Enhancement of astaxanthin accumulation using black light in Coelastrum and Monoraphidium isolated from Malaysia.

Microalgae are important microorganisms which produce potentially valuable compounds. Astaxanthin, a group of xanthophyll carotenoids, is one of the most powerful antioxidants mainly found in microalgae, yeasts, and crustaceans. Environmental stresses such as intense light, drought, high salinity, nutrient depletion, and high temperature can induce the accumulation of astaxanthin. Thus, this research aims to investigate the effect of black light, also known as long-wave ultraviolet radiation or UV-A, as a stressor on the accumulation of astaxanthin as well as to screen the antioxidant property in two tropical green algal strains isolated from Malaysia, Coelastrum sp. and Monoraphidium sp. SP03. Monoraphidium sp. SP03 showed a higher growth rate (0.66 day-1) compared to that of Coelastrum sp. (0.22 day-1). Coelastrum sp. showed significantly higher accumulation of astaxanthin in black light (0.999 g mL culture-1) compared to that in control condition (0.185 g mL-1). Similarly, Monoraphidium sp. SP03 showed higher astaxanthin content in black light (0.476 g mL culture-1) compared to that in control condition (0.363 g mL culture-1). Coelastrum sp. showed higher scavenging activity (30.19%) when cultured in black light condition, indicating a correlation between the antioxidant activity and accumulation of astaxanthin. In this study, black light was shown to possess great potential to enhance the production of astaxanthin in microalgae.

Draft Genome Sequences of Three Multidrug-Resistant Staphylococcus spp. Isolated from Hospital Wastewater in Malaysia.

Staphylococcus spp. are Gram-positive bacteria that reside within the normal microbiota of humans and animals but pose a health threat as reservoirs of antimicrobial resistance genes. Here, we present the draft genome sequences of three Staphylococcus sp. strains isolated from hospital wastewater in Malaysia that demonstrated resistance to multiple antibiotics.

Draft Genome Sequence of the Prazosin-Degrading Bacillus sp. Strain PR5, Isolated from a River Receiving Hospital and Urban Wastewater in Malaysia.

We report the complete genome sequence of Bacillus sp. strain PR5, isolated from a river receiving hospital and urban wastewater in Malaysia, which demonstrated a high capability for degrading prazosin. This genome sequence of 4,525,264 bp exhibited 41.5% GC content, 4,402 coding sequences, and 32 RNAs.

Limitation of nutrients stimulates musty odor production by Streptomyces sp. isolated from a tropical environment.

Musty odor production by actinomycetes is usually related to the presence of geosmin and 2-methylisoborneol (2-MIB), which are synthesized by enzymes encoded by the geoA and tpc genes, respectively. Streptomyces spp. strain S10, which was isolated from a water reservoir in Malaysia, has the ability to produce geosmin when cultivated in a basal salt (BS) solid medium, but no 2-MIB production occurred during growth in BS medium. Strain S10 could produce higher levels of geosmin when the phosphate concentration was limited to 0.05 mg/L, with a yield of 17.53 ± 3.12 ✕ 105 ng/L, compared with growth in BS medium. Interestingly, 2-MIB production was suddenly detected when the nitrate concentration was limited to 1.0 mg/L, with a yield of 1.4 ± 0.11 ✕ 105 ng/L. Therefore, it was concluded that phosphate- and nitrate-limiting conditions could induce the initial production of geosmin and 2-MIB by strain S10. Furthermore, a positive amplicon of geoA was detected in strain S10, but no tpc amplicon was detected by PCR analysis. Draft genome sequence analysis showed that one open reading frame (ORF) contained a conserved motif of geosmin synthase with 95% identity with geoA in Streptomyces coelicolor A3 (2). In the case of the tpc genes, it was found that one ORF showed 23% identity to the known tpc gene in S. coelicolor A3(2), but strain S10 lacked one motif in the N-terminus.

Enzymatic and genetic characterization of lignin depolymerization by Streptomyces sp. S6 isolated from a tropical environment.

