Biodegradation of organochlorine pesticides by bacteria grown in microniches of the porous structure of green bean coffee

In this paper, the authors propose a model for DDT biodegradation by bacteria grown in microniches created in the porous structure of green bean coffee. Five bacteria isolated from coffee beans, identified as Pseudomonas aeruginosa, P. putida, Stenotrophomonas maltophilia, Flavimonas oryzihabitans, and Morganella morganii. P. aeruginosa and F. oryzihabitans, were selected for pesticide degradation. Bacteria were selected according to their ability to grow on mineral media amended with: (a) glucose (10 g l⁻¹), (b) peptone (2 g l⁻¹), and (c) ground coffee beans (2 g l⁻¹). These three media were supplemented with 50 mg l⁻¹ of 1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane (DDT) and endosulfan. GC/MS analysis demonstrated that the greatest DDT removal was obtained in the medium supplemented with coffee beans, where 1,1-dichloro-2,2′-bis (4-chlorophenyl)ethylene (DDE), 1-chloro-2,2-bis (4-chlorophenyl) ethane (DDMU) and 2,2′-bis (p-chlorophenyl)ethanol (DDOH) were detected. DDMU is a product of the reductive dechlorination of DDE, which in this system could be carried out under the anaerobic conditions in microniches present in the porous structure of the coffee bean. This was supported by scanning electron microscopy. Green bean coffee could be used as a nutrient source and as a support for bacterial growth in pesticide degradation.     

Treatment of dye-rich wastewater by an immobilized thermophilic cyanobacterial strain: Phormidium sp.

The removal of Remazol Blue and Reactive Black B by the immobilized thermophilic cyanobacterial strain Phormidium sp. was investigated under thermophilic conditions in a batch system, in order to determine the optimal conditions required for the highest dye removal. In the experiments, performed at pH 8.5, with different initial dye concentrations between 9.1 mg l⁻¹ and 82.1 mg l⁻¹ and at 45 °C, calcium alginate immobilized Phormidium sp. showed high dye decolorization, with maximum uptake yields ranging from 50% to 88% at all dye concentrations tested. When the effects of high dye concentrations on dye removal were investigated, the highest uptake yield in the beads was 50.3% for 82.1 mg l⁻¹ Remazol Blue and 60.0% for 79.5 mg l⁻¹ Reactive Black B. The highest color removal was detected at 45 °C and 50 °C incubation temperatures for all dye concentrations. As the temperature decreased, the removal yield of immobilized Phormidium sp. also decreased. At about 75 mg l⁻¹ initial dye concentrations, the highest specific dye uptake measured was 41.29–41.17 mg g⁻¹ for Remazol Blue and 47.69–43.82 mg g⁻¹ for Reactive Black B at 45 °C and 50 °C incubation temperatures, respectively, after 8 days incubation.     

Pyruvate producing biocatalyst with constitutive NAD-independent lactate dehydrogenases

Production of pyruvate from lactate through biocatalysis is a valuable process for its simple composition of reaction system and convenience of recovery. Biocatalyst with lactate-induced NAD-independent lactate dehydrogenases (iLDHs) can effectively catalyze lactate into pyruvate. To reduce the cost of biocatalyst preparation caused by indispensable lactate addition, the mutants with constitutive iLDH of Pseudomonas sp. XP-M2 were screened. Mutant XP-LM exhibited high iLDHs activities in minimal salt medium with cheap substrate glucose as the carbon source. The biocatalyst (8.2 g dry cell weight l⁻¹) containing 169.8 U l⁻¹ l-iLDH was prepared with 20 g 1⁻¹ glucose. The cost-effective biocatalyst prepared from the mutant XP-LM could efficiently catalyze lactate into pyruvate with high yield (0.961 mol mol⁻¹). Based on the different thermostability of d-iLDH and l-iLDH in the biocatalyst, whole cells of the strain might also have the potential in production of pyruvate and d-lactate from racemic lactate.     

Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate

 Enzymatic hydrolysis of corncob and ethanol fermentation from cellulosic hydrolysate were investigated. After corncob was pretreated by 1% H₂SO₄ at 108 °C for 3 h, the cellulosic residue was hydrolyzed by cellulase from Trichoderma reesei ZU-02 and the hydrolysis yield was 67.5%. Poor cellobiase activity in T. reesei cellulase restricted the conversion of cellobiose to glucose, and the accumulation of cellobiose caused severe feedback inhibition to the activities of β-1,4-endoglucanase and β-1,4-exoglucanase in cellulase system. Supplementing cellobiase from Aspergillus niger ZU-07 greatly reduced the inhibitory effect caused by cellobiose, and the hydrolysis yield was improved to 83.9% with enhanced cellobiase activity of 6.5 CBU g⁻¹ substrate. Fed-batch hydrolysis process was started with a batch hydrolysis containing 100 g l⁻¹ substrate, with cellulosic residue added at 6 and 12 h twice to get a final substrate concentration of 200 g l⁻¹. After 60 h of reaction, the reducing sugar concentration reached 116.3 g l⁻¹ with a hydrolysis yield of 79.5%. Further fermentation of cellulosic hydrolysate containing 95.3 g l⁻¹ glucose was performed using Saccharomyces cerevisiae 316, and 45.7 g l⁻¹ ethanol was obtained within 18 h. The research results are meaningful in fuel ethanol production from agricultural residue instead of grain starch.     

Evaluation of distant carbon sources in biosurfactant production by a gamma ray-induced Pseudomonas putida mutant

Pseudomonas putida 33 wild strain, subjected to gamma ray mutagenesis and designated as P. putida 300-B mutant was used as microbial rhamnolipid-producer by using distant carbon sources (viz. hydrocarbons, waste frying oils ‘WFOs’, vegetable oil refinery wastes and molasses) in the minimal media under shake flask conditions. The behavior of glucose as co-substrate and growth initiator was examined. The 300-B mutant strain showed its ability to grow on all the substrates tested and produced rhamnolipid surfactants to different extents however; soybean and corn WFOs were observed to be preferred carbon sources followed by kerosene and paraffin oils, respectively. The best cell biomass (3.5 g l⁻¹) and rhamnolipids yield (4.1 g l⁻¹) were obtained with soybean WFO as carbon source and glucose as growth initiator under fed-batch cultivation showing an optimum specific growth rate (μ) of 0.272 h⁻¹, specific product yield (q_p) of 0.318 g g⁻¹ h and volumetric productivity (P_V) of 0.024 g l⁻¹ h. The critical micelle concentration of its culture supernatant was observed to be 91 mg rhamnolipids l⁻¹ and surface tension as 31.2 mN m⁻¹.     

A microcosm study on bioremediation of fenitrothion-contaminated soil using Burkholderia sp. FDS-1

Fenitrothion, a toxic organophosphorus pesticide, can build up the concentration of nitrophenolic compound in soils and hence needs to be removed. Burkholderia sp. FDS-1, a fenitrothion-degrading strain, was used in this work to study factors affecting its growth, and then evaluated for its capacity to degrade fenitrothion in soil microcosms. Minimal salt medium containing 1% (w/v) glucose was found to be a suitable carbon source for inoculum preparation. Various factors, including soil pH, temperature, initial fenitrothion concentration, and inoculum size influenced the degradation of fenitrothion. Microcosm studies performed with varying concentrations (1–200 mg kg⁻¹) of fenitrothion-spiked soils showed that strain FDS-1 could effectively degrade fenitrothion in the range of 1–50 mg kg⁻¹ soil. The addition of Burkholderia sp. FDS-1 at 2×10⁶ colony forming units g⁻¹ soil was found to be suitable for fenitrothion degradation over a temperature range of 20–40 °C and at a slight alkaline pH (7.5). The results indicate that strain FDS-1 has potential for use in bioremediation of fenitrothion and its metabolite-contaminated sites. This is a model study that could be used for decontamination of sites contaminated with other compounds.     

