•A combined heparinases depolymerized heparin libraries were built and systematically screened for the anti-pulmonary injury and fibrosis efficacy directly with Bleomycin challenged mice model.•Structural properties of the efficient components have been characterized by combination use of chip-based amide hydrophilic interaction chromatography HILIC- FT-ESI–MS, PAGE, and High performance liquid chromatography (HPLC).•The possible mechanisms that heparins exert their effects on have been primarily evaluated.Heparin has recently been shown to slow down idiopathic pulmonary fibrosis (IPF) process and improve survival of patients in some cases. To improve the anti-IPF function while minimizing their side effects, we developed heparin libraries with different structures depolymerized by single or combined heparinases, and systematically screened the efficacy of the different heparins for treatment of Bleomycin-induced pulmonary injury and fibrosis using mice model. Then we characterized the structural properties of the components capable of treating pulmonary injury and fibrosis by use of chip-based amide hydrophilic interaction chromatography (HILIC)-fourier transform (FT)-ESI–MS, polyacrylamide gel electrophoresis (PAGE), and high performance liquid chromatography (HPLC). Our results showed that the depolymerized heparins with relative higher molecular weight (I-2 and III-2) by the respective heparinase I and III protected mice from the induced pulmonary injury and fibrosis. In addition, the selected depolymerized heparins inhibited high-mobility group protein B1 (HMGB-1) expression, prevented E-cadhesin from downregulation, and reduced fibroblasts accumulation in the mouse lung tissue. Our study suggested that the depolymerized heparins of I-2 and III-2 with the most significant efficacy might target several pathways in alleviating the induced pulmonary fibrosis.

•Chondroitin B lyase-based analytical methods selectively quantify dermatan sulfate in heparin.•Simple enzymatic colorimetric method rapidly detected DS above 0.1 mg mL−1 in heparin.•LOD of the novel enzymatic SAX-HPLC method reached 1.0 μg mL−1.•Both methods exhibited good linearity, repeatability, precision and recovery.Dermatan sulfate (DS) is one of the hardest impurities to remove from heparin products due to their high structural similarity. The development of a sensitive and feasible method for quantitative detection of DS in heparin is essential to ensure the clinical safety of heparin pharmaceuticals. In the current study, based on the substrate specificity of chondroitin B lyase, ultraviolet spectrophotometric and strong anion-exchange high-performance liquid chromatographic methods were established for detection of DS in heparin. The former method facilitated analysis in heparin with DS concentrations greater than 0.1 mg mL−1 at 232 nm, with good linearity, precision and recovery. The latter method allowed sensitive and accurate detection of DS at concentrations lower than 0.1 mg mL−1, exhibiting good linearity, precision and recovery. The linear range of DS detection using the latter method was between 0.01 and 0.5 mg mL−1.

In methane-rich environments, methane-oxidizing bacteria usually occur predominantly among consortia including other types of microorganisms. In this study, artificial coal bed gas and methane gas were used to enrich mixed methanotrophic cultures from the soil of a coal mine in China, respectively. The changes in microbial community structure and function during the enrichment were examined. The microbial diversity was reduced as the enrichment proceeded, while the capacity for methane oxidation was significantly enhanced by the increased abundance of methanotrophs. The proportion of type II methanotrophs increased greatly from 7.84 % in the sampled soil to about 50 % in the enrichment cultures, due to the increase of methane concentration. After the microbial community of the cultures got stable, Methylomonas and Methylocystis became the dominant type I and type II methanotrophs, while Methylophilus was the prevailing methylotroph. The sequences affiliated with pigment-producing strains, Methylomonas rubra, Hydrogenophaga sp. AH-24, and Flavobacterium cucumis, could explain the orange appearance of the cultures. Comparing the two cultures, the multi-carbon sources in the artificial coal bed gas caused more variety of non-methanotrophic bacteria, but did not help to maintain the diversity or to increase the quantity and activity of methanotrophs. The results could help to understand the succession and interaction of microbial community in a methane-driven ecosystem.

Flexibility or rigidity of the linker between two fused proteins is an important parameter that affects the function of fusion proteins. In this study, we constructed a linker library with five elementary units based on the combination of the flexible (GGGGS) and the rigid (EAAAK) units. Molecular dynamics (MD) simulation showed that more rigid units in the linkers lead to more helical conformation and hydrogen bonds, and less distance fluctuation between the N- and C-termini of the linker. The diversity of linker flexibility of the linker library was then studied by fluorescence resonance energy transfer (FRET) of cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) fusion proteins, which showed that there is a wide range of distribution of the FRET efficiency. Dissipative particle dynamics (DPD) simulation of CFP-YFP with different linkers also gave identical results with that of FRET efficiency analysis, and we further found that the combination manner of the linker peptide had a remarkable effect on the orientation of CFP and YFP domains. Our studies demonstrated that the construction of the linker library with the widely controllable flexibility could provide appropriate linkers with the desirable characteristics to engineer the fusion proteins with the expected functions.

