Biosurfactants are one of the microbial bioproducts that are in most demand from microbial-enhanced oil recovery (MEOR). The production of biosurfactant is still a relatively high cost. Therefore, this study aims to reduce production costs by utilizing palm oil mill effluent (POME) as the main carbon source. This work examines the optimal conditions of biosurfactant production by Halomonas meridiana BK-AB4 isolated from the Bledug Kuwu mud volcano in Central Java Indonesia and studies it for EOR applications. The biosurfactant production stage was optimized by varying POME concentration, incubation time, NaCl concentration, and pH to obtain the maximum oil displacement area (ODA) values. A response surface methodology (RSM) and a central composite design (CCD) were used to identify the influence of each variable and to trace the relationship between variables. Optimum biosurfactant production was found at a POME concentration (v/v) of 16%, incubation (h) of 112, NaCl concentration (w/v) of 4.7%, pH of 6.5, with an oil displacement area of 3.642 cm. The LC-MS and FTIR analysis revealed the functional groups of carboxylic acid or esters, which indicated that the biosurfactant produced belonged to the fatty acid class. The lowest IFT value was obtained at the second and seventh-day observations at a concentration of 500 mg/L, i.e., 0.03 mN/m and 0.06 mN/m. The critical micelle concentration (CMC) of biosurfactant was about 350 mg/L with a surface tension value of about 54.16 mN/m. The highest emulsification activity (E24 = 76%) in light crude oil (naphthenic-naphthenic) and could reduce the interfacial tension between oil and water up to 0.18 mN/m. The imbibition experiment with biosurfactant results in 23.89% additional oil recovery for 60 h of observation, with the highest increase in oil recovery occurring at the 18th hour, which is 2.72%. Therefore, this bacterium and its biosurfactantshow potential, and the bacterium are suitable for use in MEOR applications.

A range of water samples from the Al-Asfar lake, Al Ahsa, Saudi Arabia were enriched with LB medium (plates and liquid medium) containing a range of salt concentrations up to 2 M NaCl. Three strains of halotolerant or moderately halophilic bacteria were isolated and identified by 16S rDNA sequencing as belonging to the genera Staphylococcus, Halobacillus and Halomonas. The first two organisms (S. warneri and Halobacillus sp.) were further charcaterized to understand their ability to grow at high salinities up to 3 M NaCl. Halobacillus sp. was shown to be moderately halophilic (optimum growth between 0.17 and 1 M NaCl), whereas S. warneri was shown to be halotolerant, optimum growth at 0.17 M NaCl. Nuclear magnetic resonance (NMR) was used to determine the compatible solutes accumulated by the two strains. Betaine was accumulated by both organisms and Halobacillus sp. also utilized glutamate at low salt concentrations. This work demonstrates the presence of moderately halophilic and halotolerant bacteria in a freshwater lake.

Objective To analyze the effect of empirical antibiotic therapy on the intestinal microbiota of VLBW (very-low-birth-weight) infants on the 28th day, to provide the reference for VLBWI intestinal flora distribution and growth rule. Methods Collected VLBW infants who were treated in NICU of our hospital from August 2016 to February 2017, the infants treated with antibiotics as antibiotics group, while other infants treated with antibiotic free as control group. Fecal samples were collected on postnatal 1st day and 28th day. Total bacterial DNA was extracted. Bacterial taxa were identified by high-throughput 16s rRNA gene sequencing with MiSeq sequencing platform. Results ①5 VLBW infants were assigned to antibiotics group and other 5 VLBW infants were assigned to control group. The fecal samples on the 1st day had no significant variation on genus level (P>0.05), without statistically significant difference in the Shannon-index / Sphingomonas (P=0.23). ②Compared with control group, antibiotics group had increased relative abundance of Acinetobacter and Halomonas in fecal samples on the 28th day, decreased relative abundance of Ureaplasma and Haemophilus, without statistically significant difference in the Shannon-index / Sphingomonas (P=0.16). Conclusion Empirical antibiotic therapy within 1 week in VLBW infants could increase intestinal Acinetobacter and Halomonas content. Key words: Antibiotics; Initial empirical therapy; VLBW infants; Gut microbiota