The conversion of lignocellulosic biomass into bioethanol or biochemical products requires a crucial pretreatment process to breakdown the recalcitrant lignin structure. This research focuses on the isolation and characterization of a lignin-degrading bacterial strain from a decaying oil palm empty fruit bunch (OPEFB). The isolated strain, identified as Streptomyces sp. S6, grew in a minimal medium with Kraft lignin (KL) as the sole carbon source. Several known ligninolytic enzyme assays were performed, and lignin peroxidase (LiP), laccase (Lac), dye-decolorizing peroxidase (DyP) and aryl-alcohol oxidase (AAO) activities were detected. A 55.3% reduction in the molecular weight (Mw) of KL was observed after 7 days of incubation with Streptomyces sp. S6 based on gel-permeation chromatography (GPC). Gas chromatography-mass spectrometry (GC-MS) also successfully highlighted the production of lignin-derived aromatic compounds, such as 3-methyl-butanoic acid, guaiacol derivatives, and 4,6-dimethyl-dodecane, after treatment of KL with strain S6. Finally, draft genome analysis of Streptomyces sp. S6 also revealed the presence of strong lignin degradation machinery and identified various candidate genes responsible for lignin depolymerization, as well as for the mineralization of the lower molecular weight compounds, confirming the lignin degradation capability of the bacterial strain.

Complete Genome Sequence of Lignin-Degrading Streptomyces sp. Strain S6, Isolated from an Oil Palm Plantation in Malaysia.

Streptomyces spp. are bacteria that are responsible for the degradation of aromatic compounds and produce secondary metabolites. Here, we present a complete genome sequence of Streptomyces sp. strain S6, which was isolated from an oil palm plantation, with a 7.8-Mbp liner chromosome, a GC content of 72%, and 4,266 coding sequences.

Elucidation of prazosin biodegradation by isolated Bacillus spp. from the tropical environment.

Prazosin (PRZ), a drug used to treat hypertensive patients, is an emergent contaminant in water systems. PRZ is an alpha-adrenergic receptor blocker used to treat anxiety, and is believed to reach the environment through human excretion, irresponsible disposal of unused medicine, and waste products from manufacturing plants. The purpose of this research was to isolate and characterize potential microbes for PRZ biodegradation and to identify the degradation pathway. After screening, isolated strain STP3 showed a capability for PRZ degradation and was chosen for further analysis. Resting cell assays with PRZ were conducted to identify the intermediate metabolites formed from biodegradation by Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) analysis. Two metabolites degraded from PRZ by STP3 were successfully found, and as these metabolites are derived from the main structure of PRZ, their presence proved PRZ degradation. Draft genome sequencing analysis of STP3 was performed to identify potential enzymes for PRZ biodegradation based on the metabolites found.

Microbial Diversity in Decaying Oil Palm Empty Fruit Bunches (OPEFB) and Isolation of Lignin-degrading Bacteria from a Tropical Environment.

Oil palm empty fruit bunches (OPEFB) are the most abundant, inexpensive, and environmentally friendly lignocellulosic biomass in Malaysia. Investigations on the microbial diversity of decaying OPEFB may reveal microbes with complex enzymes that have the potential to enhance the conversion of lignocellulose into second-generation biofuels as well as the production of other value-added products. In the present study, fungal and bacterial diversities in decaying OPEFB were identified using Illumina MiSeq sequencing of the V3 region of the 16S rRNA gene and V4 region of the 18S rRNA gene. Fungal diversity in decaying OPEFB was dominated by the phylum Ascomycota (14.43%), while most of the bacterial sequences retrieved belonged to Proteobacteria (76.71%). Three bacterial strains isolated from decaying OPEFB, designated as S18, S20, and S36, appeared to grow with extracted OPEFB-lignin and Kraft lignin (KL) as the sole carbon source. 16S rRNA gene sequencing identified the 3 isolates as Paenibacillus sp.. The molecular weight distribution of KL before and after degradation showed significant depolymerization when treated with bacterial strains S18, S20, and S36. The presence of low-molecular-weight lignin-related compounds, such as vanillin and 2-methoxyphenol derivatives, which were detected by a GC-MS analysis, confirmed the KL-degrading activities of isolated Paenibacillus strains.

Depolymerization of lignocellulose of oil palm empty fruit bunch by thermophilic microorganisms from tropical climate.

Previous studies on screening of lignin-degrading bacteria mainly focused on the ligninolytic ability of the isolated bacteria for the utilization of lignin monomers. In this study, we focused on the depolymerization of alkali lignin to prove the ability of the isolated thermophilic bacterial strains to utilize and depolymerize more than a monomer of alkali lignin within 7 days of incubation. Indigenous thermophilic bacterial isolates from the palm oil plantation were used to evaluate the depolymerization and utilization of alkali lignin. The confirmation of the bacterium-mediated depolymerization of oil palm empty fruit bunch was achieved through the removal of silica bodies, as observed with scanning electron microscopy. Stenotrophomonas sp. S2 and Bacillus subtilis S11Y were able to reduce approximately 50% and 20% of alkali lignin at 7 days of incubation without the requirement for additional carbon sources.

Whole gene transcriptomic analysis of PCB/biphenyl degrading Rhodococcus jostii RHA1.

