Methodological Considerations and Comparisons of Measurement Results for Extracellular Proteolytic Enzyme Activities in Seawater

Microbial extracellular hydrolytic enzymes that degrade organic matter in aquatic ecosystems play key roles in the biogeochemical carbon cycle. To provide linkages between hydrolytic enzyme activities and genomic or metabolomic studies in aquatic environments, reliable measurements are required for many samples at one time. Extracellular proteases are one of the most important classes of enzymes in aquatic microbial ecosystems, and protease activities in seawater are commonly measured using fluorogenic model substrates. Here, we examined several concerns for measurements of extracellular protease activities (aminopeptidases, and trypsin-type, and chymotrypsin-type activities) in seawater. Using a fluorometric microplate reader with low protein binding, 96-well microplates produced reliable enzymatic activity readings, while use of regular polystyrene microplates produced readings that showed significant underestimation, especially for trypsin-type proteases. From the results of kinetic experiments, this underestimation was thought to be attributable to the adsorption of both enzymes and substrates onto the microplate. We also examined solvent type and concentration in the working solution of oligopeptide-analog fluorogenic substrates using dimethyl sulfoxide (DMSO) and 2-methoxyethanol (MTXE). The results showed that both 2% (final concentration of solvent in the mixture of seawater sample and substrate working solution) DMSO and 2% MTXE provide similarly reliable data for most of the tested substrates, except for some substrates which did not dissolve completely in these assay conditions. Sample containers are also important to maintain the level of enzyme activity in natural seawater samples. In a small polypropylene containers (e.g., standard 50-mL centrifugal tube), protease activities in seawater sample rapidly decreased, and it caused underestimation of natural activities, especially for trypsin-type and chymotrypsin-type proteases. In conclusion, the materials and method for measurements should be carefully selected in order to accurately determine the activities of microbial extracellular hydrolytic enzymes in aquatic ecosystems; especially, low protein binding materials should be chosen to use at overall processes of the measurement

Characterization of aerobic granular sludge developed under variable and low organic loading rate

Understanding the formation of aerobic granules sludge (AGS) under the variations of organic loading rate (OLR) could give a different insight on AGS stability, which had become the bottleneck for practical application in sewage treatment. This study demonstrates the formation of AGS that had previously been stored for eight months at 5ºC in sequencing batch reactor (SBR) with sewage as substrate. Despite being redeveloped under variable OLR of 0.26 to 0.81 kg CODs/m3 d and low superficial air velocity (SAV) of 1.33 cm/s, the loose structure of AGS during storage can be recovered within 46 days of formation process. Variations in OLR intrude the formation process particularly during low OLR, resulting in longer period to achieve mature AGS or full granulation of biomass in reactor. The next-generation sequencing (NGS) analysis indicated that the shift in microbial community from Rhodocyclaceae to Comamonadaceae class for denitrification process was accommodated with the changes in the AGS size from 326 μm to more than 600 μm

Seasonal variability of anthropogenic indices of PAHs in sediment from the Kuala Selangor River, west coast Peninsular Malaysia

Rapid increase in industrialization and urbanization in the west coast of Peninsular Malaysia has led to the intense release of petroleum and products of petroleum into the environment. Surface sediment samples were collected from the Selangor River in the west coast of Peninsular Malaysia during four climatic seasons and analyzed for PAHs and biomarkers (hopanes). Sediments were soxhlet extracted and further purified and fractionated through first and second step column chromatography. A gas chromatography–mass spectrometry (GC–MS) was used for analysis of PAHs and hopanes fractions. The average concentrations of total PAHs ranged from 219.7 to 672.3 ng g−1 dw. The highest concentrations of PAHs were detected at 964.7 ng g−1 dw in station S5 in the mouth of the Selangor River during the wet inter-monsoonal season. Both pyrogenic and petrogenic PAHs were detected in the sediments with a predominance of the former. The composition of hopanes was homogeneous showing that petroleum hydrocarbons share an identical source in the study area. Diagnostic ratios of hopanes indicated that some of the sediment samples carry the crankcase oil signature

Aliphatic hydrocarbons and triterpane biomarkers in mangrove oyster ( Crassostrea belcheri ) from the west coast of Peninsular Malaysia

The Straits of Malacca is one of the world's busiest shipping routes where frequent oil spills occur. Rapid development in the west coast of Peninsular Malaysia is the other major source of petroleum pollution in this narrow waterway. In order to identify occurrence and origin of hydrocarbons in the Straits, mangrove oysters (Crassostrea belcheri) were collected from five sampling locations and analysed for n-alkanes and biomarkers. Soxhlet apparatus and two step column chromatography were used for extraction, purification and fractionation of the oysters. Petroleum origin n-alkanes were detected in majority of the sampling locations which is indicative of anthropogenic activities in this region. Using source and maturity diagnostic ratios for hopanes revealed used crankcase oil as the main source of petroleum hydrocarbons in oysters from all sampling locations except for the Pulau Merambong where signature of South East Asia crude oil (SEACO) was detected

Temporal Variation of Bacterial Respiration and Growth Efficiency in Tropical Coastal Waters ▿