Characterization of a recombinant Escherichia coli TOP10 [pQR239] whole-cell biocatalyst for stereoselective Baeyer–Villiger oxidations

This paper describes the kinetic characterization of a recombinant whole-cell biocatalyst for the stereoselective Baeyer–Villiger type oxidation of bicyclo[3.2.0]hept-2-en-6-one to its corresponding regio-isomeric lactones (−)-(1S,5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one and (−)-(1R,5S)-3-oxabicyclo[3.3.0]oct-6-en-2-one. Escherichia coli TOP10 [pQR239], expressing cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus (NCIMB 9871), was shown to be suitable for this biotransformation since it expressed CHMO at a high level, was simple to produce, contained no contaminating lactone hydrolase activity and allowed the intracellular recycle of NAD(P)H necessary for the biotransformation. A small-scale biotransformation reactor (20 ml) was developed to allow rapid collection of intrinsic kinetic data. In this system, the optimized whole-cell biocatalyst exhibited a significantly lower specific lactone production activity (55–60 μmol min⁻¹ g⁻¹ dry weight) than that of sonicated cells (500 μmol min⁻¹ g⁻¹ dry weight). It was shown that this shortfall was comprised of a difference in the pH optima of the two biocatalyst forms and mass transfer limitations of the reactant and/or product across the cell barrier. Both reactant and product inhibition were evident. The optimum ketone concentration was between 0.2 and 0.4 g l⁻¹ and at product concentrations above 4.5–5 g l⁻¹ the specific activity of the whole cells was zero. These results suggest that a reactant feeding strategy and in situ product removal should be considered in subsequent process design.     

Chromate reduction by resting cells of Achromobacter sp. Ch-1 under aerobic conditions

Chromate reduction was carried out by resting cells of Achromobacter sp. Ch-1 with lactate as electron donor under aerobic conditions. The reduction activity of the samples supplemented with lactate was two times as those without lactate. The reduction rate was influenced by initial pH and lactate concentration. Under the optimal conditions, pH 9.0 and 4000 mg l⁻¹ lactate supplement, reduction rate was 5.45 mg l⁻¹ min⁻¹. The reduction rate decreased with increasing of Cr(VI) concentrations and increased with cell densities proportionally. The maximum reduction limit of Ch-1 cells was obtained at 2107 mg l⁻¹ of Cr(VI).     

Enrichment,isolation, and characterization of phenol-degrading Pseudomonas resinovorans strain P-1 and Brevibacillus sp. strain P-6

The objectives of this research were to isolate pure phenol-degrading strains from enriched mixed cultures, monitoring the variations of species during the enrichment period. Two strains were isolated from the acclimated mixed culture. They were identified as Pseudomonas resinovorans strain P-1 and Brevibacillus sp. strain P-6. DGGE indicated that strain P. resinovorans appeared at the beginning, and maintained well during the enrichment period. The second strain, Brevibacillus sp., did not appear in the initial stage, but showed up after 2 weeks of enrichment. The optimum growth temperatures for P. resinovorans and Brevibacillus sp. were 31 and 39 °C, respectively. P. resinovorans could degrade phenol completely within 57.5 h, when the initial phenol concentration was lower than 600 mg l⁻¹. If the initial phenol concentration was lower than 200 mg l⁻¹, Brevibacillus sp. could remove phenol completely within 93.1 h. It was obvious that the phenol-degrading ability of P. resinovorans was much better than that of Brevibacillus sp. The metabolic pathway for P. resinovorans phenol degradation was assigned to the meta-cleavage activity of catechol 2,3-dioxygenase.     

Naphthalene and biphenyl oxidation by two marine Pseudomonas strains isolated from Venice Lagoon sediment

