MlrA, an Essential Enzyme for Microcystins and Nodularin on First Step Biodegradation in Microcystin-Degrading Bacteria.

Microcystin-degrading bacteria first degrade microcystins by microcystinase A (MlrA) to cleave the cyclic structure of microcystins at the Adda-Arg site of microcystin-LR, microcystin-RR, and microcystin-YR, but the cleavage of the other microcystins was not clear. In our study, the microcystin-degrading bacterium Sphingopyxis sp. C-1 as wild type and that of mlrA-disrupting mutant, Sphingopyxis sp. CMS01 were used for microcystins biodegradation. The results showed MlrA degraded microcystin-LA, microcystin-LW, microcystin-LY, microcystin-LF, and nodularin. MlrA could cleave the Adda-L-amino acid site.

A device for assessing microbial activity under ambient hydrostatic pressure: The in situ microbial incubator (ISMI).

Microbes in the dark ocean are exposed to hydrostatic pressure increasing with depth. Activity rate measurements and biomass production of dark ocean microbes are, however, almost exclusively performed under atmospheric pressure conditions due to technical constraints of sampling equipment maintaining in situ pressure conditions. To evaluate the microbial activity under in situ hydrostatic pressure, we designed and thoroughly tested an in situ microbial incubator (ISMI). The ISMI allows autonomously collecting and incubating seawater at depth, injection of substrate and fixation of the samples after a preprogramed incubation time. The performance of the ISMI was tested in a high-pressure tank and in several field campaigns under ambient hydrostatic pressure by measuring prokaryotic bulk 3H-leucine incorporation rates. Overall, prokaryotic leucine incorporation rates were lower at in situ pressure conditions than under to depressurized conditions reaching only about 50% of the heterotrophic microbial activity measured under depressurized conditions in bathypelagic waters in the North Atlantic Ocean off the northwestern Iberian Peninsula. Our results show that the ISMI is a valuable tool to reliably determine the metabolic activity of deep-sea microbes at in situ hydrostatic pressure conditions. Hence, we advocate that deep-sea biogeochemical and microbial rate measurements should be performed under in situ pressure conditions to obtain a more realistic view on deep-sea biotic processes.

Limited carbon cycling due to high-pressure effects on the deep-sea microbiome.

Deep-sea microbial communities are exposed to high-pressure conditions, which has a variable impact on prokaryotes depending on whether they are piezophilic (that is, pressure-loving), piezotolerant or piezosensitive. While it has been suggested that elevated pressures lead to higher community-level metabolic rates, the response of these deep-sea microbial communities to the high-pressure conditions of the deep sea is poorly understood. Based on microbial activity measurements in the major oceanic basins using an in situ microbial incubator, we show that the bulk heterotrophic activity of prokaryotic communities becomes increasingly inhibited at higher hydrostatic pressure. At 4,000 m depth, the bulk heterotrophic prokaryotic activity under in situ hydrostatic pressure was about one-third of that measured in the same community at atmospheric pressure conditions. In the bathypelagic zone-between 1,000 and 4,000 m depth-~85% of the prokaryotic community was piezotolerant and ~5% of the prokaryotic community was piezophilic. Despite piezosensitive-like prokaryotes comprising only ~10% (mainly members of Bacteroidetes, Alteromonas) of the deep-sea prokaryotic community, the more than 100-fold metabolic activity increase of these piezosensitive prokaryotes upon depressurization leads to high apparent bulk metabolic activity. Overall, the heterotrophic prokaryotic activity in the deep sea is likely to be substantially lower than hitherto assumed, with major impacts on the oceanic carbon cycling.

Draft Genome Sequences of Multidrug-Resistant Escherichia coli Strains Isolated from River Water in Malaysia.

Antimicrobial resistance has become a primary concern in clinical and public health. Escherichia coli is one of the bacteria that carries and disseminates antimicrobial resistance genes to the community. Here, we report the draft genome sequence of three multidrug-resistant E. coli strains that were isolated from river water in Malaysia.

Temperature affects growth, geosmin/2-methylisoborneol production, and gene expression in two cyanobacterial species.