•Heparin depolymerization characteristics of Hep I/II/III were compared systemically.•Heparin depolymerization by different heparinase combinations were elucidated.•A novel combinatorial enzymatic method for LMWHs production was established.•HepIII and HepI is the best for maintaining high anti-IIa activity (75.7 ± 4.21 IU/mg).•A action pattern of HepI and HepII on heparin was excluded.Enzymatic depolymerization of heparin by heparinases is promising for production of low molecular weight heparins (LMWHs) as anticoagulants, due to its mild reaction conditions and high selectivity. Here, different heparinase combinations were used to depolymerize heparin. Heparinase I and heparinase II can depolymerize heparin more efficiently than heparinase III, respectively, but heparinase III was the best able to protect the anticoagulant activities of LMWHs. Heparinase III and heparinase I/II combinations were able to efficiently depolymerize heparin to LMWHs with higher anticoagulant activity than the LMWHs produced by the respective heparinase I and heparinase II. HepIII and HepI is the best combination for maintaining high anti-IIa activity (75.7 ± 4.21 IU/mg) at the same Mw value. Furthermore, considering both the changes in molecular weight and anticoagulant activity, the action patterns of heparinase I and heparinase II were found not to follow the exolytic and processive depolymerizing mechanism from the reducing end of heparin.

By the combination of fusion protein technology and nanoparticle surface modification technology, a maltose-modified magnetic support has been successfully developed for the integration of separation/purification, and immobilization of maltose binding protein–Heparinase I fusion enzyme (MBP–HepA) for enzymatic reaction and recycle. HepA is an important enzyme to degrade heparin and heparan sulfate to produce low molecular weight heparin (LMWH). This magnetic support may play an important role for process simplification and cost reduction on the industrial production of LMWH. Further, this magnetic support also can be used for the immobilization of other MBP fusion proteins, which implies that this magnetic support may have wide applications in biotechnology and enzyme engineering.

An NADH dehydrogenase encoded by the nuo cluster was isolated and impaired by knocking out the nuoB gene in Enterobacter aerogenes to examine its effect on hydrogen production. Three nuoB-deleted mutant strains were constructed from the wild-type strain E. aerogenes IAM1183 and two recombinant strains, IAM1183-A (ΔhycA) and IAM1183-O (ΔhybO), from which the hycA and hybO genes had already been deleted previously, respectively. Compared with the performance of the wild-type strain, the overall hydrogen production of the mutants IAM1183-B (ΔnuoB), IAM1183-AB (ΔhycA/ΔnuoB) and IAM1183-BO (ΔhybO/ΔnuoB) was increased by 49.2%, 54.0%, and 52.4% in batch culture, respectively. The hydrogen yields from glucose by the three mutants IAM1183-B, IAM1183-AB, IAM1183-BO were 1.38, 1.49, and 1.39 mol H2/mol glucose, respectively, while it was 1.16 mol H2/mol glucose in the wild-type strain. Metabolic flux analysis indicated that all three mutants exhibited reduced fluxes to lactate production, and enhanced fluxes toward the generation of hydrogen, acetate, ethanol, succinate and 2,3-butanediol. Both the formate pathway and the NADH pathway contributed to increased hydrogen production in the mutant strains. The assay of 4 NADH-mediated enzyme activities (H2ase, LDH, ADH and BDDH) was in accordance with the redistributions of the metabolic fluxes in the mutant strains.Highlights► An NADH dehydrogenase encoded by the nuo cluster was isolated in Enterobacter aerogenes. ► Deleting the nuoB gene resulted in a substantial increase in hydrogen production. ► Reduced fluxes to lactate were found in all three nuoB-deleted mutant strains. ► Changes in NADH-mediated enzyme activities agreed with metabolic redistributions.

