Environmental microbes utilize four degradation pathways for the oxidation of n-alkanes. Although the enzymes degrading n-alkanes in different microbes may vary, enzymes functioning in the first step in the aerobic degradation of alkanes all belong to the alkane hydroxylases. Alkane hydroxylases are a class of enzymes that insert oxygen atoms derived from molecular oxygen into different sites of the alkane terminus (or termini) depending on the type of enzymes. In this review, we summarize the different types of alkane hydroxylases, their degrading steps, and compare typical enzymes from various classes with regard to their three-dimensional structures, in order to provide insights into how the enzymes mediate their different roles in the degradation of n-alkanes and what determines their different substrate ranges. Through the above analyzes, the degrading mechanisms of enzymes can be elucidated and molecular biological methods can be utilized to expand their catalytic roles in the petrochemical industry or in bioremediation of oil-contaminated environments.

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Rapid contamination of drinking water in distribution and storage systems can occur due to pressure drop, backflow, cross-connections, accidents, and bio-terrorism. Small volumes of a concentrated contaminant (e.g., wastewater) can contaminate large volumes of water in a very short time with potentially severe negative health impacts. The technical limitations of conventional, cultivation-based microbial detection methods neither allow for timely detection of such contaminations, nor for the real-time monitoring of subsequent emergency remediation measures (e.g., shock-chlorination). Here we applied a newly developed continuous, ultra high-frequency flow cytometry approach to track a rapid pollution event and subsequent disinfection of drinking water in an 80-min laboratory scale simulation. We quantified total (TCC) and intact (ICC) cell concentrations as well as flow cytometric fingerprints in parallel in real-time with two different staining methods. The ingress of wastewater was detectable almost immediately (i.e., after 0.6% volume change), significantly changing TCC, ICC, and the flow cytometric fingerprint. Shock chlorination was rapid and detected in real time, causing membrane damage in the vast majority of bacteria (i.e., drop of ICC from more than 380 cells μl-1 to less than 30 cells μl-1 within 4 min). Both of these effects as well as the final wash-in of fresh tap water followed calculated predictions well. Detailed and highly quantitative tracking of microbial dynamics at very short time scales and for different characteristics (e.g., concentration, membrane integrity) is feasible. This opens up multiple possibilities for targeted investigation of a myriad of bacterial short-term dynamics (e.g., disinfection, growth, detachment, operational changes) both in laboratory-scale research and full-scale system investigations in practice.

As microbiological parameters are important in monitoring the correct operation of wastewater treatment plants and controlling the microbiological quality of wastewater, the abundances of total bacteria (including intact and damaged bacterial cells) and total viruses in wastewater were investigated using a combination of ultrasonication and flow cytometry. The comparisons between flow cytometry (FCM) and other cultivation-independent methods (adenosine tri-phosphate (ATP) analysis for bacteria enumeration and epifluorescence microscopy (EFM) for virus enumeration) gave very similar patterns of microbial abundance changes, suggesting that FCM is suitable for targeting and obtaining reliable counts for bacteria and viruses in wastewater samples. The main experimental results obtained were: (1) effective removal of total bacteria in wastewater, with a decrease from an average concentration of 1.74 × 10(8)counts ml(-1) in raw wastewater to 3.91 × 10(6)counts ml(-1) in the effluent, (2) compared to influent raw wastewater, the average concentration of total viruses in the treated effluent (3.94 × 10(8)counts ml(-1)) exhibited no obvious changes, (3) the applied FCM approach is a rapid, easy, and convenient tool for understanding the microbial dynamics and monitoring microbiological quality in wastewater treatment processes.

To date, few aquatic microbial ecology studies have discussed the variability of the microbial community in exorheic river ecosystems on both the spatial and seasonal scales. In this study, we examined the spatio-temporal variation of bacterioplankton community composition in an anthropogenically influenced exorheic river, the Haihe River in Tianjin, China, using pyrosequencing analysis of 16S rRNA genes. It was verified by one-way ANOVA that the spatial variability of the bacterioplankton community composition over the whole river was stronger than the seasonal variation. Salinity was a major factor leading to spatial differentiation of the microbial community structure into riverine and estuarial parts. A high temperature influence on the seasonal bacterial community variation was only apparent within certain kinds of environments (e.g., the riverine part). Bacterial community richness and diversity both exhibited significant spatial changes, and their seasonal variations were completely different in the two environments studied here. Furthermore, riverine bacterial community assemblages were subdivided into urban and rural groups due to changes in the nutritional state of the river. In addition, the nutrient-loving group including Limnohabitans, Hydrogenophaga, and Polynucleobacter were abundant in the urbanized Haihe River, indicating the environmental factors in these anthropogenic waterbodies heavily influence the core freshwater community composition.

Residual chlorine is often required to remain present in public drinking water supplies during distribution to ensure water quality. It is essential to understand how bacteria respond to long-term chlorine exposure, especially with the presence of assimilable organic carbon (AOC). This study aimed to investigate the effects of chlorination on Pseudomonas aeruginosa in low AOC medium by both conventional plating and culture-independent methods including flow cytometry (FCM) and quantitative PCR (qPCR). In a simulated chlorinated system using a bioreactor, membrane damage and DNA damage were measured by FCM fluorescence fingerprint. The results indicated membrane permeability occurred prior to DNA damage in response to chlorination. A regrowth of P. aeruginosa was observed when the free chlorine concentration was below 0.3 mg/L. The bacterial response to long-term exposure to a constant low level of free chlorine (0.3 mg/L) was subsequently studied in detail. Both FCM and qPCR data showed a substantial reduction during initial exposure (0–16 h), followed by a plateau where the cell concentration remained stable (16–76 h), until finally all bacteria were inactivated with subsequent continuous chlorine exposure (76–124 h). The results showed three-stage inactivation kinetics for P. aeruginosa at a low chlorine level with extended exposure time: an initial fast inactivation stage, a relatively stable middle stage, and a final stage with a slower rate than the initial stage. A series of antibiotic resistance tests suggested long-term exposure to low chlorine level led to the selection of antibiotic-resistant P. aeruginosa. The combined results suggest that depletion of residual chlorine in low AOC medium systems could reactivate P. aeruginosa, leading to a possible threat to drinking water safety.

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