A sediment sample from Venice Lagoon was found to be contaminated with 475 mg Kg⁻¹ polycyclic aromatic hydrocarbons (PAHs). Naphthalene was the principal pollutant at 26% of total PAHs. Two strains of Pseudomonas SN1 and SB1 were isolated from sediment amended with 2% naphthalene. 16S rRNA gene sequence analysis indicated that the two strains have about 99% nucleotide identity with strains of the genus Pseudomonas, and are very close to Pseudomonas stutzeri. Their metabolic profiles showed significant nutritional differences, the most significant of which was that SN1 grows in marine mineral medium spiked with naphthalene and SB1 grows with biphenyl as sole carbon and energy sources. Pseudomonas sp. SN1 had a doubling time of 3.1 h with 2% naphthalene and SB1 had a doubling time of 19.5 h with 2% biphenyl. Strain SN1 oxidised naphthalene at 564±32 mg O₂ l⁻¹ d⁻¹ and SB1 oxidised biphenyl at 426±25 mg O₂ l⁻¹ d⁻¹ in respirometry reaction vessels under controlled conditions. Screening of the two strains for dioxygenase genes involved in the first step of the two hydrocarbon degradation pathways, by polymerase chain reaction, showed naphthalene dioxygenase in SN1 and biphenyl dioxygenase in SB1. The strains each have a different catechol 2,3-dioxygenase responsible for cleavage of the aromatic ring.     

Effect of soybean oil and Tween 80 on the production of botryosphaeran by Botryosphaeria rhodina MAMB-05

The addition of soybean oil and Tween 80 was evaluated with the objective of increasing the production of botryosphaeran, an exopolysaccharide (EPS) of the (1 → 3;1 → 6)-β-d-glucan type produced by the fungus Botryosphaeria rhodina MAMB-05. Factorial design and analysis by response surface methodology was developed to select the main factors that would affect and enhance EPS production. The optimized culture conditions were: 40 g l⁻¹ glucose with 10 ml l⁻¹ soybean oil, and 4.5 ml l⁻¹ Tween 80, during 72 h cultivation at 28 °C (180 rpm) and initial pH 5.7. The predicted result for botryosphaeran production was 8.22 ± 1.36 g l⁻¹, and compared with the experimental value of 7.74 ± 0.13 g l⁻¹. Partial characterization of the botryosphaeran produced under the optimized conditions showed one type of polysaccharide with β-glycosidic linkages containing glucose as monosaccharide.     

In situ quantification of microcarrier animal cell cultures using near-infrared spectroscopy

In-line monitoring tools are still required to understand and control animal cell processes, particularly in the case of vaccine production. Here, in situ near-infrared spectroscopy (NIRS) quantification of components in culture media was performed using microcarrier-based cultivations of adherent Vero cells. Because microcarriers were found to interfere with NIRS spectra acquisition, a suitable and innovative in situ calibration was developed for bioreactor cultures. A reliable and accurate NIRS technique for the quantification of glucose and lactate was established, with a calibration standard error of 0.30 and 0.21 g l⁻¹, respectively. The robustness of this method was evaluated by performing NIRS calibration with operating conditions similar to those of industrial processes, including parameters such as microcarrier concentrations, cell seeding states and changes in analyte concentration due to feed and harvest strategies. Based on this calibration procedure, the predicted analyte concentrations in unknown samples was measured by NIRS analyses with an accuracy of 0.36 g l⁻¹ for glucose and 0.29 g l⁻¹ for lactate.     

Characterization of inducible cold-active β-glucosidases from the psychrotolerant bacterium Shewanella sp. G5 isolated from a sub-Antarctic ecosystem

The psychrotolerant bacterium Shewanella sp. G5 was used to study differential protein expression on glucose and cellobiose as carbon sources in cold-adapted conditions. This strain was able to growth at 4 °C, but reached the maximal specific growth rate at 37 °C, exhibiting similar growing rates values with glucose (μ: 0.4 h⁻¹) and cellobiose (μ: 0.48 h⁻¹). However, it grew at 15 °C approximately in 30 h, with specific growing rates of 0.25 and 0.19 h⁻¹ for cellobiose and glucose, respectively. Thus, this temperature was used to provide conditions related to the environment where the organism was originally isolated, the intestinal content of Munida subrrugosa in the Beagle Channel, Fire Land, Argentina. Cellobiose was reported as a carbon source more frequently available in marine environments close to shore, and its degradation requires the enzyme β-glucosidase. Therefore, this enzymatic activity was used as a marker of cellobiose catabolism. Zymogram analysis showed the presence of cold-adapted β-glucosidase activity bands in the cell wall as well as in the cytoplasm cell fractions. Two-dimensional gel electrophoresis of the whole protein pattern of Shewanella sp. G5 revealed 59 and 55 different spots induced by cellobiose and glucose, respectively. Identification of the quantitatively more relevant proteins suggested that different master regulation schemes are involved in response to glucose and cellobiose carbon sources. Both, physiological and proteomic analyses could show that Shewanella sp. G5 re-organizes its metabolism in response to low temperature (15 °C) with significant differences in the presence of these two carbon sources.     