Biohydrogen and subsequent biomethane generation from biomass is a promising strategy for renewable energy supply, because this combination can lead to higher energy recovery efficiency and faster fermentation than single methane fermentation. Microbial consortium control by retaining hydrogen-producers through the addition of microbial carriers is an alternative to constructing hydrogen-producing reactors. Here we report the use of carbon nanotubes (CNTs) as microbial carriers to enhance microbial retention and the production of biohydrogen. Laboratory-scale upflow anaerobic sludge blanket (UASB) reactors with CNTs at 100 mg/L achieved a maximal hydrogen production rate of 5.55 L/L/d and a maximal hydrogen yield of 2.45 mol/mol glucose. Compared to frequently used activated carbon (AC) particles, CNTs resulted in quicker startup and better performance of hydrogen fermentation in UASB reactors. Scanning electron microscopy (SEM) and pyrosequencing results revealed that the reactor with CNTs led to a high proportion of hydrogen-producing bacteria among the microbial consortium, which endowed the microbes with strong flocculation capacity and hydrogen productivity.Highlights► We report carbon nanotubes (CNTs) as a carrier for biohydrogen production. ► CNTs resulted in greater hydrogen production in UASB than activated carbon. ► SEM showed different microbial morphologies induced with different carriers. ► Pyrosequencing proved a high proportion of hydrogen-producing bacteria on CNTs.

To improve the hydrogen productivity and examine the hydrogen evolution mechanism of Clostridium paraputrificum, roles of formate in hydrogen evolution and effects of introducing formate-originated NADH regeneration were explored. The formate-decomposing pathway for hydrogen production was verified to exist in C. paraputrificum. Then NAD+-dependent formate dehydrogenase FDH1 gene (fdh1) from Candida boidinii was overexpressed, which regenerate more NADH from formate to form hydrogen by NADH-mediated pathway. With fdh1 overexpression, the hydrogen yield via NADH-involving pathway increased by at least 59 % compared with the control. Accompanied by the change of hydrogen metabolism, the whole cellular metabolism was redistributed greatly.

Violacein is a bacteria-originated indolocarbazole pigment with potential applications due to its various bioactivities such as anti-tumor, antiviral, and antifungal activities. However, stable mass production of this pigment is difficult due to its low productivities and the instability of wild-type violacein-producing strains. In order to establish a stable and efficient production system for violacein, the violacein synthesis pathway from a new species of Duganella sp. B2 was reconstructed in different bacterial strains including Escherichia coli, Citrobacter freundii, and Enterobacter aerogenes by using different vectors. The gene cluster that encodes five enzymes involved in the violacein biosynthetic pathway was first isolated from Duganella sp. B2, and three recombinant expression vectors were constructed using the T7 promoter or the alkane-responsive promoter PalkB. Our results showed that violacein could be stably synthesized in E. coli, C. freundii, and E. aerogenes. Interestingly, we found that there were great differences between the different recombinant strains, not only in the protein expression profiles pertaining to violacein biosynthesis but also in the productivity and composition of crude violacein. Among the host strains tested, the crude violacein production by the recombinant C. freundii strain reached 1.68 g L−1 in shake flask cultures, which was 4-fold higher than the highest production previously reported in flask culture by other groups. To the best of our knowledge, this is the first report on the efficient production of violacein by genetically engineered strains.

Experiments involving the addition of external nicotinamide adenine dinucleotide, reduced form (NADH) or nicotinamide adenine dinucleotide (NAD+) have been designed to examine how the hydrogen in Enterobacter aerogenes is liberated by NADH or NAD+. The addition of external NADH or NAD+ was found to regulate hydrogen production by E. aerogenes in resting cells, batch cultures, and chemostat cultures. Particularly in chemostat cultivation, with the external addition of NADH, hydrogen production via the NADH pathway was decreased, while that via the formate pathway was increased; in the end, the overall hydrogen p was decreased. The addition of NAD+, on the other hand, gave the opposite results. The membrane-bound hydrogenase was found to play a central role in regulating hydrogen production. The occurrence of NADH oxidation (NAD+ reduction) on the cell membrane resulted in an electron flow across the membrane; this changed the oxidation state and metabolic pattern of the cells, which eventually affected the hydrogen evolution.

To examine perturbation effects of formate pathway on hydrogen productivity in Enterobacter aerogenes (Ea), formate dehydrogenase FDH-H gene (fdhF) and formate hydrogen lyase activator protein FHLA gene (fhlA) originated from Escherichia coli, were overexpressed in the wild strain Ea, its hycA-deleted mutant (A) by knockout the formate hydrogen lyase repressor and hybO-deleted mutant (O) by knockout of the uptake hydrogenase, respectively. Overexpression of fdhF and fhlA promoted cell growth and volumetric hydrogen production rates of all the strains, and the hydrogen production per gram cell dry weight (CDW) for Ea, A and O was increased by 38.5%, 21.8% and 5.25%, respectively. The fdhF and fhlA overexpression improved the hydrogen yield per mol glucose of strains Ea and A, but declined that of strain O. The increase of hydrogen yield of the strain Ea with fdhF and fhlA expression was mainly attributed to the increase of formate pathway, while for the mutant A, the improved hydrogen yield with fdhF and fhlA expression was mainly due to the increase of NADH pathway. Analysis of the metabolites and ratio of ethanol-to-acetate showed that the cellular redox state balance and energy level were also changed for these strains by fdhF and fhlA expression. These findings demonstrated that the hydrogen production was not only dependent on the hydrogenase genes, but was also affected by the regulation of the whole metabolism. Therefore, fdhF and fhlA expression in different strains of E. aerogenes could exhibit different perturbation effects on the metabolism and the hydrogen productivity.

