Mesophilic and thermophilic fermentation of activated sludge for volatile fatty acids production: focusing on anaerobic degradation of carbohydrate and protein.

The volatile fatty acids (VFAs) productions, as well as particulate organics decomposition, soluble chemical oxygen demand (SCOD) yield, and the VFAs production pathways from mesophilic and thermophilic anaerobic fermentation in waste activated sludge were investigated. Batch experiments showed that the decomposition rate of volatile suspended solids (VSS), particulate carbohydrate (P-C) and particulate protein (P-P) followed the first-order kinetic model at different temperatures. However, the intermediates, accumulated in the process of protein or carbohydrate digestion had a more significant inhibitory effect on the production of VFAs during the mesophilic anaerobic acidification process. The production of VFAs by thermophilic anaerobic fermentation is 2086.05 mg COD/L, which is about twice the production under mesophilic conditions. Among them, the concentration and proportion of high molecular weight organic acids such as isobutyric acid (320.29 mgCOD/L) and isovaleric acid (745.75 mgCOD/L) are relatively high. Then 13C stable isotope labelling experiment demonstrated that, the decomposition of carbohydrates yields 77% acetic acid and 86% butyric acid, while protein breakdown produces 85% propionic acid and 99% valeric acid. This confirms that carbohydrates are more favourable for the formation of even-carbon organic acids, while proteins tend to yield odd-carbon organic acids. Additionally, this helps refine the pathway for valeric acid formation during anaerobic acidogenesis.

ELISA-Based Method for Variant-Independent Detection of Total Microcystins and Nodularins via a Multi-immunogen Approach.

Required routine monitoring of microcystins (MCs) and nodularins (NODs) in water samples, as posed by U.S. EPA Unregulated Contaminant Monitoring Rule 4, demands cost-effective, reliable, and sensitive detection methods. To target as many MC and NOD variants as possible, we developed an indirect competitive enzyme-linked immunosorbent assay (ELISA) with group-specific monoclonal antibodies for variant-independent detection of total MCs and NODs. In this ELISA method, the mice monoclonal antibodies presenting both high affinities and broad-spectrum recognition capabilities against MCs and NODs were self-produced by designing MC hapten-based multi-immunogens to minimize specificity for the particular variant. Their high affinities and variant-independent binding capabilities against MCs and NODs were validated by both wet lab and in silico methods. The developed ELISA method achieved a limit of detection of below 0.3 µg/L for 13 MC/NOD variants, well with the reported best cross-reactivities of 60-127% relative to MC-LR. As a case study, this ELISA method was used to map the variations of intracellular and extracellular total MCs/NODs in the Luoma Lake drinking water source, China, in July, 2020. Its capability to measure total MCs/NODs with high sensitivity and high throughput in a simple and affordable way would truly be a disruptive technology capable of changing our understanding of bloom/toxin dynamics and having obvious implications for monitoring efforts.

Light-sheet skew rays enhanced U-shaped fiber-optic fluorescent immunosensor for Microcystin-LR.

A novel U-shaped fiber-optic evanescent-wave fluorescent immunosensor was designed that exploits light-sheet excitation of skew rays in a passive fiber for sensitive microcystin-LR (MC-LR) detection in real-time. In particular, a light sheet comprising a thin plane of light can be concentrated into exciting the optimum ray group, resulting in enhanced interaction between light and fluorophores. Meanwhile, skew rays excited by transmitting light into an optical fiber with an angle offset allow a much higher number of total-internal-reflections with increased interaction length along the fiber interface, which strengthens the light-matter interactions. Under the optimal angle offset, the proposed evanescent wave fluorescent immunosensor is the first demonstration of integrating light-sheet skew rays and a U-shaped fiber-optic probe for enhanced sensitivity. The results show that fluorescence sensitivity of the U-shaped fiber-optic probe with light-sheet skew rays excitation is 16 times higher than that of collimated skew rays excitation. Combined with this newly designed light-sheet skew rays enhanced U-shaped fiber-optic fluorescent immunosensor, a sensitive and real-time MC-LR detection method was established based on the indirect competitive immunoassay principle. Real environmental water samples spiked with MC-LR were determined by the immunosensor with recovery rates between 85% and 112%. The present system could be an alternative tool for the on-site environmental monitoring, in-field food safety assurance and clinical diagnostics. It also advances the fiber-optic sensors field in terms of experimental design.