Cyanobacterial blooms accompanied by taste and odor (T&O) compounds affect the recreational function and safe use of drinking water. Geosmin and 2-methylisoborneol (2-MIB) are the most common T&O compounds. In this study, we investigated the effect of temperature on geosmin and 2-MIB production in Dolichospermum smithii and Pseudanabaena foetida var. intermedia. More specifically, transcription of one geosmin synthase gene (geoA) and two 2-MIB synthase genes (mtf and mtc) was explored. Of the three temperatures (15, 25, and 35 °C) tested, the maximum Chl-a content was determined at 25 °C in both D. smithii and P. foetida var. intermedia. The maximum total geosmin concentration (19.82 µg/L) produced by D. smithii was detected at 25 °C. The total 2-MIB concentration (82.5 µg/L) produced by P. foetida var. intermedia was the highest at 35 °C. Besides, the lowest Chl-a content and minimum geosmin/2-MIB concentration were observed at 15 °C. There was a good positive correlation between geosmin/2-MIB concentration and Chl-a content. The expression levels of the geoA, mtf, and mtc genes at 15 °C were significantly higher than those at 25 and 35 °C. The transcription of the mtf and mtc genes in P. foetida var. intermedia was higher at 35 °C than at 25 °C. The results highlight unfavorable temperature can increase the potential of geosmin/2-MIB synthesis from the gene expression level in cyanobacteria. This study could provide basic knowledge of geosmin/2-MIB production by cyanobacteria for better understanding and management of T&O problems in drinking water.

Dynamics of the prokaryotic and eukaryotic microbial community during a cyanobacterial bloom.

Toxic cyanobacterial blooms frequently develop in eutrophic freshwater bodies worldwide. Microcystis species produce microcystins (MCs) as a cyanotoxin. Certain bacteria that harbor the mlr gene cluster, especially mlrA, are capable of degrading MCs. However, MC-degrading bacteria may possess or lack mlr genes (mlr+ and mlr- genotypes, respectively). In this study, we investigated the genotype that predominantly contributes to biodegradation and cyanobacterial predator community structure with change in total MC concentration in an aquatic environment. The 2 genotypes coexisted but mlr+ predominated, as indicated by the negative correlation between mlrA gene copy abundance and total MC concentration. At the highest MC concentrations, predation pressure by Phyllopoda, Copepoda, and Monogononta (rotifers) was reduced; thus, MCs may be toxic to cyanobacterial predators. The results suggest that cooperation between MC-degrading bacteria and predators may reduce Microcystis abundance and MC concentration.

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.

Effects of elevated nitrogen on the growth and geosmin productivity of Dolichospermum smithii.

Geosmin is one of the most common earthy-musty odor compounds, which is mainly produced by cyanobacteria in surface water. Nitrogen (N) is an important factor affecting the growth of cyanobacteria and its secondary metabolites production due to the eutrophication. In this study, we compared the effects of elevated N on the growth and geosmin productivity of Dolichospermum smithii NIES-824 (synonym Anabaena smithii NIES-824), aiming to better understand the mechanisms involved and give an important and fundamental knowledge to solve off-flavor problem. Results show that elevated N concentration promoted more chlorophyll a (Chl-a) production, whereas the geosmin synthesis decreased, revealing a possible competitive correlation between the Chl-a concentration and geosmin production of D. smithii NIES-824. The majority of geosmin (> 90%) was retained intracellularly during the 28 days of cultivation. The qRT-PCR analysis demonstrates that the expression level of the geosmin synthase gene (geoA) was constitutive and decreased at the higher N concentration during the exponential growth phase of cyanobacterial cells. Furthermore, the decrease of geoA expression during the decline phase suggested that geoA transcription was closely related to cell activity and isoprenoid productivity.

Toxic cyanobacteria and microcystin dynamics in a tropical reservoir: assessing the influence of environmental variables.