A 5431-bp DNA fragment partially encoding the formate hydrogen lyase (FHL) gene cluster hycABCDE was isolated and identified from Enterobacter aerogenes IAM1183 chromosomal DNA. All the five putative gene products showed a high degree of homology to the reported bacterial FHL proteins. The gene hycA, encoding the FHL repressor protein, and hybO, encoding the small subunit of the uptake hydrogenase, were targeted for genetic knockout for improving the hydrogen production. The pYM-Red recombination system was adopted to form insertional mutations in the E. aerogenes genome, thereby creating mutant strains of IAM1183-A (△hycA), IAM1183-O (△hybO), and IAM1183-AO (△hycA/△hybO double knockout). The hydrogen production experiments with these mutants showed that the maximum specific hydrogen productivities of IAM1183-A, IAM1183-O, and IAM1183-AO were 2879.466 ± 38.59, 2747.203 ± 13.25 and 3372.019 ± 4.39 (ml h−1 g−1dry cell weight), respectively, higher than that of the wild strain (2321.861 ± 15.34 ml h−1 g−1dry cell weight). The total H2 yields by the three mutants IAM1183-A, IAM1183-O and IAM1183-AO were 0.73, 0.78, and 0.83 mol-H2/mol glucose, respectively, while the wild-type IAM1183 was only 0.65 mol-H2/mol glucose. The metabolites of the mutants including acetate, ethanol, 2,3-butanediol and succinate were all increased compared with that of the wild type, implying the changed metabolic flux by the mutation. In the fermentor cultivation with IAM1183△hycA/△hybO, the total hydrogen volume after 16 h cultivation reached 4.4 L, while that for the wild type was only 2.9 L.

Slow growth and relatively low cell densities of methanotrophs have limited their uses in industrial applications. In this study, a novel method for rapid cultivation of Methylosinus trichosporium OB3b was studied by adding a water-immiscible organic solvent in the medium. Paraffin oil was the most effective at enhancing cell growth and final cell density. This is at least partially due to the increase of methane gas transfer between gas and medium phases since methane solubility is higher in paraffin than in water/nitrate minimal salt medium. During cultivation with paraffin oil at 5% (v/v) in the medium, M. trichosporium OB3b cells also showed higher concentrations of the intermediary metabolites, such as formic acid and pyruvic acid, and consumed more methane compared with the control. Paraffin as methane vector to improve methanotroph growth was further studied in a 5-L fermentor at three concentrations (i.e., 2.5%, 5%, and 10%). Cell density reached about 14 g dry weight per liter with 5% paraffin, around seven times higher than that of the control (without paraffin). Cells cultivated with paraffin tended to accumulate around the interface between oil droplets and the water phase and could exist in oil phase in the case of 10% (v/v) paraffin. These results indicated that paraffin could enhance methanotroph growth, which is potentially useful in cultivation of methanotrophs in large scale in industry.

An expression system for NAD+-dependent formate dehydrogenase gene (fdh1), from Candida boidinii, was constructed and cloned into Enterobacter aerogenes IAM1183. With the fdh1 expression, the total H2 yield was attributed to a decrease in activity of the lactate pathway and an increase of the formate pathway flux due to the NADH regeneration. Analysis of the redox state balance and ethanol-to-acetate ratio in the fdhl-expressed strain showed that increased reducing power arose from the reconstruction of NADH regeneration pathway from formate thereby contributing to the improved H2 production.