Unconventional Split Aptamers Cleaved at Functionally Essential Sites Preserve Biorecognition Capability.

Split aptamers (SPAs) are a pair of oligonucleotide fragments generated by cleaving a long parent aptamer. SPAs have many compelling advantages over the parent aptamer such as sandwich target binding, optimized concise structure, and low cost. However, only a limited number of SPAs have been developed so far because the traditional theory restricts the splitting to the functionally dispensable site that many parent aptamers do not possess. In this work, the traditional mechanism and hypothesis that SPAs can also be generated by splitting the parent aptamer at the functionally essential site while still preserving the biorecognition capability are challenged. To prove the hypothesis, three SPAs with Broken initial small-molecule binding Pockets (BPSPAs) are discovered and their binding capabilities are validated both in the wet lab and in silico. An allosteric binding mechanism of BPSPAs, in which a new binding pocket is formed upon the target binding, is revealed by all-atom microsecond-scale molecular dynamics simulations. Our work highlights the important role of MD simulations in predicting the ligand binding potency with functional nucleic acids at the molecular level. The findings will greatly promote discovery of new SPAs and their applications in molecular sensing in many fields.

Multitag-Regulated Cascade Reaction: A Generalizable Ultrasensitive MicroRNA Biosensing Approach for Cancer Prognosis.

Ultrasensitive PCR-free microRNA (miR) analysis based on biosensors with enzyme-free nucleic acid amplification and reusable surface has great clinical significance in cancer prognosis. However, building such a biosensing strategy has long been challenging due to uncontrollable miR-triggered cascade amplifiers and insufficient sensing surface regeneration capability. To meet the challenge, for the first time, a general approach, named enzyme-free multitag-regulated cascade reaction (MCR), is developed to fabricate reliable trace miR biosensors. As a proof of concept, miR let-7a is detected on an evanescent wave fluorescent optical-fiber biosensing platform. The size and morphology of well-formed MCR assemblies (∼1 µm in length) are characterized by atomic force microscopy. This MCR method achieves a 30 000-fold improved sensitivity (detection limit 0.8 fM) compared to the MCR-free system and can detect abnormal urinary miR levels in lung cancer patients. Moreover, the biosensor is robust enough to be reused for over 100 cycles, which greatly reduces the cost of single detection. In sum, MCR is developed as a generalizable ultrasensitive miR biosensing approach for cancer prognosis, which opens a broad field for facile enzyme-free biosensing applications by nucleic acid assembling regulation.

Triple functional small-molecule-protein conjugate mediated optical biosensor for quantification of estrogenic activities in water samples.

Establishing biosensors to map a comprehensive picture of potential estrogen-active chemicals remains challenging and must be addressed. Herein, we describe an estrogen receptor (ER)-based evanescent wave fluorescent biosensor by using a triple functional small-molecule-protein conjugate as a signal probe for the determination of estrogenic activities in water samples. The signal probe, consisting of a Cy5.5-labelled streptavidin (STV) moiety and a 17ß-estradiol (E2) moiety, acts simultaneously as signal conversion, signal recognition and signal report elements. When xenoestrogens compete with the E2 moiety of conjugate in binding to the ER, the unbound conjugates are released, and their STV moiety binds with desthiobiotin (DTB) modified on the optical fiber via the STV-DTB affinity interactions. Signal probe detection is accomplished by fluorescence emission induced by an evanescent field, which positively relates with the estrogenic activities in samples. Quantification of estrogenic activity expressed as E2 equivalent concentration (EEQ) can be achieved with a detection limit of 1.05 µg/L EEQ by using three times standard deviation of the mean blank values and a linear calibration range from 20.8 to 476.7 µg/L EEQ. The optical fiber system is robust enough for hundreds of sensing cycles. The biosensor-based determination of estrogenic activities in wastewater samples obtained from a full-scale wastewater treatment plant is consistent with that measured by the two-hybrid recombinant yeast bioassay.

A photoregulated split aptaswitch for small molecules with improved sensitivity.

Here we show the design of a photoregulated split aptaswitch (PSA) for the challenging small-molecule biorecognition and its sensing application. The PSA specifically binds with the target in a binary reaction, and can easily rule out false positive signals by azobenzene photoisomerization, exhibiting an approximately 50-fold improved sensitivity over a control assay.