Effects of feed sugar concentration on continuous ethanol fermentation of cheese whey powder solution (CWP)

Cheese whey powder (CWP) solution with different CWP or sugar concentrations was fermented to ethanol in a continuous fermenter using pure culture of Kluyveromyces marxianus (DSMZ 7239). Sugar concentration of the feed CWP solution varied between 55 and 200 g l⁻¹ while the hydraulic residence time (HRT) was kept constant at 54 h. Ethanol formation, sugar utilization and biomass formation were investigated as functions of the feed sugar concentration. Percent sugar utilization and biomass concentrations decreased and the effluent sugar concentration increased with increasing feed sugar concentrations especially for the feed sugar contents above 100 g l⁻¹. Ethanol concentration and productivity (DP) increased with increasing feed sugar up to 100 g l⁻¹ and then decreased with further increases in the feed sugar content. The highest ethanol concentration (3.7%, v v⁻¹) and productivity (0.54 gE l⁻¹ h⁻¹) were obtained with the feed sugar content of 100 g l⁻¹ or 125 g l⁻¹. The ethanol yield coefficient (Y_P/S) was also maximum (0.49 gE gS⁻¹) when the feed sugar was between 100 and 125 g l⁻¹. The growth yield coefficient (Y_X/S) decreased steadily from 0.123 to 0.063 gX gS⁻¹ when the feed sugar increased from 55 to 200 g l⁻¹ due to adverse effects of high sugar contents on yeast growth. The optimal feed sugar concentration maximizing the ethanol productivity and sugar utilization was between 100 and 125 g l⁻¹ under the specified experimental conditions.     

Isolation and bioelectrochemical characterization of novel fungal sources with oxidasic activity applied in situ for the cathodic oxygen reduction in microbial fuel cells

Brazilian filamentous fungi Rhizopus sp. (SIS-31), Aspergillus sp. (SIS-18) and Penicillium sp. (SIS-21), sources of oxidases were isolated from Caatinga's soils and applied during the in situ cathodic oxygen reduction in fuel cells. All strains were cultivated in submerged cultures using an optimized saline medium enriched with 10 g L⁻¹ of glucose, 3.0 g L⁻¹ of peptone and 0.0005 g L⁻¹ of CuSO₄ as enzyme inducer. Parameters of oxidase activity, glucose consumption and microbial growth were evaluated. In-cell experiments evaluated by chronoamperometry were performed and two different electrode compositions were also compared. Maximum current densities of 125.7, 98.7 and 11.5 μA cm⁻² were observed before 24 h and coulombic efficiencies of 56.5, 46.5 and 23.8% were obtained for SIS-31, SIS-21 and SIS-18, respectively. Conversely, maximum power outputs of 328.73, 288.80 and 197.77 mW m⁻³ were observed for SIS-18, SIS-21 and SIS-31, respectively. This work provides the primary experimental evidences that fungi isolated from the Caatinga region in Brazil can serve as efficient biocatalysts during the oxygen reduction in air-cathodes to improve electricity generation in MFCs.     

In situ quantification of microcarrier animal cell cultures using near-infrared spectroscopy

In-line monitoring tools are still required to understand and control animal cell processes, particularly in the case of vaccine production. Here, in situ near-infrared spectroscopy (NIRS) quantification of components in culture media was performed using microcarrier-based cultivations of adherent Vero cells. Because microcarriers were found to interfere with NIRS spectra acquisition, a suitable and innovative in situ calibration was developed for bioreactor cultures. A reliable and accurate NIRS technique for the quantification of glucose and lactate was established, with a calibration standard error of 0.30 and 0.21 g l⁻¹, respectively. The robustness of this method was evaluated by performing NIRS calibration with operating conditions similar to those of industrial processes, including parameters such as microcarrier concentrations, cell seeding states and changes in analyte concentration due to feed and harvest strategies. Based on this calibration procedure, the predicted analyte concentrations in unknown samples was measured by NIRS analyses with an accuracy of 0.36 g l⁻¹ for glucose and 0.29 g l⁻¹ for lactate.     