By constructing the expression system for fusion protein of GFPmut1 (a green fluorescent protein mutant) with the hyperthermophilic xylanase obtained from Dictyoglomus thermophilum Rt46B.1, the effects of temperature on the fluorescence of GFP and its relationship with the activities of GFP-fused xylanase have been studied. The fluorescence intensities of both GFP and GFP-xylanase have proved to be thermally sensitive, with the thermal sensitivity of the fluorescence intensity of GFP-xylanase being 15% higher than that of GFP. The lost fluorescence intensity of GFP inactivated at high temperature of below 60°C in either single or fusion form can be completely recovered by treatment at 0°C. By the fluorescence recovery of GFP domain at low temperature, the ratios of fluorescence intensity to xylanase activity (Rgfp/Axyl) at 15°C and 37°C have been compared. Even though the numbers of molecules of GFP and xylanase are equivalent, the Rgfp/Axyl ratio at 15°C is ten times of that at 37°C. This is mainly due to the fact that lower temperature is more conducive to the correct folding of GFP than the hyperthermophilic xylanase during the expression. This study has indicated that the ratio of GFP fluorescence to the thermophilic enzyme activity for the fusion proteins expressed at different temperatures could be helpful in understanding the folding properties of the two fusion partners and in design of the fusion proteins.

Fluorescent proteins (FPs), such as green fluorescent protein (GFP) and its variants, are well-developed visible markers for analyzing bioprocesses. Accurate measurement of fluorescence emitted from FPs in whole cells is complicated by the inner filter effect (IFE), which is caused by intracellular light absorption and scattering by cell particles. The IFE causes nonlinearity between fluorescence intensity and fluorophore concentrations in FP-harboring cells and can significantly influence the accuracy of FP-based analysis, especially at high cell densities. A mathematical model based on detection of fluorescence intensity using a fluorescence spectrophotometer was developed to provide a simple correction for the IFE in fluorescence intensity detection in high-density cultures. The parameters of this model were determined in three different FP-harboring bacterial strains to give the “real fluorescence” intensity without the IFE. Using these parameters, accurate analysis of FP-labeled Escherichia coli at high cell density in pure culture and in mixed cultures with fluorescent and nonfluorescent strains was easily and successfully achieved.

A flow-based immunoassay system utilizing secondary-antibody coated microbeads and Cy5-secondary antibody for signal production was successfully developed to quantitate target bacteria with a kinetic exclusion assay (KinExA™ 3000 Instrument). It directly measured the concentration of unliganded antibody separated from the equilibrated mixture of antibody and bacteria through a 0.2 μm polyethersulfone membrane, enabling it to quantify the concentration of bacteria. The novel method demonstrated the qualities of rapidness, sensitivity, high accuracy and reproducibility, and ease to perform. Detection of Pseudomonas aeruginosa and Staphylococcus aureus was accomplished with low detection limits of 4.10 × 106 and 5.20 × l04 cells/mL, respectively, with an assay time of less than 15 min. The working ranges for quantification were 4.10 × l06 to 1.64 × l010 cells/mL for P. aeruginosa, and 5.20 × l04 to 1.04 × l09 cells/mL for S. aureus. It yielded an assay with at least 10-fold greater sensitivity than ELISA and could correctly assess the concentration of predominant bacterium spiked in the mixture of P. aeruginosa and S. aureus. With this reliable platform, the average amount of antibody bound by one cell in the maximum capability could be further provided: (1.6–2.5) × l05 antibodies for one P. aeruginosa cell and (2.2–2.7) × l08 antibodies for one S. aureus cell. The KinExA system is flexible to determine different kinds of bacteria conveniently by using anti-mouse IgG as the same immobilizing agent. However, a higher specificity of the antibodies to the target bacteria will be required for the use of this system with higher detection sensitivity.

In this paper, microbubble technology was employed to increase the mass transfer rate of ozone and enhance the ozone oxidation of practical textile wastewater. Experiments were performed using a microbubble generator and a bubble contactor, which is commonly used in ozonation system, for comparison. The microbubble generator produced a milky and high intensity microbubble solution, which could reach a higher oxygen transfer rate at a lower input gas flow rate. A volumetric oxygen transfer rate (VOTR) of 0.086–0.413 kg/m3 h and a total mass transfer coefficient of 0.1072–0.4859 min−1 were obtained at airflow rates of 0.02–1.5 dm3/min. During the ozonation of practical textile wastewater by using the microbubble system, the input ozone could be almost completely utilized, and the rate of decolorization and organic reduction were much faster than those of the bubble contactor. For the practical textile wastewater tested, the time required for 80% removal of color was about 140 and 280 min by ozone microbubble and conventional bubbles, respectively. The chemical oxygen demand (COD) removal efficiency in the microbubble system was higher by 20%. The results revealed that in addition to the enhancement of the mass transfer of ozone, microbubbles could improve the oxidation of actual textile wastewater.