Nucleic acid functionalized fiber optic probes for sensing in evanescent wave: optimization and application.

Nucleic acid functionalized evanescent wave fiber optic (EWFO) biosensors have attracted much attention due to their remarkable advantages in both device configuration and sensing performance. One critical technique in EWFO biosensor fabrication is its surface modification, which requires (1) minimal nonspecific adsorption and (2) high-quality DNA immobilization to guarantee satisfactory sensing performances. Focusing on these two requirements, a series of optimizations have been conducted in this work to develop reliable DNA-functionalized EWFO probes. Firstly, the surface planeness of EWFO probes were found to be greatly improved by a novel HF/HNO3 mixture etching solution. Both atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) were conducted to investigate the morphological structures and surface chemical compositions. Besides, EWFO sensing performances adopting moderate immobilization of irrelevant DNA were investigated for optimization purposes. Furthermore, a split aptamer based sandwich-type EWFO sensor was developed using adenosine (Ade) as the model target (LOD = 25 µM). To the best of our knowledge, this study is the first case to focus on the optimization of etching solution compositions in the fabrication of combination tapered fibers, which provides experimental basis for the understanding of the silica-etching mechanism using HF/HNO3 mixture solution and may further inspire related researches.

Real-Time Study of Rapid Spread of Antibiotic Resistance Plasmid in Biofilm Using Microfluidics.

Gene transfer in biofilms is known to play an important role in antibiotic resistance dissemination. However, the process remains poorly understood. In this study, microfluidics with time-lapse imaging was used for real-time monitoring of plasmid-mediated horizontal gene transfer (HGT) in biofilms. Pseudomonas putida KT2440 harboring an antibiotic resistance plasmid RP4 was chosen as the donor while Escherichia coli and activated sludge bacteria were used as the recipient cells. Dynamic features of the transfer process, including the transfer rate, cell growth rate and kinetic changes of the transfer frequency, were determined. It was found that the routes for gene transfer strongly depend on the structure and composition of a biofilm. While intraspecies HGT is essential to initiate a transfer event, the secondary retransfer from transconjugants to the same species is more efficient and can cause cascading gene spread in single-strain biofilms. For the activated sludge biofilm, only small and scattered colonies formed and vertical gene transfer appears to be the dominant route after initial intraspecies transfer. Furthermore, more than 46% of genera in the activated sludge were permissive to plasmid RP4, many of which are associated with human pathogens. These phenomena imply early prevention and interruptions to biofilm structure could provide an effect way to inhibit rapid antibiotic resistance gene spread and reduce the likelihood of catastrophic events associated with antibiotic resistance.

TriPleX™ waveguide-based fluorescence biosensor for multichannel environmental contaminants detection.

In order to realize the multi-analyte assays for environmental contaminants, an optical biosensor utilizing laser-induced fluorescence-based detection via the binding of biomolecules to the surface of an integrated TriPleX™ waveguide chip on a glass substrate (fused silica, FS) is described. As far as we know, this is the first demonstration of using the TriPleX™ technology to fabricate the waveguide chip on a FS substrate. The sensor consists of 32 individually addressable sensor patches, which were formed on the chip surface by exploiting 3 Y-junction splitters, creating four equal rows of eight evanescently excited windows in parallel. The basic low-loss SiO2/Si3N4 TriPleX™ waveguide configuration in combination with on-chip spotsize convertors allows for both high fiber-to-chip coupling efficiency and enables at the same time individually optimized high chip surface intensity and low patch-to-patch deviation. Moreover, the complementary metal-oxide-semiconductor compatible fabrication of waveguide chip allows for its mass production at low cost. By taking MC-LR, 2,4-D, atrazine and BPA as the model analytes, the as-proposed waveguide based biosensor was proven sensitive with the detection limits of 0.22 µg/L for MC-LR, 1.18 µg/L for 2, 4-D, 0.2 µg/L for atrazine and 0.06 µg/L for BPA. Recoveries of the biosensor towards simultaneous detection of MC-LR, 2, 4-D, atrazine and BPA in spiked real water samples varied from 84% to 120%, indicating the satisfactory accuracy of the established technology.