Petroleum hydrocarbon pollution has been a major environmental challenge in the coastal areas, Niger-Delta, Nigeria. In this study, culture dependent and molecular techniques were used to monitor bioremediation over a-64 day period in seven microcosms setup in 2.5 L stirred tank bioreactors with each tank containing either Poultry droppings (BPOUT), NPK fertilizer (BNPK), Cow dung (BCD) or Urea fertilizer (BUREA). One bioreactor (BAUG) was bioaugmented while two others served as unamended (BUNa) and heat-killed (BHKD) controls. A decrease in petroleum hydrocarbon concentration and a concomitant increase in biomass was observed in all treatments at varying levels. BNPK (97.2%; 97.1%) showed highest reduction percentage while BHKD (82.34%, 81.3%) was the least for total petroleum hydrocarbon and polycyclic aromatic hydrocarbon amongst all treatment. Screening of isolates for aromatic hydrocarbon ring cleavage functional gene (catechol 2,3-dioxygenase) revealed that catechol 2,3-dioxygenase (C23D0) gene was detected in the following genera: Pseudomonas spp. (3), Rhodococcus sp. (2), Bacillus spp.(2)., Achromobacter sp., Serratia sp., Aeromonas sp., Micrococcus sp. and Acinetobacter sp. Sequences obtained from amplification of 16S rRNA gene gave a total number of 24 hydrocarbon utilizing bacterial species which showed 96-100% similarity with those deposited in GenBank and are identified as Brevundimonas naejangsanensis, Pseudomonas pseudoalcaligenes, Pseudomonas spp. (6), Aquitalea magnusonii, Achromobacter sp., Halomonas lutea, Pseudomonas aeruginosa (8), Shewanella sp, Achromobacter sp., Gordonia sp., Sphingobacterium sp. and Bacillus sp. Our result revealed that these extant indigenous bacterial population in the crude oil-polluted sediment habour the relevant aromatic hydrocarbon ring cleavage genes (catechol 2,3-dioxygenase) and may have a key role in bioremediation of crude oil-polluted sediment.

Despite known risks of inappropriate disposal of biomedical solid waste; most cities in developing countries are still disposing unsorted and untreated solid biomedical waste in common dumpsites. While many studies reported the presence of pathogens in fresh biomedical waste from hospitals, none has reported on the abundance and diversity of bacterial community in aged solid biomedical waste from a common dumpsite. A qualitative survey was done to identify types of solid biomedical waste on the dumpsite. Soils, sludge or washings of biomedical wastes were sampled. Total DNA was extracted and v4 region of 16S rRNA amplicons were sequenced using an Illumina MiSeq platform. A total of 1,706,442 sequences from 15 samples passed quality control. The number of sequences per sample ranged from 70664 to 174456 (mean 121765, SD 35853). Diversity was high with an InvSimpson index of 63 (Range 5 – 496, SD 121). Thirty five phyla were identified, but only 9 accounted for 96% of all sequences. The dominant phyla were Proteobacteria 37.4%, Firmicutes 34.4%, Bacteroidetes 14.1 %, Actinobacteria 5.6% and Chloroflex 1.7%. Catchall analysis predicted a mean of 9399 species per sample. Overall, 31402 operational taxonomic units (OTUs) were detected, however, only 19.8% (6,202) OTUs were found more than ten times. The most predominant OTUs were Proteinclasticum (10.4%), Acinetobacter (6.9), Halomonas (3.9), Pseudomonas (1.7%), Escherichia/Shigella 1.5% and Planococcus (1.3%). Proteiniclasticum spp and Acinetobacter spp were found in 67% (10/15) of all samples at relative abundance of 1%. Taxonomic-to-phenotype mapping revealed the presence of 36.2% related to bacteria involved in dehalogenation, 11.6% degraders of aromatic hydrocarbons, 14.8% chitin degraders, 8.5% chlorophenol degradation and Atrazine metabolism 8.3%. Taxonomy-to human pathogen mapping found 34% related to human pathogens and 39.4% were unknown. Conclusions There’s rich and diverse bacterial community in aged solid biomedical waste. Some of the predominant OTUs are related to bacteria of industrial use. We found a good number of OTUs mapping to human pathogens. Most of OTUs mapped to unknown metabolism and also to group unknown whether they human pathogens or not. To our knowledge, this is the first reports on bacteria related to industrial use from solid biomedical waste. This finding will facilitate to design further research using functional metagenomics to better understand the potential of bacteria from aged solid biomedical waste.

In order to reveal the diversity of sea ice bacterial communities in polar and sub-polar regions, we investigated 2 drifting ice floes, one from the Australian side of the Southern Ocean and the other from the Sea of Okhotsk. We extracted bacterial DNA from sea ice and constructed 221 16S rDNA clone libraries including 109 clones from the Antarctic sea ice and 112 from the Okhotsk sea ice. The phylogenetic analysis of 16S rDNA sequences showed that Roseobacter and Sulfitobacter (Alphaproteobacteria), Psychrobacter, Halomonas, and Pseudoalteromonas (Gammaproteobacteria) were frequent in the Antarctic sea ice; Colwellia, Psychromonas, and Glaciecola (Gammaproteobacteria) and Polaribacter (Bacteroidetes) were major genera in the Okhotsk sea ice. While Alphaproteobacteria and Gammaproteobacteria were abundant in both samples, Bacteroidetes were detected only in the Okhotsk sea ice. Comparing the bacterial diversity of our samples with that of other studies, bacterial communities in sea ice were similar to one another at the phylum level, whereas their populations were quite different at the genus level. We also tried to detect antimicrobial and heavy metal resistance genes in our samples but didn’t identified. Our results provide additional information about the bacterial communities in sea ice.