For biomass production of Spirulina platensis as feedstock of fermentation, the culture characteristics of three typical mutants of 3-A10, 3-B2 and 4-B3 generated by atmospheric and room temperature plasmas (ARTP) mutagenesis were systematically studied by using CO2 aeration culture system and compared with the wild strain. The specific growth rate of wild strain in the pure air aeration culture system exhibited a 76.2% increase compared with static culture, while the specific growth rates of the 3-A10, 3-B2 and 4-B3 in pure air aeration culture system were increased by 114.4%, 95.9% and 88.2% compared with their static cultures. Compared with static culture, the carbohydrate contents of wild strain, 3-A10, 3-B2 and 4-B3 in pure air aeration culture system dropped plainly by 51.0%, 79.3%, 85.5% and 26.1%. Increase of CO2 concentration enhanced carbohydrate content and productivity. Based on the carbohydrate productivity, the optimal inlet of CO2 concentration in aeration culture was determined to be 12% (v/v). Under this condition, 3-B2 exhibited the highest carbohydrate content (30.7%), CO2 fixation rate (0.120gCO2·g−1·d−1) and higher growth rate (0.093 g L−1·d−1), while 3-A10 showed the highest growth rate (0.118 g L−1·d−1) and higher CO2 fixation rate (0.117gCO2·g−1·d−1) but low carbohydrate content (24.5%), and 4-B3 showed the highest chlorophyll (Chl) content (3.82 mg·g−1). The most outstanding mutant by static culture in terms of growth rate and carbohydrate productivity (3-B2), was also demonstrated by CO2 aeration culture system. This study revealed that the ARTP mutagenesis could generate the S. platensis mutants suitable for CO2 aeration culture aiming at biomass production.

Green fluorescent protein (GFP) and its variants/homolog proteins are generally called as GFP-like fluorescent proteins (FPs), which are widely used as visible molecular tools for monitoring a wide range of biological processes due to their capability of simple, accurate and real time quantification. The FPs-based molecular and visible quantification tools are giving more impact on bioprocess engineering, enabling the biomolecule-level dynamic information to be linked with the process-level events. In this review, different applications of FPs in biological engineering with emphasis on rapid molecular bioprocess quantification, such as quantification of the transcription efficiency, the protein production, the protein folding efficiency, the cell concentration, the intracellular microenvironments and so on, would be first introduced. The challenges of using FPs with respect to actual bioprocess applications for the precise quantification including the interaction of FPs and the fused partner proteins, the maturation of FPs, the inner filter effect and sensing technology were then discussed. Finally, the future development for the FPs used in molecular bioprocess quantification would be proposed.

In order to study the effects of ionic surfactants on bacterial luciferase, the cationic surfactant dodecyltrimethylammonium biomide (DTAB) and anionic surfactant sodium dodecylsulfate (SDS) were chosen. For comparison with bacterial luciferase, α-amylase was used since these two enzymes have similar electrostatic potential and charged active sites. After the enzymes were treated with the surfactants, the catalytic properties of bacterial luciferase and α-amylase were assayed, and fluorescence spectroscopy and circular dichroism (CD) were used to analyze the alteration of the protein structure. The results showed that when the DTAB concentration was low, the cationic surfactant DTAB enhanced the enzymatic activities of bacterial luciferase and α-amylase. On the other hand, the anionic surfactant SDS did not alter the enzymatic activity. The main interaction of cationic surfactant DTAB and the negatively charged surface of the proteins was the ionic interaction, which could alter the environment for the enzyme to work when the DTAB/enzyme molar ratio was low. However, at high cationic surfactant concentration, the ionic interaction and hydrophobic interaction might destroy the secondary and tertiary structures of the proteins, leading to the loss of enzymatic activities.

A microbubble ozonation process for enhancing sludge solubilization was proposed and its performance was evaluated in comparison to a conventional ozone bubble contactor. Microbubbles are defined as bubbles with diameters less than several tens of micrometers. Previous studies have demonstrated that microbubbles could accelerate the formation of hydroxyl radicals and hence improve the ozonation of dyestuff wastewater. The results of this study showed that microbubble ozonation was effective in increasing ozone utilization and improving sludge solubilization. For a contact time of 80 min, an ozone utilization efficiency of more than 99% was obtained using the microbubble system, while it gradually decreased from 94% to 72% for the bubble contactor. The rate of microbial inactivation was obviously faster in the microbubble system. At an ozone dose of 0.02 g O3 g−1 TSS, about 80% of microorganisms were inactivated in the microbubble system, compared with about 50% inactivation for the bubble contactor. Compared to the bubble contactor, more than two times of COD and total nitrogen, and eight times of total phosphorus content were released from the sludge into the supernatant by using the microbubble system at the same ozone dosage. The application of microbubble technology in ozonation processes may provide an effective and low cost approach for sludge reduction.