Toxic cyanobacterial blooms (TCBs) have become a growing concern worldwide. The present study investigated the dynamic of toxic cyanobacteria and microcystin (MC) concentrations in the Tri An Reservoir (TAR), a tropical system in Vietnam, with quantitative real-time polymerase chain reaction (qPCR) and high-performance liquid chromatography (HPLC), respectively. The results of the qPCR quantification revealed that Microcystis was the dominant group and the primary MC producer in the TAR. Potentially toxigenic cyanobacteria varied from 1.2 × 104 to 1.58 × 107 cells/mL, and the mean proportion of toxic Microcystis to that of the total toxic cyanobacteria varied from 21 to 88%. Microcystin concentrations in raw water and sediment samples often peaked during June to October as blooms occurred and varied from 0.27 to 6.59 µg/L and from 1.79 to 544.9 ng/g in wet weight, respectively. The results of this study indicated that conditions favoring Microcystis proliferation lead to the selection of more toxic genotypes. Water temperature and light availability were not driving factor in the formation of TCBs in the TAR. However, the high loads of total nitrogen (TN), phosphate, and total phosphorus (TP) into the water via rainfall runoff in combination with a high total suspended solid (TSS) and decreased water level during the early months of the rainy seasons did lead to a shift in Microcystis blooms and higher proportions of toxic genotypes of Microcystis in the TAR. This research may provide more insight into the occurrence mechanism of TCBs in tropical waters. The strategy to control TCB problems in tropical regions should be focused on these limnological and hydrological parameters, in addition to a reduction in nitrogen and phosphorus loading.

Coupling biogas recirculation with FeCl3 addition in anaerobic digestion system for simultaneous biogas upgrading, phosphorus conservation and sludge conditioning.

The high costs involved in sewage sludge treatment and disposal in wastewater treatment plants (WWTPs) not only bring about improper sludge disposal and thus environmental pollutions, but also limit the investment on construction of WWTPs, especially in rural areas or low-income regions. This comparative study examined the effect of biogas recirculation coupled with chemical addition in a semi-continuous anaerobic digester for sludge treatment, which was proven to achieve biogas upgrading, phosphorus conservation and sludge conditioning simultaneously, largely reducing the sludge treatment cost. Results show that FeCl3 addition coupling biogas recirculation can improve sludge dewaterability by 94% in comparison to 75% by equivalent MgCl2 addition, and 97% phosphorus in digestate can be conserved in solid with formation of vivianite-like crystals. Biogas recirculation can enhance CH4 yield and content by 13% and 11%, respectively, likely attributable to the increased relative abundances of both hydrogenotrophic Methanomicrobiales and acetoclastic Methanosarcinales.

Draft Genome Sequence of the Microcystin-Degrading Bacterium Novosphingobium sp. Strain MD-1.

This report describes the whole-genome sequence of a microcystin-degrading bacterium, Novosphingobium sp. strain MD-1, isolated from a lake in Japan. The Novosphingobium sp. strain MD-1 genome had a total length of 4,617,766 bp. Moreover, strain MD-1 showed a conserved microcystin-degrading gene cluster (mlrA to mlrF), similar to Sphingopyxis sp. strain C-1.

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.

Effects of levofloxacin exposure on sequencing batch reactor (SBR) behavior and microbial community changes.

The adaptation mechanisms of bacterial community for nitrogen removal performance exposed to fluctuated levels of levofloxacin (LVX) during wastewater treatment in SBRs were investigated. Although LVX is completely synthetic, the results of minimum inhibitory concentration (MIC, 32 mg-LVX/L) and minimum bactericidal concentration (MBC, 512 mg-LVX/L) of the sampled sludge showed that the LVX resistance/tolerance for bacterial growth has already existed in the actual wastewater treatment plants (WWTPs). The key bacteria, i.e. Nitrosomonas sp. (ammonia-oxidizing bacteria), Nitrospira sp. (nitrite-oxidizing bacteria) and Thauera sp. (the predominant denitrifiers), decreased with LVX exposure, and the recovery of biological process in the reactor was disturbed due to LVX exposure. However, after stopping exposure their population was quickly increased and thus the performance was recovered. The results of the non-metric multidimensional scaling and microbial community by sequencing showed the LVX concentration was a crucial factor to the change of bacterial communities and controlled the quantitative evolution of the communities in our systems. This effect was more pronounced as the LVX concentration was higher. The results suggested the removal of residual antibiotics to accomplish under no effect concentration before biological treatment is important to suppress emerging and increasing of the antibiotic resistant bacteria in WWTPs.

Response of microcystin biosynthesis and its biosynthesis gene cluster transcription in Microcystis aeruginosa on electrochemical oxidation.

