization by a single bacterial strain.     

Ecofriendly degradation of sulfonated diazo dye C.I. Reactive Green 19A using Micrococcus glutamicus NCIM-2168

Micrococcus glutamicus NCIM-2168 exhibited complete decolorization and degradation of C.I. Reactive Green 19A (an initial concentration of 50 mg l⁻¹) within 42 h at temperature 37 °C and pH 8, under static condition. Extent of mineralization was determined with total organic carbon (TOC) and chemical oxygen demand (COD) measurement, showing a satisfactory reduction of TOC (72%) and COD (66%) within 42 h. Enzyme studies shows involvement of oxidoreductive enzymes in decolorization/degradation process. Analytical studies of the extracted metabolites confirmed the significant degradation of Reactive Green 19A into various metabolites. The microbial toxicity and phytotoxicity assay revealed that the degradation of Reactive Green 19A produced nontoxic metabolites. In addition, the M. glutamicus strain was applied to decolorize a mixture of ten reactive dyes showing a 63% decolorization (in terms of decrease in ADMI value) within 72 h, along with 48% and 42% reduction in TOC and COD under static condition.     

Staphylococcus sp. KW-07 contains nahH gene encoding catechol 2,3-dioxygenase for phenanthrene degradation and a test in soil microcosm

We isolated three species of phenanthrene-degrading bacteria from oil-contaminated soils and marine sediment, and assessed the potential use of these bacteria for bioremediation of soils contaminated by polycyclic aromatic hydrocarbons (PAHs). Based on 16S rDNA sequences, these bacteria were Staphylococcus sp. KW-07 and Pseudomonas sp. CH-11 from soil, and Ochrobactrum sp. CH-19 from the marine sediment. By PCR amplification, catechol 2,3-dioxygenase genes (nahH genes) mediating PAH degradation in the chromosome of Staphylococcus sp. KW-07 and Ochrobactrum sp. CH-19, and in plasmid DNA of Pseudomonas sp. CH-11 were detected. All isolates had a similar optimal growth temperature (25 °C) and optimal growth pH (7.0) in a minimal salt medium (MSM) with 0.1% (w/v) phenanthrene as the sole source of carbon and energy. Pseudomonas sp. CH-11 and Staphylococcus sp. KW-07 degraded 90% of added phenanthrene in 3 days and Ochrobactrum sp. CH-19 degraded 90% of the phenanthrene in 7 days under laboratory batch culture conditions. However, Staphylococcus sp. KW-07 was the most effective among the three strains in degradation of phenanthrene in soil. After inoculation of 1 × 10¹¹ cells of Staphylococcus sp. KW-07, over 90% degradation of 0.1% phenanthrene (0.1 g/100 g soil) was achieved after 1 month at 25 °C. The results collectively suggest that the Staphylococcus sp. KW-07 strain isolated may be useful in bioremediation of PAH-contaminated soils.     

Direct conversion of inulin and extract of tubers of Jerusalem artichoke into single cell oil by co-cultures of Rhodotorula mucilaginosa TJY15a and immobilized inulinase-producing yeast cells

In this study, it was found that the immobilized inulinase-producing cells of Pichia guilliermondii M-30 could produce 169.3 U/ml of inulinase activity while the free cells of the same yeast strain only produced 124.3 U/ml of inulinase activity within 48 h. When the immobilized inulinase-producing yeast cells were co-cultivated with the free cells of Rhodotorula mucilaginosa TJY15a, R. mucilaginosa TJY15a could accumulate 53.2% oil from inulin in its cells and cell dry weight reached 12.2 g/l. Under the similar conditions, R. mucilaginosa TJY15a could accumulate 55.4% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 12.8 g/l within 48 h. When the co-cultures were grown in 2 l fermentor, R. mucilaginosa TJY15a could accumulate 56.6% (w/w) oil from the extract of Jerusalem artichoke tubers in its cells and cell dry weight reached 19.6 g/l within 48 h. Over 90.0% of the fatty acids from the yeast strain TJY15a grown in the extract of Jerusalem artichoke tubers was C_16:0, C_18:1 and C_18:2, especially C_18:1 (50.6%).     

