Occurrence of aerobic denitrifying bacteria in integrated fixed film activated sludge system.

Denitrification can be enhanced in the Integrated Fixed Film Activated System (IFAS) system by integrating media into the anoxic or aerobic zone. The simultaneous nitrification and denitrification (SND) in the biofilm layers has been reported in the aerobic zone of the IFAS system. In this study, two IFAS systems with Bioweb® media installed in the anoxic or aerobic reactor were operated in parallel to evaluate both anoxic denitrification or aerobic SND in the biofilm layers enhanced by fixed film media at three different nitrite and nitrate recirculation (NR) ratios of 75%, 100%, and 125%. The results revealed that both IFAS systems achieved the same organic and nitrogen removal efficiencies without statistically significant difference. The NR ratio of 125% enhanced slightly the denitrification in the anoxic zones of both systems. The media increased the anoxic denitrification at the NR ratio of 100%. The SND in the biofilm was found insignificant in both systems. It was evident that suspended-growth microorganisms stored substrates internally in the cells under anoxic conditions due to insufficient retention time. The aerobic denitrifiers including Chryseobacterium sp., Klebsiella pneumonia, and Pseudomonas aeruginosa were abundant in both IFAS systems providing aerobic denitrification with storage products as carbon sources. In summary, the denitrification in the anoxic zone and SND in the biofilm of the aerobic zone, both were enhanced by the fixed film media, did not contribute significantly to the IFAS system for the biological nitrogen removal because of microbial storage products and aerobic denitrification of several aerobic denitrifiers.

Removal of Cr(VI) from synthetic wastewater by adsorption onto coffee ground and mixed waste tea.

We attempted to recycle mixed waste tea and coffee ground as alternative low-cost adsorbents for Cr(VI) removal. The adsorption parameters optimized were: initial Cr(VI) concentration (10-30 mg L-1), contact time (180 min), adsorbent dose (2.0 g L-1), initial pH (2.0), temperature (30-50 °C), and agitation speed (250 rpm). Freundlich isotherm was found better fitted with a high correlation coefficient (R2 = 0.97 for mixed waste tea and 0.92 for coffee ground) than to Langmuir model (R2 = 0.89 for mixed waste tea and 0.86 for coffee ground) for the 10-250 mg L-1 concentration range. Analysis of kinetic studies indicated that Cr(VI) adsorption by both adsorbents was consistent with the pseudo-second-order kinetic model with a good R2 and Marquardt's present standard deviation (MPSD) values. Experimental data demonstrated a sorption capacity of 94.34 mg g-1 of mixed waste tea and 87.72 mg g-1 of coffee ground. Scanning electron microscopy (SEM), Transmission electron microscopy (TEM) and Energy dispersive X-ray spectroscopy (EDS) revealed the noticeable chromium accumulation on the adsorbent surfaces after adsorption. Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) studies showed that carbon and oxygen functional groups on the surface of both adsorbents involved in Cr(VI) adsorption. The adsorbents could be reused four times. Large-scale operation using 100 L of packed-bed reactor showed the breakthrough time of adsorption for mixed waste tea of 30 min in 100 mg L-1 Cr(VI) concentration. These results suggested that mixed waste tea and coffee ground be considered as alternative adsorbent for Cr(VI) removal.

Cultivable Methylobacterium species diversity in rice seeds identified with whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis.

Methylobacterium species are methylotrophic bacteria that widely inhabit plant surfaces. In addition to studies on methylotrophs as model organisms, research has also been conducted on their mechanism of plant growth promotion as well as the species-species specificity of plant-microbe interaction. We employed whole-cell matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (WC-MS) analysis, which enables the rapid and accurate identification of bacteria at the species level, to identify Methylobacterium isolates collected from the rice seeds of different cultivars harvested in Japan, Thailand, and Kenya. Rice seeds obtained from diverse geographical locations showed different communities of Methylobacterium species. We found that M. fujisawaense, M. aquaticum, M. platani, and M. radiotolerans are the most frequently isolated species, but none were isolated as common species from 18 seed samples due to the highly biased communities in some samples. These findings will contribute to the development of formulations containing selected species that promote rice growth, though it may be necessary to customize the formulations depending on the cultivars and farm conditions.