This study gives the first picture of whole RNA-Sequencing analysis of a PCB-degrading microbe, Rhodococcus jostii RHA1. Genes that were highly expressed in biphenyl-grown cells, compared with pyruvate-grown cells, were chosen based on the Reads Per Kilobase Million (RPKM) value and were summarized based on the criteria of RPKM ≥100 and fold change ≥2.0. Consequently, 266 total genes were identified as genes expressed particularly for the degradation of biphenyl. After comparison with previous microarray data that identified highly-expressed genes, based on a fold change ≥2.0 and p-value ≤0.05, 62 highly-expressed genes from biphenyl-grown cells were determined from both analytical platforms. As these 62 genes involve known PCB degradation genes, such as bph, etb, and ebd, the genes identified in this study can be considered as essential genes for PCB/biphenyl degradation. In the 62 genes, eleven genes encoding hypothetical proteins were highly expressed in the biphenyl-grown cells. Meanwhile, we identified several highly-expressed unannotated DNA regions on the opposite strand. In order to verify the encoded proteins, two regions were cloned into an expression vector. A protein was successfully obtained from one region at approximately 25 kDa from the unannotated strand. Thus, the genome sequence with transcriptomic analysis gives new insight, considering re-annotation of the genome of R. jostii RHA1, and provides a clearer picture of PCB/biphenyl degradation in this strain.

Molecular characterization of multi-drug resistant Escherichia coli isolates from tropical environments in Southeast Asia.

The emergence of antibiotic resistance among multidrug-resistant (MDR) microbes is of growing concern, and threatens public health globally. A total of 129 Escherichia coli isolates were recovered from lowland aqueous environments near hospitals and medical service centers in the vicinity of Kuala Lumpur, Malaysia. Among the eleven antibacterial agents tested, the isolates were highly resistant to trimethoprim-sulfamethoxazole (83.7%) and nalidixic acid (71.3%) and moderately resistant to ampicillin and chloramphenicol (66.7%), tetracycline (65.1%), fosfomycin (57.4%), cefotaxime (57.4%), and ciprofloxacin (57.4%), while low resistance levels were found with aminoglycosides (kanamycin, 22.5%; gentamicin, 21.7%). The presence of relevant resistance determinants was evaluated, and the genotypic resistance determinants were as follows: sulfonamides (sulI, sulII, and sulIII), trimethoprim (dfrA1 and dfrA5), quinolones (qnrS), ß-lactams (ampC and blaCTX-M), chloramphenicol (cmlA1 and cat2), tetracycline (tetA and tetM), fosfomycin (fosA and fosA3), and aminoglycosides (aphA1 and aacC2). Our data suggest that multidrug-resistant E. coli strains are ubiquitous in the aquatic systems of tropical countries and indicate that hospital wastewater may contribute to this phenomenon.

Removal efficiency of Gram-positive and Gram-negative bacteria using a natural coagulant during coagulation, flocculation, and sedimentation processes.

Staphylococcus sp. as Gram-positive and Escherichia coli as Gram-negative are bacterial pathogens and can cause primary bloodstream infections and food poisoning. Coagulation, flocculation, and sedimentation processes could be a reliable treatment for bacterial removal because suspended, colloidal, and soluble particles can be removed. Chemical coagulants, such as alum, are commonly used. However, these chemical coagulants are not environmentally friendly. This present study evaluated the effectiveness of coagulation, flocculation, and sedimentation processes for removing Staphylococcus sp. and E. coli using diatomite with standard jar test equipment at different pH values. Staphylococcus sp. demonstrated 85.61% and 77.23% significant removal in diatomite and alum, respectively, at pH 5. At pH 7, the removal efficiency decreased to 79.41% and 64.13% for Staphylococcus sp. and E. coli, respectively. At pH 9, there was a decrease in Staphylococcus sp. after adding diatomite or alum compared with that of E. coli. The different removal efficiencies of the Gram-positive and Gram-negative bacteria could be owing to the membrane composition and different structures in the bacteria. This study indicates that diatomite has higher efficiency in removing bacteria at pH 5 and can be considered as a potential coagulant to replace alum for removing bacteria by the coagulation process.

Importance of Soil Temperature for the Growth of Temperate Crops under a Tropical Climate and Functional Role of Soil Microbial Diversity.