Multiple regeneration pathways in Spathoglottis plicata Blume — A study in vitro

Asymbiotic germination of immature seeds (embryos), and mature seeds and micropropagation of Spathoglottis plicata were described. Effects of three nutrition media namely, Murashige & Skoog (MS); Phytamax (PM); and Phyto-Technology orchid seed sowing medium (P₇₂₃), two carbon sources such as glucose and sucrose at 2–3% (w/v), two plant growth regulators such as 6-benzylaminopurine (BAP; 0.5–3.0 mg l^− 1) and α-naphthalene acetic acid (NAA; 0.5–2.0 mg l^− 1) and peptone (2.0 g l^− 1) were examined on seed germination, early protocorm development and micropropagation. The maximum germination of mature seeds (95%) was recorded in PM medium supplemented with 2% (w/v) sucrose + 2.0 g l^− 1 peptone. For germination of embryos P₇₂₃ medium supplemented with 1.0 mg l^− 1 BAP proved best. Multiple shoot buds or protocorm-like bodies (PLBs) were produced from stem segments of in vitro raised seedlings. Both direct organogenesis and embryogenesis were observed and the morphogenetic response was initiated by different concentrations and combinations of PGRs. The optimum PGR combination for maximal PLB regeneration was 1.0 mg l^− 1 NAA + 2.5 mg l^− 1 BAP, while 1.0 mg l^− 1 NAA + 1.0 mg l^− 1 BAP for shoot bud development. Strong and stout root system was induced in half strength PM medium supplemented with 0.5 mg l^− 1 IAA. The well-rooted plantlets were transferred to pots containing a potting mixture composed of saw dust, coconut coir, humus, and coal pieces at 1:1:1:2 (w/w) with 80% survival in outside environment and flowered after two years of transfer.     

Enantioselective oxidation of racemic lactic acid to d-lactic acid and pyruvic acid by Pseudomonas stutzeri SDM

d-Lactic acid and pyruvic acid are two important building block intermediates. Production of d-lactic acid and pyruvic acid from racemic lactic acid by biotransformation is economically interesting. Biocatalyst prepared from 9 g dry cell wt l^?1 of Pseudomonas stutzeri SDM could catalyze 45.00 g l^?1 dl-lactic acid into 25.23 g l^?1 d-lactic acid and 19.70 g l^?1 pyruvic acid in 10 h. Using a simple ion exchange process, d-lactic acid and pyruvic acid were effectively separated from the biotransformation system. Co-production of d-lactic acid and pyruvic acid by enantioselective oxidation of racemic lactic acid is technically feasible.     

Improvement of succinate production by overexpression of a cyanobacterial carbonic anhydrase in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing cyanobacterium Anabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). In strain BL21 (DE3) bearing ecaA, the activity of CA was 21.8 U mg⁻¹ protein, whereas non-detectable CA activity was observed in the control strain. Meanwhile, the activity of phosphoenolpyruvate carboxylase (PEPC) increased from 0.2 U mg⁻¹ protein to 1.13 U mg⁻¹ protein. The recombinant bearing ecaA reached a succinate yield of 0.39 mol mol⁻¹ glucose at the end of the fermentation. It was 2.1-fold higher than that of control strain which was just 0.19 mol mol⁻¹ glucose. EcaA gene was also introduced into E. coli DC1515, which was deficient in glucose phosphotransferase, lactate dehydrogenase and pyruvate:formate lyase. Succinate yield can be further increased to 1.26 mol mol⁻¹ glucose. It could be concluded that the enhancement of the supply of HCO₃⁻ in vivo by ecaA overexpression is an effective strategy for the improvement of succinate production in E. coli.