Genome shuffling enhances lipase production of thermophilic Geobacillus sp.

Thermostable lipases are potential enzymes for biocatalytic application. In this study, the lipase production of Geobacillus sp. CF03 (WT) was improved by genome shuffling. After two rounds of genome shuffling, one fusant strain (FB1) achieved increase lipase activity from the populations generated by ultraviolet irradiation and ethyl methylsulfonate (EMS) mutagenesis. The growth rate and lipase production of FB1 increased highest by 150 and 238 %, respectively, in comparison to the wild type. The fusant enzyme had a significant change in substrate specificity but still prefers the long-chain length substrates. It had an optimum activity at 60 °C, pH at 7.0-8.0, with p-nitrophenyl palmitate (C16) as a substrate and retained about 50 % of their activity after 15 min at 70 °C, pH 8.0. Furthermore, the fusant lipase showed the preference of sesame oil, waste palm oil, and canola oil. Therefore, the genome shuffling strategy has been successful to strain improvement and selecting strain with multiple desirable characteristics.

Polyvinyl alcohol and polyethylene glycol form polymer bodies in the periplasm of Sphingomonads that are able to assimilate them.

Scanning electron microscopy (SEM) shows remarkable morphological surface changes in Sphingopyxis sp. 113P3 cells grown in polyvinyl alcohol (PVA) but not in Luria-Bertani medium (LB) (Hu et al. in Arch Microbiol 188: 235-241, 2007). However, transmission electron microscopy showed no surface changes in PVA-grown cells and revealed the presence of polymer bodies in the periplasm of PVA-grown cells, which were not observed in LB-grown cells. The presence of polymer bodies was supported by low-vacuum SEM observation of PVA- and LB-grown cells of strain 113P3, and the presence of similar polymer bodies was also found when Sphingopyxis macrogoltabida 103 and S. terrae were grown in polyethylene glycol (PEG). The extraction of PVA and PEG from the periplasmic fraction of cells using a modified Anraku and Heppel method and their analysis by MALDI-TOF mass spectrometry strongly suggested that the polymer bodies are composed of PVA and PEG, respectively, in Sphingopyxis sp. 113P3 (PVA degrader) and Sphingopyxis macrogoltabida 103 or S. terrae (PEG degraders). PEG-grown S. macrogoltabida 103 and S. terrae showed higher transport of (14)C-PEG 4000 than LB-grown cells. Recombinant PegB (TonB-dependent receptor-like protein consisting of a barrel structure) interacted with PEG 200, 4000 and 20000, suggesting that the barrel protein in the outer membrane contributes to the transport of PEG into the periplasm.

Isolation and characterization of Kluyvera georgiana strain with the potential for acrylamide biodegradation.

Worldwide contamination by acrylamide, a neurotoxicant and carcinogen in animals, is becoming a significant problem. We isolated three novel acrylamide-degrading bacteria from domestic wastewater in Chonburi, Thailand. Using biochemical characteristics and 16S rRNA gene sequencing, the strains were identified as Klebsiella pneumoniae, Kluyvera georgiana and Enterococcus faecalis. K. georgiana strain No. 2 was selected for further characterization due to its degradation potential of high concentrations of acrylamide at the mesophilic temperatures. The strain grew well in the presence of acrylamide at concentrations to 0.5 % (w/v), pH 5.0 to 7.0 and 37°C. Degradation of acrylamide to acrylic acid began after 30 min of cultivation as a biomass-dependent manner. Mass balance analysis revealed 92.3 % conversion of acrylamide to acrylic acid and two lower polarity compounds. Strain No. 2 degraded many aliphatic amides but not iodoacetamide and thioacetamide. High degradation level (>80 %) was found with propionamide, cyanoacetamide and acetamide. Moderate degradation was obtained in the order of formamide > butyramide > lactamide > urea while sodium azide provided 34 % degradation. These findings render this novel bacterium attractive for biodegradation of acrylamide and other aliphatic amides in the environment.