An Optical Biosensor-Based Quantification of the Microcystin Synthetase A Gene: Early Warning of Toxic Cyanobacterial Blooming.

The monitoring and control of toxic cyanobacterial strains, which can produce microcystins, is critical to protect human and ecological health. We herein reported an optical-biosensor-based quantification of the microcystin synthetase A (mcyA) gene so as to discriminate microcystin-producing strains from nonproducing strains. In this assay, the mcyA-specific ssDNA probes were designed in silico with an on-line tool and then synthesized to be covalently immobilized on an optical-fiber surface. Production of fluorescently modified target DNA fragment amplicons was accomplished through the use of Cy5-tagged deoxycytidine triphosphates (dCTPs) in the polymerase chain reaction (PCR) method, which resulted in copies with internally labeled multiple sites per DNA molecule and delivered great sensitivity. With a facile surface-based hybridization process, the PCR amplicons were captured on the optical-fiber surface and were induced by an evanescent-wave field into fluorescence emission. Under the optimum conditions, the detection limit was found to be 10 pM (S/N ratio = 3) and equaled 103 gene copies/mL. The assay was triumphantly demonstrated for PCR amplicons of mcyA detection and showed satisfactory stability and reproducibility. Moreover, the sensing system exhibited excellent selectivity with quantitative spike recoveries from 87 to 102% for M. aeruginosa species in the mixed samples. There results confirmed that the method would serve as an accurate, cost-effective, and rapid technique for in-field testing of toxic Microcystis sp. in water, giving early information for water quality monitoring against microcystin-producing cyanobacteria.

Screening Criteria for Qualified Antibiotic Targets in Unmodified Gold Nanoparticles-Based Aptasensing.

In designing unmodified gold nanoparticles-based aptasensing (uGA) assays for antibiotics, we find that some antibiotics can adsorb directly on gold nanoparticles (GNP) regardless of the presence of aptamers, which have been long overlooked in the past. Some adsorptions, however, would strongly disturb the charge distribution on the GNP surface, break up the static colloidal profile, and thus generate false positive colorimetric signals. To identify antibiotics qualified for uGA assays, we established two rational screening criteria for antibiotic targets relying on their oil-water partition coefficients (log P values) and net physiological charges: log P > 0 and charge ≤0. A good agreement of the GNP color change was obtained between the two criteria-based predictions and the actual tests using six representative antibiotics. The proposed criteria help to shed light on GNP-target interactions, which is significant for developing novel GNP-based colorimetric assays with high reliability.

Free-Energy-Driven Lock/Open Assembly-Based Optical DNA Sensor for Cancer-Related microRNA Detection with a Shortened Time-to-Result.

Quantification of cancer biomarker microRNAs (miRs) by exquisitely designed biosensors with a short time-to-result is of great clinical significance. With immobilized capture probes (CPs) and fluorescent-labeled signal probes (SPs), surface-involved sandwich-type (SST) biosensors serve as powerful tools for rapid, highly sensitive, and selective detection of miR in complex matrices as opposed to the conventional techniques. One key challenge for such SST biosensors is the existence of false-negative signals when the amount of miRs exceeds SPs in solution phase for a surface with a limited number of CP. To meet this challenge, a dynamic lock/open DNA assembly was designed to rationally program the pathway for miR/SP hybrids. Based on secondary structure analysis and free-energy assessment, a "locker" strand that partially hybridizes with target miR by two separated short arms was designed to stabilize target miR, preventing possible false-negative signals. The strategy was demonstrated on a fiber-based fluorescent DNA-sensing platform. CP/miR/SP sandwiches formed on the fiber surface would generate fluorescent signals for quantitative analysis. The developed SST biosensor was able to detect miR Hsa let-7a with a detection limit of 24 pM. The applicability of this free-energy-driven lock/open assembly-based optical DNA sensor was further confirmed with spiked human urine and serum samples.

Isoelectric Bovine Serum Albumin: Robust Blocking Agent for Enhanced Performance in Optical-Fiber Based DNA Sensing.