Extremophile bacteria are able to survive in harsh life conditions, such as high or low temperatures (thermophiles and psychrophiles, respectively), high pressure (barophiles), high or low pH values (acidophiles or alkalophiles), environments characterized by high salt concentrations (halophiles). Structural features of the macromolecules belonging to the external layer are fundamental in adaptation mechanisms, e.g. it is well known that halophiles membrane phospholipids showed an increased negative charge density, while in psychrophiles these molecules display shorter acyl chains and higher unsaturation degree. In Gram-negative bacteria, 75% of the outer membrane is constituted by lipopolysaccharides (LPSs). Consequently they play a key role in the adaptation and survival in extreme life conditions. Nevertheless, very few LPSs isolated from extremophilic bacteria has been characterized so far. LPS are constituted by three covalently linked regions: - lipid A, which is the glycolipidic portion of the macromolecule. It is the most conservative region between bacteria belonging to the same genus and represents the minimal endotoxic structural motif; - core region, which is an oligosaccharidic portion where it is possible to find LPSs peculiar monosaccharides, such as heptoses and Kdo (3-deoxy oct-2-oulosonic acid); - O-chain, which is the polysaccharidic region, not always expressed by the bacterium. Moreover, O-chain is highly variable even among bacteria belonging to the same species. Beside the structural characterization of LPS, aimed at adaptation mechanisms comprehension, also their biological activity is worth being investigated. In fact extremophilic bacteria are rarely found to be pathogen, so they are source of lipid A with potential anti-inflammatory (antagonist) or adjuvant activity. During this phD work, the LPSs from three haloalkaliphilic and two psychrophilic bacteria has been investigated. Each LPS has been extracted from dried cells, then purified and analysed by chemical analysis, NMR spectroscopy and mass spectrometry. As for the haloalkaliphilic bacteria, the LPSs belonging to Halomonas alkaliantarctica strain CRSS, Halomonas stevensii strain S18214 and Salinivibrio sharmensis strain BAGT were completely characterized. By comparing the structures obtained, especially for core oligosaccharides, it is possible to speculate that they are all characterized by high negative charge density, due to phosphate groups, usually linked to Kdo and lipid A saccharidic residues, or to uronic acids. Such structural elements contribute to the tightness of the outer-membrane and decrease the ion permeability, due to the association of LPS molecules through divalent cations (Ca2+ and Mg2+). Moreover, lipid A structural characterization of lipid A from H. stevensii and S. sharmensis has been carried out. Both the psychrophilic bacteria Pseudoalteromonas haloplanktis strain TAB 23 and Colwellia psychrerythraea strain 34H expressed a rough-LPS. The core and lipid A structures were obtained. Moreover, biological assays on both lipid A were performed. The structural common features of these two bacteria are the high negative charge density and the lack of the O-chain. The first helps membrane permeability, allowing bacterial survival in marine environment, where these microorganisms are often isolated. The second characteristic was found in all known LPS from psychrophiles and can be explained as a consequence of cell economy: O-chain biosynthesis is an energy-demanding process avoided by the organisms in low-temperature life conditions. As for the lipid A structures, they both share the presence of short and unsaturated fatty acids chains, as already found in psychrophilic bacteria membrane phospholipids. Moreover, the TNFα production was not elicited in both cases, and for P. haloplanktis TAB 23 lipid A an inhibitory activity was found. These results led to a deeper knowledge of halo- and cold adaptation mechanisms in Gram-negative bacteria. Moreover, molecules with different and interesting physicochemical and biological properties has been isolated and characterized. It is evident that extremophilic bacteria are an important source of biomolecules of which probably nowadays only the peak of the iceberg is known.