We investigated the temporal variation of bacterial production, respiration, and growth efficiency in the tropical coastal waters of Peninsular Malaysia. We selected five stations including two estuaries and three coastal water stations. The temperature was relatively stable (averaging around 29.5°C), whereas salinity was more variable in the estuaries. We also measured dissolved organic carbon and nitrogen (DOC and DON, respectively) concentrations. DOC generally ranged from 100 to 900 μM, whereas DON ranged from 0 to 32 μM. Bacterial respiration ranged from 0.5 to 3.2 μM O2 h−1, whereas bacterial production ranged from 0.05 to 0.51 μM C h−1. Bacterial growth efficiency was calculated as bacterial production/(bacterial production + respiration), and ranged from 0.02 to 0.40. Multiple correlation analyses revealed that bacterial production was dependent upon primary production (r2 = 0.169, df = 31, and P < 0.02) whereas bacterial respiration was dependent upon both substrate quality (i.e., DOC/DON ratio) (r2 = 0.137, df = 32, and P = 0.03) and temperature (r2 = 0.113, df = 36, and P = 0.04). Substrate quality was the most important factor (r2 = 0.119, df = 33, and P = 0.04) for the regulation of bacterial growth efficiency. Using bacterial growth efficiency values, the average bacterial carbon demand calculated was from 5.30 to 11.28 μM C h−1. When the bacterial carbon demand was compared with primary productivity, we found that net heterotrophy was established at only two stations. The ratio of bacterial carbon demand to net primary production correlated significantly with bacterial growth efficiency (r2 = 0.341, df = 35, and P < 0.001). From nonlinear regression analysis, we found that net heterotrophy was established when bacterial growth efficiency was <0.08. Our study showed the extent of net heterotrophy in these waters and illustrated the importance of heterotrophic microbial processes in coastal aquatic food webs

Long-term comparison of dissolved nitrogen species in tropical estuarine and coastal water systems

Long-term observation of aquatic habitat is invaluable as it reveals trends that are not evident from shorter-term measurements. In this study, we assessed measurements of dissolved nitrogen species over a 12-year period (2004-2015) at Port Klang estuary and Port Dickson coastal water. Total suspended solids (TSS) was also measured, and increased at 6-9 mg L-1 yr(-1). In contrast, total dissolved nitrogen (TDN) did not increase but was higher at Port Klang (8-320 mu M) relative to Port Dickson (2-30 mu M). Chl a concentration was also higher at Port Klang (5.18 +/- 7.79 mu g L-1) than at Port Dickson (2.27 +/- 1.39 mu g L-1). Among the dissolved nitrogen species, we found that ammonium (NH4) was predominant at Port Klang where dissolved inorganic nitrogen (DIN) contributed 71 +/- 3% of TDN. At Port Dickson, dissolved organic nitrogen (DON) predominated, contributing 92 +/- 7% of TDN. When we calculated the nitrogen loading at Port Klang, dissolved nitrogen load increased over time (1.13 +/- 0.29 kg d(-1) for TDN, 1.11 +/- 0.29 kg d(-1) for DIN, and 0.15 +/- 0.06 kg d(-1) for DON). We showed that the nitrogen loading increase at Port Klang was not due to climatic forcing but was most probably due to urbanization occurring upstream that inevitably increased river discharge rates (4.04 +/- 0.12 m(3) s(-1) yr(-1))

Analysis of Water Treatment by Moringa oleifera Bioflocculant Prepared Via Supercritical Fluid Extraction

In this study, we used supercritical fluid extraction to prepare Moringa oleifera as a bioflocculant and studied its efficacy in water treatment. Relative to the conventional solvent extraction method, supercritical fluid extraction produced 42% more bioflocculant and was a more efficient method with both time (about seven hours less) and cost savings (about USD30 less). The bioflocculant produced via supercritical fluid extraction was also smaller (18±5 µm) and more similar in size (Coefficient of Variation, CV = 28%) as opposed to conventional solvent extraction (23±8 µm, CV = 35%). It was able to reduce more than 95% of turbidity and up to 60% bacterial population. Its performance in reducing selected heavy metals from water samples was also generally better than aluminium sulfate or alum. Our study showed that with the exception of cost restrictions, M. oleifera bioflocculant produced via supercritical fluid extraction has the potential to replace alum in water treatment plants

Influence of Precursor Concentration and Temperature on the Formation of Nanosilver in Chemical Reduction Method

Nanosilver particles (NSPs) were produced by the reduction of silver nitrate using glucose as reducer, poly (vinyl pyrrolidone) as stabilizer and sodium hydroxide as reaction enhancer. Two parameters were investigated which are silver nitrate concentration (0.1 M, 0.5 M and 1.0 M) and reaction temperature (60°C and 80°C). Through spectral analysis using ultraviolet-visible spectrophotometer (UV-vis), all the samples recorded the maximum peak in the range of 384-411 nm which verified the formation of NSPs. TEM images showed the nanoparticles have spherical shape with the size range of 25-39 nm. Particle size and zeta potential analysis recorded the hydrodynamic size of nanoparticles in the range of 85-105 nm and the zeta potential ranging from -25 to -30 mV, under the pH value of 8. X-ray diffraction analysis showed that the NSPs have face center cubic (FCC) structure. All the produced NSPs surprisingly showed ferromagnetic-like behaviour based on the magnetization curves. FTIR result confirmed the presence of poly (vinyl pyrrolidone) on the NSPs surface. Furthermore, at the reaction temperature 60°C, the crystallite size, physical size as well as hydrodynamic size increased as the precursor concentration increased from 0.1 M to 0.5 M. However, as the precursor concentration further increases to 1.0 M, the size become smaller due to incomplete reduction process. In contrast, at 80°C, the sizes was gradually increased as the precursor concentration increases up to 1.0 M. In terms of controlled precursor concentration, the crystallite size and physical size become smaller as the temperature increases