Biodegradation of acrylamide by Enterobacter aerogenes isolated from wastewater in Thailand.

A widespread use of acrylamide, probably a neurotoxicant and carcinogen, in various industrial processes has led to environmental contamination. Fortunately, some microorganisms are able to derive energy from acrylamide. In the present work, we reported the isolation and characterization of a novel acrylamide-degrading bacterium from domestic wastewater in Chonburi, Thailand. The strain grew well in the presence of acrylamide as 0.5% (W/V), at pH 6.0 to 9.0 and 25 degrees C. Identification based on biochemical characteristics and 16S rRNA gene sequence identified the strain as Enterobacter aerogenes. Degradation of acrylamide to acrylic acid started in the late logarithmic growth phase as a biomass-dependent pattern. Specificity of cell-free supernatant towards amides completely degraded butyramide and urea and 86% of lactamide. Moderate degradation took place in other amides with that by formamide > benzamide > acetamide > cyanoacetamide > propionamide. No degradation was detected in the reactions of N,N-methylene bisacrylamide, sodium azide, thioacetamide, and iodoacetamide. These results highlighted the potential of this bacterium in the cleanup of acrylamide/amide in the environment.

Isolation and characterization of a novel thermophilic-organic solvent stable lipase from Acinetobacter baylyi.

The benzene tolerant Acinetobacter baylyi isolated from marine sludge in Angsila, Thailand could constitutively secrete lipolytic enzymes. The enzyme was successfully purified 21.89-fold to homogeneity by ammonium sulfate precipitation and gel-permeable column chromatography with a relative molecular mass as 30 kDa. The enzyme expressed maximum activity at 60 degrees C and pH 8.0 with p-nitrophenyl palmitate as a substrate and found to be stable in pH and temperature ranging from 6.0-9.0 to 60-80 degrees C, respectively. A study on solvent stability revealed that the enzyme was highly resisted to many organic solvents especially benzene and isoamyl alcohol, but 40% inhibited by decane, hexane, acetonitrile, and short-chain alcohols. Lipase activity was completely inhibited in the presence of Fe(2+), Mn(2+), EDTA, SDS, and Triton X-100 while it was suffered detrimentally by Tween 80. The activity was enhanced by phenylmethylsulfonyl fluoride (PMSF), Na(+), and Mg(2+) and no significant effect was found in the presence of Ca(2+) and Li(+). Half of an activity was retained by Ba(2+), Ag(+), Hg(+), Ni(2+), Zn(2+), and DTT. The enzyme could hydrolyze a wide range of p-nitrophenyl esters, but preferentially medium length acyl chains (C(8)-C(12)). Among natural oils and fats, the enzyme 11-folds favorably catalyzed the hydrolysis of rice bran oil, corn oil, sesame oil, and coconut oil in comparison to palm oil. Moreover, the transesterification activity of palm oil to fatty acid methyl esters (FAMEs) revealed 31.64 +/- 1.58% after 48 h. The characteristics of novel A. baylyi lipase, as high temperature stability, organic solvent tolerance, and transesterification capacity from palm oil to FAMEs, indicate that it could be a vigorous biocatalyzer in the prospective fields as bioenergy industry or even in organic synthesis and pharmaceutical industry.

Involvement of PEG-carboxylate dehydrogenase and glutathione S-transferase in PEG metabolism by Sphingopyxis macrogoltabida strain 103.