Surface blocking is a well-known process for reducing unwanted nonspecific adsorption in sensor fabrication, especially important in the emerging field where DNA/RNA applied. Bovine serum albumin (BSA) is one of the most popular blocking agents with an isoelectric point at pH 4.6. Although it is widely recognized that the adsorption of a blocking agent is strongly affected by its net charge and the maximum adsorption is often observed under its isoelectric form, BSA has long been perfunctorily used for blocking merely in neutral solution, showing poor blocking performances in the optical-fiber evanescent wave (OFEW) based sensing toward DNA target. To meet this challenge, we first put forward the view that isoelectric BSA (iep-BSA) has the best blocking performance and use an OFEW sensor platform to demonstrate this concept. An optical-fiber was covalently modified with amino-DNA, and further coupled with the optical system to detect fluorophore labeled complementary DNA within the evanescent field. A dramatic improvement in the reusability of this DNA modified sensing surface was achieved with 120 stable detection cycles, which ensured accurate quantitative bioassay. As expected, the iep-BSA blocked OFEW system showed enhanced sensing performance toward target DNA with a detection limit of 125 pM. To the best of our knowledge, this is the highest number of regeneration cycles ever reported for a DNA immobilized optical-fiber surface. This study can also serve as a good reference and provide important implications for developing similar DNA-directed surface biosensors.

Integrated optical waveguide-based fluorescent immunosensor for fast and sensitive detection of microcystin-LR in lakes: Optimization and Analysis.

Nowadays, biosensor technologies which can detect various contaminants in water quickly and cost-effectively are in great demand. Herein, we report an integrated channel waveguide-based fluorescent immunosensor with the ability to detect a maximum of 32 contaminants rapidly and simultaneously. In particular, we use waveguide tapers to improve the efficiency of excitation and collection of fluorescent signals in the presence of fluorophore photobleaching in a solid surface bioassay. Under the optimized waveguide geometry, this is the first demonstration of using such a type of waveguide immunosensor for the detection of microcystin-LR (MC-LR) in lake water. The waveguide chip was activated by (3-Mercaptopropyl) trimethoxysilane/N-(4-maleimidobutyryloxy) succinimide (MTS/GMBS) for immobilization of BSA-MC-LR conjugate, which was confirmed to have uniform monolayer distribution by atomic force microscopy. All real lake samples, even those containing MC-LR in the sub-microgram per liter range (e.g. 0.5 µg/L), could be determined by the immunosensor with recovery rates between 84% and 108%, confirming its application potential in the measurement of MC-LR in real water samples.

Effects of graphite nanoparticles on nitrification in an activated sludge system.

Graphite nanoparticles (GNPs) might result in unexpected effects during their transportation and transformation in wastewater treatment systems, including strong thermo-catalytic and catalytic effects and microbial cytotoxicity. In particular, the effects of GNPs on the nitrification process in activated sludge systems should be addressed. This study aimed to estimate the influence of GNPs on the nitrification process in a short-term nitrification reactor with exposure to different light sources. The results indicated that GNPs could only improve the efficiency of photothermal transformation slightly in the activated sludge system because of its photothermal effects under the standard illuminant (imitating 1 × sun). However, even with better photothermal effects, the nitrification efficiency still decreased significantly with GNP dosing under the standard illuminant, which might result from stronger cytotoxic effects of GNPs on the nitrifying bacteria. The disappearance of extracellular polymeric substances (EPS) around bacterial cells was observed, and the total quantity of viable bacteria decreased significantly after GNP exposuring. Variation in bacterial groups primarily occurred in nitrifying microbial communities, including Nitrosomonas sp., Nitrosospira sp., Comamonas sp. and Bradyrhizobiace sp. Nitrifiers significantly decreased, while the phyla Gammaproteobacteria, Deinocccus, and Bacteroidetes exhibited greater stability during GNP treatment.

Facile screening of potential xenoestrogens by an estrogen receptor-based reusable optical biosensor.