Objectives To identify the infrequent strains in clinical isolates by broad-spectrum PCR amplification and direct sequencing targeting the bacterial 16S rRNA gene. Methods Total 48 clinical isolates and 7 false-positive blood culture samples were collected from 7 different hospitals or institutions from December 2010 to September 2011.The bacterial 16S rRNA gene were amplified and sequenced by universal prime sets of 27f-1492r and 27f-1525r，and MicroSeq 500 16S rRNA gene kit.The homology analysis was used by the Basic Local Alignment Search Tool, and comparing to gene sequence of the type strain.provided by the List of Prokaryotic names with Standing in Nomenclature.The criteria for the bacterial identification was interpreted according to the Clinical and Laboratory Standards Institute （CLSI） MM18-A. Results All of the 48 cultured strains were succeeded amplifying and sequencing the targeted 16S rRNA genes.According to the criteria of CLSI MM18-A, total 35 strains were specified to the species level, 11 strains were specified to the genus level, and the other 2 strains were specified to possible novel genus and species.Combining the analysis the sequence of other housekeeping gene with the results of biochemical results, total 42 strains can be specified to the species level, including some clinical important pathogens, such as Streptobacillus, Capnocytophaga, Nocardia, Mycobacterium, Roseomonas and Campylobacter.Two false-positive blood culture samples were managed to amplify 16S rRNA genes and finally identified as Streptococcus pneumoniae.We also identified one novel subspecies of Campylobacter fetus, and some new valid-published species, such as Acinetobacter parvus,Mycobacterium phocaicum,Roseomonas mucosa and Halomonas johnsoniae. Conclusions The 16S rRNA gene sequence based identification has unique advantages over the phenotypic methods.It is universal to almost of all the bacteria, and can provide the genetic classified information. It is very suitable for the clinical infrequent and special bacterial cultures, such as the slow-growing, fastidious, or un-cultured bacteria.（Chin J Lab Med,2012,35:612-619） Key words: RNA，ribosomal，16S; Polymerase chain reaction; Sequence analysis; Bacteriological techniques

Hypersaline produced water is a high volume waste stream that is typically contaminated by toxic low molecular weight aromatic compounds exemplified by phenol. Aromatic compound degrading Halomonas were isolated from hypersaline produced water obtained from offshore operations in Brazil, which had a chemical oxygen demand (COD) of 4300 mg/L. These isolates were able to aerobically degrade the oxygenated aromatics phenol, benzoic acid, para-hydroxybenzoic acid and some isolates were shown to produce aromatic dioxygenase activities associated with highly conserved aromatic degradation pathways utilized by a broad range of aromatic degrading bacteria. The presence of aromatic degrading bacteria in the hypersaline produced water suggested that the COD content could be reduced by bioremediation using the indigenous microbial population by the addition of nutrients. Using this approach a variety of nitrogen, phosphorous and carbons sources were identified that individually or in combination significantly improved the reduction in COD after aerobic incubation. These results demonstrate the potential of applying bioremediation to undiluted hypersaline produced water for COD reduction and aromatic compound removal.

The wide use of ion-exchange processes to remove perchlorate from drinking water creates an urgency for the regeneration or treatment of perchlorate-laden ion-exchange resins and/or regenerant brines. The use of biological processes with a salt-tolerant culture NP30 has been demonstrated as a promising cost-effective approach. In this study, the kinetics and ecology of NP30 were studied. A pure culture was isolated from the mixed culture, identified and characterized. Perchlorate–laden ion-exchange resins were effectively regenerated by the mixed culture in laboratory batch reactors. A numerical model was developed to describe the regeneration process and for design predictions. A unique “resin phase” regeneration, in which the culture degraded perchlorate on the resin instead of only what desorbed into the bulk medium, was proposed in the model. The model generated an acceptable correlation to experimental data and the degradation from the “resin phase” accounted for the majority of the perchlorate removal. The microbial composition of NP30 and the changes during a pilot plant experiment treating perchlorate- and nitrate-laden ion-exchange brine were analyzed using DGGE (denaturing gradient gel electrophoresis) and FISH (fluorescence in situ hybridization). Halomonas was the dominant (>18%) nitrate-reducing organism and Azoarcus/Denitromonas was the dominant (>22%) perchlorate-reducing organism. A shift towards nitrate-reducing organisms with time in the reactors was observed and attributed to the non-obvious perchlorate reduction seen in operation data. A pure salt-tolerant, perchlorate-reducing strain P4B1 (Marinobacter multirespiro sp. nov. proposed name) was successfully isolated from the mixed culture. P4B1 could grow in the presence of 1.8%-10.2% NaCl. A molar Mg²⁺/Na⁺ ratio of ~0.11 optimized the perchlorate degradation and cell growth when perchlorate was the sole electron acceptor. It could use perchlorate, nitrate and oxygen as electron acceptors. P4B1 preferred perchlorate to nitrate as the electron acceptor. A perchlorate reductase, which is only induced by perchlorate, is active in both perchlorate and nitrate reduction. When nitrate was used as the sole electron acceptor, the strain eventually lost the ability to reduce nitrate. The maximum specific substrate utilization rate (Vm) and the half saturation coefficient (Ks) for P4B1 were determined to be 0.050 ±0.007 mg ClO₄⁻/mg VSS-hr and 22±12 mg ClO₄⁻/L respectively.

Identification and bioactivities analysis of <I>Halomonas</I> sp. from seawater

KEYWORDS: ionic strengthbacteriacurium(III)europium(III)adsorptionHalomonas elongatecell surfacehydrophobicity