A soil cooling system that prepares soil for temperate soil temperatures for the growth of temperate crops under a tropical climate is described herein. Temperate agriculture has been threatened by the negative impact of temperature increases caused by climate change. Soil temperature closely correlates with the growth of temperate crops, and affects plant processes and soil microbial diversity. The present study focuses on the effects of soil temperatures on lettuce growth and soil microbial diversity that maintains the growth of lettuce at low soil temperatures. A model temperate crop, loose leaf lettuce, was grown on eutrophic soil under soil cooling and a number of parameters, such as fresh weight, height, the number of leaves, and root length, were evaluated upon harvest. Under soil cooling, significant differences were observed in the average fresh weight (P<0.05) and positive development of the roots, shoots, and leaves of lettuce. Janthinobacterium (8.142%), Rhodoplanes (1.991%), Arthrospira (1.138%), Flavobacterium (0.857%), Sphingomonas (0.790%), Mycoplana (0.726%), and Pseudomonas (0.688%) were the dominant bacterial genera present in cooled soil. Key soil fungal communities, including Pseudaleuria (18.307%), Phoma (9.968%), Eocronartium (3.527%), Trichosporon (1.791%), and Pyrenochaeta (0.171%), were also recovered from cooled soil. The present results demonstrate that the growth of temperate crops is dependent on soil temperature, which subsequently affects the abundance and diversity of soil microbial communities that maintain the growth of temperate crops at low soil temperatures.

Characterization of Musty Odor-Producing Actinomycetes from Tropics and Effects of Temperature on the Production of Musty Odor Compounds.

Geosmin and 2-methylisoborneol (MIB) outbreaks in tropical water bodies, such as Southeast Asia, by actinomycetes have not yet been elucidated in detail. Six Streptomyces isolates from lowland environments in Malaysia were selected and evaluated for their odor production under different temperatures. The gene responsible for the production of geosmin, geoA, was detected in all isolates, while only two isolates harbored tpc, which is responsible for 2-MIB production. This result suggested that geosmin and 2-MIB synthesis pathway genes already existed in the environment in the Tropics of Southeast Asia. Furthermore, our isolates produced musty odor compounds at 30°C, and differences were observed in musty odor production between various temperatures. This result indicated the potential for odor episodes in water bodies of the tropical countries of Southeast Asia throughout the year due to the mean annual ambient temperature of 27°C in the lowlands.

Recombinant protein expression of Moringa oleifera lectin in methylotrophic yeast as active coagulant for sustainable high turbid water treatment.

The natural coagulant Moringa oleifera lectin (MoL) as cationic protein is a promising candidate in coagulation process of water treatment plant. Introducing the gene encoding MoL into a host, Pichia pastoris, to secrete soluble recombinant protein is assessed in this study. Initial screening using PCR confirmed the insertion of MoL gene, and SDS-PAGE analysis detected the MoL protein at 8 kDa. Cultured optimization showed the highest MoL protein at 520 mg/L was observed at 28 °C for 144 h of culturing by induction in 1% methanol. Approximately, 0.40 mg/mL of recombinant MoL protein showed 95 ± 2% turbidity removal of 1% kaolin suspension. In 0.1% kaolin suspension, the concentration of MoL at 10 µg/mL exhibits the highest turbidity reduction at 68 ± 1%. Thus, recombinant MoL protein from P. pastoris is an effective coagulant for water treatment.

Phenotypic and genetic characterization of multidrug-resistant Staphylococcus aureus in the tropics of Southeast Asia.

Antibiotic resistance has become a major public health problem throughout the world. The presence of antibiotic-resistant bacteria such as Staphylococcus aureus and antibiotic resistance genes (ARGs) in hospital wastewater is a cause for great concern today. In this study, 276 Staph. aureus isolates were recovered from hospital wastewater samples in Malaysia. All of the isolates were screened for susceptibility to nine different classes of antibiotics: ampicillin, ciprofloxacin, gentamicin, kanamycin, erythromycin, vancomycin, trimethoprim and sulfamethoxazole, chloramphenicol, tetracycline and nalidixic acid. Screening tests showed that 100 % of Staph.aureus isolates exhibited resistance against kanamycin, vancomycin, trimethoprim and sulfamethoxazole and nalidixic acid. Additionally, 91, 87, 50, 43, 11 and 8.7 % of isolates showed resistance against erythromycin, gentamicin, ciprofloxacin, ampicillin, chloramphenicol and tetracycline, respectively. Based on these results, 100 % of isolates demonstrated multidrug-resistant (MDR) characteristics, displaying resistance against more than three classes of antibiotics. Of 276 isolates, nine exhibited resistance to more than nine classes of tested antibiotics; these were selected for antibiotic susceptibility testing and examined for the presence of conserved ARGs. Interestingly, a high percentage of the selected MDR Staph.aureus isolates did not contain conserved ARGs. These results indicate that non-conserved MDR gene elements may have already spread into the environment in the tropics of Southeast Asia, and unique resistance mechanisms against several antibiotics may have evolved due to stable, moderate temperatures that support growth of bacteria throughout the year.