The apparent increase in hormone-induced cancers and disorders of the reproductive tract has led to a growing demand for new technologies capable of screening xenoestrogens. We reported an estrogen receptor (ER)-based reusable fiber biosensor for facile screening estrogenic compounds in environment. The bioassay is based on the competition of xenoestrogens with 17ß-estradiol (E2) for binding to the recombinant receptor of human estrogen receptor α (hERα) protein, leaving E2 free to bind to fluorophore-labeled anti-E2 monoclonal antibody. Unbound anti-E2 antibody then binds to the immobilized E2-protein conjugate on the fiber surface, and is detected by fluorescence emission induced by evanescent field. As expected, the stronger estrogenic activity of xenoestrogen would result in the weaker fluorescent signal. Three estrogen-agonist compounds, diethylstilbestrol (DES), 4-n-nonylphenol (NP) and 4-n-octylphenol (OP), were chosen as a paradigm for validation of this assay. The rank order of estrogenic potency determined by this biosensor was DES>OP>NP, which were consistent with the published results in numerous studies. Moreover, the E2-protein conjugate modified optical fiber was robust enough for over 300 sensing cycles with the signal recoveries ranging from 90% to 100%. In conclusion, the biosensor is reusable, reliable, portable and amenable to on-line operation, providing a facile, efficient and economical alternative to screen potential xenoestrogens in environment.

Microbial Character Related Sulfur Cycle under Dynamic Environmental Factors Based on the Microbial Population Analysis in Sewerage System.

The undesired sulfur cycle derived by microbial population can ultimately causes the serious problems of sewerage systems. However, the microbial community characters under dynamic environment factors in actual sewerage system is still not enough. This current study aimed to character the distributions and compositions of microbial communities that participate in the sulfur cycle under the dynamic environmental conditions in a local sewerage system. To accomplish this, microbial community compositions were assessed using 454 high-throughput sequencing (16S rDNA) combined with dsrB gene-based denaturing gradient gel electrophoresis. The results indicated that a higher diversity of microbial species was present at locations in sewers with high concentrations of H2S. Actinobacteria and Proteobacteria were dominant in the sewerage system, while Actinobacteria alone were dominant in regions with high concentrations of H2S. Specifically, the unique operational taxonomic units could aid to characterize the distinct microbial communities within a sewerage manhole. The proportion of sulfate-reducing bacteria, each sulfur-oxidizing bacteria (SOB) were strongly correlated with the liquid parameters (DO, ORP, COD, Sulfide, NH3-N), while the Mycobacterium and Acidophilic SOB (M&A) was strongly correlated with gaseous factors within the sewer, such as H2S, CH4, and CO. Identifying the distributions and proportions of critical microbial communities within sewerage systems could provide insights into how the microbial sulfur cycle is affected by the dynamic environmental conditions that exist in sewers and might be useful for explaining the potential sewerage problems.

Simultaneous di-oxygenation and denitrification in an internal circulation baffled bioreactor.

An internal circulation baffled bioreactor was employed to realize simultaneous di-oxygenation of phthalic acid (PA) and denitrification of nitrate, which require aerobic and anoxic conditions, respectively. Adding a small concentration of succinate as an exogenous electron donor stimulated PA di-oxygenation, which produced readily oxidizable downstream products whose oxidation also enhanced denitrification of nitrate; succinate addition also stimulated denitrification. Depending on the concentration of PA, addition of 0.17 mM succinate increased the PA removal rate by 25 and 42%, while the corresponding nitrate removal rate was increased by 73 and 51%. UV/H2O2 advanced oxidation of PA had the same effects as adding succinate, since succinate is generated by UV/H2O2; this acceleration effect was approximately equivalent to adding 0.17 mM succinate.

Utilization of unmodified gold nanoparticles for label-free detection of mercury (II): Insight into rational design of mercury-specific oligonucleotides.

Colorimetric detection of mercury (II) with the use of DNA oligonucleotides and unmodified gold nanoparticles (AuNPs) as indicators has been extensively studied. This study provides in-depth insights into the rational design of mercury-specific oligonucleotides (MSO) in the biosensing system. The leftover bases of MSO, as a result of the formation of T-Hg2+-T base pairs, can adsorb on the AuNPs and hinder their aggregation at concentrations of salt. This phenomenon was directly verified by the changes in particle sizes characterized by dynamic light scattering for the first time. Based on these findings, we proposed a rational design for the MSO with approximately 20-fold improvement in detection sensitivity. The detection limit of the proposed assay decreased to 15nM with a linear working range from 50nM to 300nM for Hg2+. The cross-reactivity against eight other metal ions was negligible compared with the response to Hg2+. Considering the diverse applications of AuNPs with oligonucleotides, this study can serve as a good reference and provides important implications in sensing and DNA-directed nanoparticle assembly.