Phosphorus removal from wastewater is of great importance. In the present study, ferric chloride was selected as the coagulant, and tannic acid (TA), a natural polymer, as the coagulant aid to develop an effective coagulation process with the emphasis of phosphorus recovery from different types of wastewater. The results showed that TA can accelerate the settling speed by forming flocs with large size, reduce the residual Fe(III) to eliminate the yellow color caused by Fe(III), and slightly increase the phosphorus removal efficiency. The precipitate formed by TA-aided coagulation showed the advantage of releasing phosphorus faster than ferric phosphate, indicating the possibility of phosphorus recovery from wastewater as slow release fertilizer. To further understand the structural characteristics of the precipitate, analytical techniques such as Raman spectroscopy, X-ray photoelectron spectroscopy and matrix-assisted laser desorption ionization-time of flight mass spectrometry were employed. The analytical results indicated that TA–Fe–P complex was formed during the coagulation/flocculation processes. Solid phase in the precipitate consisted of TA–Fe–P complex, Fe–TA complex and/or ferric hydroxyphosphate.

Effects of different microaerophilic conditions on cell growth, glucose consumption, hydrogen production and cellular metabolism of wild Enterobacter aerogenes strain and polyphosphate kinase (PPK) overexpressing strain were systematically studied in this paper, using NaH2PO4 as the phosphate sources. Under different microaerophilic conditions, PPK-overexpressing strain showed better cell growth, glucose consumption and hydrogen production than the wild strain. In the presence of limited oxygen (2.1%) and by PPK overexpression, the hydrogen production per liter of culture, the hydrogen production per cell and the hydrogen yield per mol of glucose increased by 20.1%, 12.3% and 10.8%, respectively, compared with the wild strain under strict anaerobic conditions. Metabolic analysis showed that the increase of the total hydrogen yield was attributed to the improvement of NADH pathway. The result of more reductive cellular oxidation state balance also further demonstrated that, under proper initial microaerophilic conditions and by PPK overexpression, the cell could adjust the cellular redox states and make more energy flow into hydrogen production pathways.

Purification of the total DNA extracted from activated sludge samples was studied. The effects of extraction buffers and lysis treatments (lysozyme, sodium dodecyl sulfate (SDS), sonication, mechanical mill and thermal shock) on yield and purity of the total DNA extracted from activated sludge were investigated. It was found that SDS and mechanical mill were the most effective ways for cell lysis, and both gave the highest DNA yields, while by SDS and thermal shock, the purest DNA extract could be obtained. The combination of SDS with other lysis treatment, such as sonication and thermal shock, could apparently increase the DNA yields but also result in severe shearing. For the purification of the crude DNA extract, polyvinyl polypyrrolidone was used for the removal of humic contaminants. Cetyltrimethyl ammonium bromide, potassium acetate and phenol/chloroform were used to remove proteins and polysaccharides from crude DNA. Crude DNA was further purified by isopropanol precipitation. Thus, a suitable protocol was proposed for DNA extraction, yielding about 49.9 mg (total DNA)/g volatile suspended solids, and the DNA extracts were successfully used in PCR amplifications for 16S rDNA and 16S rDNA V3 region. The PCR products of 16S rDNA V3 region allowed the DGGE analysis (denatured gradient gel electrophoresis) to be possible.

The extensive prospects of violacein in the pharmaceutical industry have attracted increasing interest. However, the fermentation levels of violacein are currently inadequate to meet the demands of industrial production. This study was undertaken to develop an efficient process for the production of violacein by recombinant Citrobacter freundii. The effects of dissolved oxygen (DO) and pH on cell growth and violacein production in batch cultures were investigated first. When the DO and pH of the medium were controlled at around 25% and 7.0, respectively, the biomass and concentration of violacein were maximized. Based on the consumption of nutrients in the medium observed during batch culture, a fed-batch fermentation strategy with controlled DO and pH was implemented. By continuously feeding glycerol, NH4Cl, and l-tryptophan at a constant feeding rate of 16 mL h−1, the final concentration of violacein reached 4.13 g L−1, which was 4.09-fold higher than the corresponding batch culture, and the maximal dry cell weight (DCW) and average violacein productivity obtained for the fed-batch culture were 3.34 g DCW L−1 and 82.6 mg L−1 h−1, respectively. To date, this is the first report on the efficient production of violacein by genetically engineered strains in a fermentor.Highlights► A violacein productivity of 82.6 mg L−1 h−1 was obtained by the implementation of a fed-batch fermentation strategy. ► It is the first time that an efficient production of violacein by genetically engineered strains was studied with the highest productivity ever reported. ► Violacein could be produced by recombinant Citrobacter freundii economically and efficiently using glycerol as the sole carbon source and endogenous l-tryptophan as the precursor. ► We found that the production of violacein partly leads to the increase of intracellular pH.