Electrocoagulation using a rotated anode: A novel reactor design for textile wastewater treatment.

This paper investigates the optimum operational conditions of a novel rotated bed electrocoagulation (EC) reactor for the treatment of textile wastewater. The effect of various operational parameters such as rotational speed, current density (CD), operational time (RT), pH, temperature, and inter-electrode distance (IED) on the pollutant removal efficiency were examined. In addition, the consumption of aluminum (Al) and electrical energy, as well as operating costs at optimum conditions were also calculated. The results indicated that the optimum conditions for the treatment of textile wastewater were achieved at CD = 4 mA/cm(2), RT = 10 min, rotational speed = 150 rpm, pH = 4.57, temperature = 25 °C, and IED = 1 cm. The electrode consumption, energy consumption, and operating costs were 0.038 kg/m(3), 4.66 kWh/m(3) and 0.44 US$/m(3), respectively. The removal efficiencies of chemical oxygen demand (COD), biological oxygen demand (BOD), total suspended solid (TSS), turbidity and color were 97.10%, 95.55%, 98%, 96% and 98.50%, respectively, at the first 10 min of reaction time, while the phenol compound of the wastewater was almost entirely removed (99.99%). The experimental results confirm that the new reactor design with rotated anode impellers and cathode rings provided high treatment efficiency at a reduced reaction time and with lower energy consumption.

Identification of novel extracellular protein for PCB/biphenyl metabolism in Rhodococcus jostii RHA1.

Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.

Multi-residue analytical methodology-based liquid chromatography-time-of-flight-mass spectrometry for the analysis of pharmaceutical residues in surface water and effluents from sewage treatment plants and hospitals.

An analytical method that facilitated the analysis of 11 pharmaceuticals residue (caffeine, prazosin, enalapril, carbamazepine, nifedipine, levonorgestrel, simvastatin, hydrochlorothiazide, gliclazide, diclofenac-Na, and mefenamic acid) with a single pre-treatment protocol was developed. The proposed method included an isolation and concentration procedure using solid phase extraction (Oasis HLB), a separation step using high-performance liquid chromatography, and a detection procedure that applies time-of-flight mass spectrometry. The method was validated for drinking water (DW), surface water (SW), sewage treatment plant (STP) influent and effluent, and hospital (HSP) influent and effluent. The limits of quantification were as low as 0.4, 1.6, 5, 3, 2.2 and 11 ng/L in DW, SW, HSP influent and effluent, STP effluent, and STP influent, respectively. On average, good recoveries higher than 75% were obtained for most of the target analytes in all matrices. Matrix effect was evaluated for all samples matrices. The proposed method successfully determined and quantified the target compounds in raw and treated wastewater of four STPs and three hospitals in Malaysia, as well as in two SW sites. The results showed that a number of the studied compounds pose moderate to high persistency in sewage treatment effluents as well as in the recipient rivers, namely; caffeine, simvastatin, and hydrochlorothiazide. Ten out of 11 compounds were detected and quantified in 13 sampling points. Caffeine was detected with the highest level, with concentrations reaching up to 9099 ng/L in STP influent.

Assessing the ecological risk of polycyclic aromatic hydrocarbons in sediments at Langkawi Island, Malaysia.

Tourism-related activities such as the heavy use of boats for transportation are a significant source of petroleum hydrocarbons that may harm the ecosystem of Langkawi Island. The contamination and toxicity levels of polycyclic aromatic hydrocarbon (PAH) in the sediments of Langkawi were evaluated using sediment quality guidelines (SQGs) and toxic equivalent factors. Ten samples were collected from jetties and fish farms around the island in December 2010. A gas chromatography/flame ionization detector (GC/FID) was used to analyse the 18 PAHs. The concentration of total PAHs was found to range from 869 ± 00 to 1637 ± 20 ng g⁻¹ with a mean concentration of 1167.00 ± 24 ng g⁻¹, lower than the SQG effects range-low (3442 ng g⁻¹). The results indicated that PAHs may not cause acute biological damage. Diagnostic ratios and principal component analysis suggested that the PAHs were likely to originate from pyrogenic and petrogenic sources. The toxic equivalent concentrations of the PAHs ranged from 76.3 to 177 ng TEQ/g d.w., which is lower compared to similar studies. The results of mean effects range-median quotient of the PAHs were lower than 0.1, which indicate an 11% probability of toxicity effect. Hence, the sampling sites were determined to be the low-priority sites.