Virulence of Hypocreales fungi to pecan aphids (Hemiptera: Aphididae) in the laboratory

There is need for efficacious biocontrol agents for aphids in commercial orchards. As a preliminary step to this end we determined the virulence of several Hypocreales fungi to pecan aphids. In the first experiment we tested the virulence of Isaria fumosorosea (ARSEF 3581) blastospores to three pecan aphids Monellia caryella, Melanocallis caryaefoliae, and Monelliopsis pecanis under laboratory conditions. Rates of 1 × 10⁷ or 1 × 10⁸ spores per ml were applied in 2 ml via a spray tower to 90 mm Petri dishes containing 10 aphids each. Mortality and mycosis were determined after 24, 48 and 72 h. Treatment effects were observed by 48 h post-application, and by 72 h the higher application rate caused >90% mortality and mycosis in M. caryella and M. caryaefoliae, whereas <70% was observed in M. pecanis.We conducted two subsequent experiments (Experiments 2 and 3), using the same methodology, to compare the virulence of several Hypocreales species and strains against the aphid of primary economic concern to most pecan growers, M. caryaefoliae. In Experiment 2, we compared blastospores and conidia of two I. fumosorosea strains (ARSEF 3581 and ATCC 20874 [= strain 97]). The blastospores of ARSEF 3581 and conidia of ATCC 20874 showed higher virulence than other treatments and thus were included in Experiment 3, which also compared the virulence of conidia of Beauveria bassiana (GHA strain) and Metarhizium anisopliae (F52 strain). Results in Experiment 3 indicated the highest virulence in I. fumosorosea 3581 blastospores and M. anisopliae (F52) followed by I. fumosorosea (20874) conidia. The detection of pathogenicity to pecan aphids establishes the potential for commercial usage and additional study. Results reported here will narrow treatments to test in future greenhouse and field trials.     

Enhancement of fibrinolytic enzyme production from Bacillus subtilis via immobilization process onto radiation synthesized starch/dimethylaminoethyl methacrylate hydrogel

Hydrogel matrices based on starch and dimethylaminoethyl methacrylate (Starch/DMAEMA) were synthesized including γ-irradiation as a clean initiator. The prepared hydrogels were characterized in terms of their gel fraction yield, degree of equilibrium swelling. The prepared hydrogels were examined as carriers for immobilization of Bacillus subtilis that has the ability to secrete an extracellular fibrinolytic enzyme that degrades fibrin. Scanning electron microscope (SEM) analysis showed proliferation of the bacterial cells entrapped inside the polymeric matrix. The immobilization process increases the production time of fibrinolytic enzyme up to 120 h instead of 96 h for the free cells. The optimum temperature of activity broadened and a significant shift in the pH optima was observed upon immobilization. The reusability of immobilized cells under repeated batch fermentation conditions was also investigated. At the optimum production conditions, immobilization of B. subtilis cells onto Starch/DMAEMA resulted in a four fold increase in enzyme activity.     

Biohydrogen production by immobilized Chlorella sp. using cycles of oxygenic photosynthesis and anaerobiosis

Hydrogen production was studied using immobilized green alga Chlorella sp. through a two-stage cyclic process where immobilized cells were first incubated in oxygenic photosynthesis followed by anaerobic incubation for H₂ production in the absence of sulfur. Chlorella sp. used in this study was capable of generating H₂ under immobilized state in agar. The externally added glucose enhanced H₂ production rates and total produced volume while shortened the lag time required for cell adaptation prior to H₂ evolution. The rate of hydrogen evolution was increased as temperature increased, and the maximum evolution rate under 30 mM glucose was 183 mL/h/L and 238 mL/h/L at 37 °C and 40 °C, respectively. In order to continue repeated cycles of H₂ production, at least two days of photosynthesis stage should be allowed for cells to recover H₂ production potential and cell viability before returning to H₂ production stage again.     

Enhancement of cell growth rate by light irradiation in the cultivation of Rhodotorula glutinis

A yeast, Rhodotorula glutinis, is regarded as a potential microbial oil producer, due to its high lipid content. The flask results of this study indicated that irradiation could increase the growth of R. glutinis compared to that of a batch without irradiation. Further 5-l fermenter results confirmed that irradiation could greatly enhance the cells’ growth rate and total lipid productivity. The maximum lipid productivity obtained in the fed-batch operation with 3 LED (light emitting diode) lamps was 0.39 g/l h as compared to 0.34 g/l h in the batch with 3 LED lamps and 0.19 g/l h in the batch without irradiation. Conclusively, the irradiation could significantly increase the cells’ growth rate, which, in turn, could be applied to the commercialized production of biodiesel from single cell oils.