•The scale-up fermentation of mixed methane-oxidizing bacteria was achieved.•A culture with 2.69 g L−1 DCW was obtained in a 600 L fermenter after 25 h.•Growth, MMO activity and community maintained during the scale-up.The mixed methane-oxidizing bacteria, which can keep the unique catalytic properties of methanotrophs and overcome the general limitations of slow growth and low activity of the pure methanotrophic culture, have promising applications for biotechnology. In this study, scale-up fermentation for the mixed methane-oxidizing bacteria was carried out from 5 L (working volume 3 L) to 100 L (working volume 65 L) and then to 600 L (working volume 350 L) fermenter, and the mixed bacterial concentration with 2.69 g L−1 dry cell weight was obtained in a 600 L fermenter after 25 h culture. The existence of the coexisting bacteria together with methanotrophs could make a positive contribution to the bacterial growth and MMO activity of the mixed culture. During the scale-up cultivation, the growth, methane-oxidizing capacity, and community of the mixed bacteria were maintained, indicating that it would be possible for large-scale preparation of the mixed methane-oxidizing bacteria.

•Major advances in the development of anti-inflammatory low molecular weight heparins (LMWHs), preclinical and clinical studies as well as possible underlying molecular mechanisms of actions have been summarized.•The activities of commercially available LMWHs have been compared to highlight different anti-inflammatory effects in LMWHs produced using different manufacturing processes.•The importance of structure-activity relationship (SAR) studies on the non-anticoagulant effects of LMWHs was stressed, and strategies for exploring new clinical indications were discussed.Low molecular weight heparins (LMWHs) are produced by chemical or enzymatic depolymerization of unfractionated heparin (UFH). Besides their well-known anticoagulant effects, LMWHs have also been reported to exhibit numerous anti-inflammatory properties. Previous studies have, however, shown that different production processes result in unique structural characteristics of LMWHs. The structural variations may help explain the different therapeutic spectrums in disease treatment for non-anticoagulant effects. In the present review, we summarize major advances in understanding and exploiting the anti-inflammatory disorder activities of LMWHs, based on mechanistic studies, preclinical experiments and clinical trials. We highlight differences in these activities of commercially available LMWHs produced using different manufacturing processes. We stress the importance of structure-activity relationship (SAR) studies on the non-anticoagulant effects of LMWHs and discuss strategies for exploring new clinical indications.

Metabolic engineering is recognized as one of the most important technologies for improving fermentative hydrogen yield. A vector with hydrogenase genes (hoxEFUYH) from Synechocystis sp. PCC 6803 under an alkB promoter was constructed, and introduced into Escherichia coli DH5α to alter the hydrogen metabolism with glucose as the sole carbon source. The recombinant strain reached a highest hydrogen yield of 1.89 mol/mol glucose, which was 95% of the theoretical hydrogen yield of E. coli. Hydrogenase activities for hydrogen evolution were increased and formic acid assimilation was accelerated with the expression of hoxEFUYH. The expression of hoxEFUYH suppressed the transcription of native hydrogenase 1 and hydrogenase 2, which were responsible for hydrogen uptake activity, while it had no influence on the transcription of the hydrogenase 3. Moreover, as the electron donor of HoxEFUYH is NADH, the expressed HoxEFUYH expanded the substrate specificity of the hydrogen-evolving hydrogenase in E. coli.Highlights► We constructed a vector with hydrogenase genes from Synechocystis sp. PCC 6803, and introduced it into Escherichia coli DH5α to alter the hydrogen metabolism with glucose. ► The recombinant strain reached a yield of 1.89 mol/mol glucose, 95% of the theoretical hydrogen yield of E. coli. ► The expression of hoxEFUYH suppressed the transcription of native dehydrogenase, while it had no influence on the hydrogenase 3. ► The expressed HoxEFUYH expanded the substrate specificity of the hydrogen-evolving hydrogenase in E. coli.

By the combination of fusion protein technology and nanoparticle surface modification technology, a maltose-modified magnetic support has been successfully developed for the integration of separation/purification, and immobilization of maltose binding protein–Heparinase I fusion enzyme (MBP–HepA) for enzymatic reaction and recycle. HepA is an important enzyme to degrade heparin and heparan sulfate to produce low molecular weight heparin (LMWH). This magnetic support may play an important role for process simplification and cost reduction on the industrial production of LMWH. Further, this magnetic support also can be used for the immobilization of other MBP fusion proteins, which implies that this magnetic support may have wide applications in biotechnology and enzyme engineering.