Microcystin-LR (MC-LR), which is one of the most commonly found microcystins (MCs) in fresh water, has been proved to be a potential tumour promoter and classified as 2B by the International Agency for Research on Cancer. MC-LR decomposition and inhibition of MC-LR production in Microcystis aeruginosa were investigated under electrolysis condition using an electrolysis cell consisting of Ti/Pt electrodes and Nafion membrane. The relationship between the decrease in MC-LR concentration and transcription of MC-LR synthesis gene clusters was determined by performing real-time reverse transcription polymerase chain reaction (RT-qPCR) to monitor changes in the levels of transcription encoding mcyB and mcyD (cDNA to DNA) in M. aeruginosa NIES 1086 under electrolysis condition and three different conditions (i.e. oxygenated, air aerated and unaerated) as controls. Cell density decreased from day 2 under electrolysis than under the three controls. Intracellular MC-LR concentration was approximately 33 fg cell-1 under electrolysis from days 4 to 8, while those in the other conditions ranged in 40-50 fg cell-1. The mcyB transcription continuously decreased from day 2 to nondetectable level in day 6 under electrolysis, while this transcription was stabilised under the three controls. This result suggested that oxidative stress, such as hydroxyl radicals, played an important role in the down-regulation of mcyB and mcyD gene transcription level and the MC-LR concentration and cell density of M. aeruginosa.

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.

Response of algal-bacterial granular system to low carbon wastewater: Focus on granular stability, nutrients removal and accumulation.

The effect of influent chemical oxygen demand to nitrogen (COD/N) ratio on the granular stability, nutrients removal and accumulation of the algal-bacterial AGS was investigated. Two sequencing batch reactors were operated under different influent COD/N ratio, i.e., R1 (control, COD/N = 8) and R2: (COD/N = 8, 4, 2, and 1 through stepwise decrease of COD). Results showed that the integrity coefficient of the granules in R2 stabilized at 0.7-5.4% during the whole operation. Significantly enhanced dissolved inorganic carbon (DIC) uptake and the faster growth of algae indicated the great potential for reduction in greenhouse gases (GHGs) emission by using the algal-bacterial AGS system. The algal-bacterial AGS biomass contained high phosphorus (P) and N contents as well as extremely high P bioavailability (up to 98%) which could be easily used for resource recovery. Loosely bound extracellular polymeric substances (LB-EPS) might be the key factor to control the deterioration of granular stability in this system.

Fertilizer potential of liquid product from hydrothermal treatment of swine manure.

Compared with composting, hydrothermal treatment (HTT) technology can dramatically shorten the duration for manure waste treatment. This study firstly investigated the effect of HTT on solubilization of N, P and organics from swine manure, and then evaluated the phytotoxicity of liquid product from hydrothermally treated manure by seed germination test. Results show that 98% of N in manure could be converted into soluble form after HTT at 200 °C for 60 min. Soluble P in hydrothermally treated manure (at 150 °C for 60 min) was 2.7 times that in raw manure. The germination indices (GI) were all greater than 100% when the liquid product (from HTT at 150 °C for 60 min) or its diluted samples being used. Results from this study suggest that HTT could be a promising technology for producing safe and value-added liquid fertilizers from swine manure.

An overview of the accumulation of microcystins in aquatic ecosystems.

Cyanotoxins produced by toxic cyanobacteria pose a major, worldwide environmental threat to freshwater ecosystems. Microcystins (MCs) are considered to be the most hazardous groups. Indeed, some of the largest aquatic ecosystems on the earth are being contaminated with MCs. Questions have arisen regarding their transfer and bioaccumulation in natural environment. This review summarizes the present state of knowledge regarding toxic cyanobacteria and MCs, with a specific focus on their distribution in different components of aquatic ecosystems. Their accumulation in water columns, aquatic animals, plants, and sediments is summarized. MCs have been contaminating all areas of the aquatic ecosystems. Of these, the water column was the most contaminated with MCs and served as an intermediate transmission substation. Via this route, MCs could enter to other stations such as sediment, animals, aquatic and terrestrial plants. Therefore, the use of water contaminated with MCs may induce food chain contaminations with considerable health risks.