Sphingopyxis terrae and the Sphingopyxis macrogoltabida strains 103 and 203 are able to degrade polyethylene glycol (PEG). They possess the peg operon, which is responsible for the conversion of PEG to PEG-carboxylate-coenzyme A (CoA). The upstream (3.0 kb) and downstream (6.5 kb) regions of the operon in strain 103 were cloned and sequenced. The structure was well conserved between S. macrogoltabida strain 203 and S. terrae, except that two sets of transposases are absent in strain 203. The downstream region contains the genes for PEG-carboxylate dehydrogenase (PCDH), glutathione S-transferase (GST), tautomerase, and a hypothetical protein. The genes for pcdh and gst were transcribed constitutively and monocistronically, indicating that their transcription is independent of the operon regulation. PCDH and GST were expressed in Escherichia coli and characterized biochemically. PCDH is a homotetramer of 64-kDa subunits and contains one molecule of flavin adenine dinucleotide per subunit. The enzyme dehydrogenates PEG-carboxylate to yield glyoxylate, suggesting that the enzyme is the third enzyme involved in PEG degradation. GST is a homodimer of 28-kDa subunits. GST activity was noncompetitively inhibited by acyl-CoA and PEG-carboxylate-CoA, suggesting the interaction of GST with them. The proposed role for GST is to buffer the toxicity of PEG-carboxylate-CoA.

Structure and conservation of a polyethylene glycol-degradative operon in sphingomonads.

Sphingopyxis terrae, and Sphingopyxis macrogoltabida strains 103 and 203, can degrade polyethylene glycols (PEGs). They differ in the following respects: (i) different substrate specificities (chain length) of assimilable PEG, (ii) PEG-inducible or constitutive PEG-degradative proteins, and (iii) symbiotic or axenic degradation of PEG. S. terrae was able to incorporate PEG 6000, but strain 103 could not incorporate more than PEG 4000, suggesting that the difference in assimilable PEG chain length depends on the ability to take up substrate. PEG-degradative genes (pegB, C, D, A, E and R) from these strains were cloned. Their primary structures shared a high homology of more than 99 %. The peg genes encode a TonB-dependent receptor (pegB), a PEG-aldehyde dehydrogenase (pegC), a permease (pegD), a PEG dehydrogenase (pegA) and an acyl-CoA ligase (pegE), and in the opposite orientation, an AraC-type transcription regulator (pegR). The peg operon was flanked by two different sets of transposases. These three strains contained large plasmids and the operon was located in one of the large plasmids in S. terrae. The peg genes could be detected in other PEG-degrading sphingomonads. These results suggest that the peg genes have evolved in a plasmid-mediated manner. An insertion of a transposon gene (pegF) between pegD and pegA in strain 203 was found, which caused the constitutive expression of pegA in this strain.

Dual regulation of a polyethylene glycol degradative operon by AraC-type and GalR-type regulators in Sphingopyxis macrogoltabida strain 103.

The genes for polyethylene glycol (PEG) catabolism (pegB, C, D, A and E) in Sphingopyxis macrogoltabida strain 103 were shown to form a PEG-inducible operon. The pegR gene, encoding an AraC-type regulator in the downstream area of the operon, is transcribed in the reverse direction. The transcription start sites of the operon were mapped, and three putative sigma(70)-type promoter sites were identified in the pegB, pegA and pegR promoters. A promoter activity assay showed that the pegB promoter was induced by PEG and oligomeric ethylene glycols, whereas the pegA and pegR promoters were induced by PEG. Deletion analysis of the pegB promoter indicated that the region containing the activator-binding motif of an AraC/XylS-type regulator was required for transcription of the pegBCDAE operon. Gel retardation assays demonstrated the specific binding of PegR to the pegB promoter. Transcriptional fusion studies of pegR with pegA and pegB promoters suggested that PegR regulates the expression of the pegBCDAE operon positively through its binding to the pegB promoter, but PegR does not bind to the pegA promoter. Two specific binding proteins for the pegA promoter were purified and identified as a GalR-type regulator and an H2A histone fragment (histone-like protein, HU). The binding motif of a GalR/LacI-type regulator was found in the pegA and pegR promoters. These results suggested the dual regulation of the pegBCDAE operon through the pegB promoter by an AraC-type regulator, PegR (PEG-independent), and through the pegA and pegR promoters by a GalR/LacI-type regulator together with HU (PEG-dependent).