Zhong Hu

Isolation, characterization of Rhodococcus sp. P14 capable of degrading high-molecular-weight polycyclic aromatic hydrocarbons and aliphatic hydrocarbons

Rhodococcus sp. P14 was isolated from crude oil-contaminated sediments. This strain was capable of utilizing three to five rings polycyclic aromatic hydrocarbons (PAHs) including phenanthrene (Phe), pyrene (Pyr), and benzo[a]pyrene (BaP) as a sole carbon and energy source. After cultivated with 50mg/L of each PAH, strain P14 removed 43% Phe, 34% Pyr and 30% BaP in 30 d. Four different hydroxyphenanthrene products derived from Phe by strain P14 (1,2,3,4-hydroxyphenanthrene) were detected using SPME-GC-MS. Strain P14 also was capable of degrading mineral oil with n-alkanes of C17 to C21 carbon chain length. Compared with glucose-grown cells, PAHs-grown cells had decreased contents of shorter-chain length fatty acids (≤ C16:0), increased contents of C18:0, Me-C19:0 and disappeared odd-number carbon chain fatty acids. The contents of unsaturated C19:1, Me-C19:0 increased and C18:0 decreased in mineral oil-grown cells. At the same time, the strain P14 tended to float when cultivated in mineral oil-supplemented liquid medium. The degradation capability of P14 to alkane and PAHs and its floating characteristics will be very helpful for futures application in oil-spill bioremediation.

Production and purification of agarase from a marine agarolytic bacterium Agarivorans sp. HZ105

Aims:  Isolation and characterization of an agarase‐producing bacterium Agarivorans sp. HZ105.

Identification and agglutination properties of hemocyanin from the mud crab (Scylla serrata)

Infectious diseases have significantly delayed the growth of crab aquaculture. Identification of the immune molecules and characterization of the defense mechanisms will be pivotal to the reduction of these diseases. Hemocyanin is an important non-specific immune protein present in the hemolymph of both mollusks and arthropods. However, little is known about the hemocyanin from the mud crab Scylla serrata. In this study, we identified the S. serrata hemocyanin using affinity proteomics and investigated its agglutinative properties. The results showed that S. serrata hemocyanin consists of five subunits with molecular weights of 70, 72, 75, 76 and 80 kDa, respectively. It demonstrated agglutination activities against seven bacterial species at concentrations ranging from 7.5 to 30 μg/ml. Agglutination was inhibited by 50-200 mM of N-acetylneuraminic acid, α-d-glucose, d-galactose and d-xylose. The 76 kDa subunit was identified as the protein that primarily binds bacterial cells and we speculate that it functions as the agglutinating subunit. We showed that outer membrane proteins (Omp) of bacteria could completely inhibit agglutination and that the agglutination activities of hemocyanin against Escherichia coli ▵OmpA and ▵OmpX mutants were significantly decreased, suggesting that these two Omps may be important ligands of hemocyanin. Together, the data collectively suggests that the 76 kDa subunit of S. serrata hemocyanin mediates agglutination through recognition of OmpA and OmpX proteins in bacteria.

The intestinal microbial diversity in mud crab (Scylla paramamosain) as determined by PCR‐DGGE and clone library analysis

To identify the intestinal microbial diversity in mud crab and to investigate the bacterial difference in the intestinal microbiology between wild crabs (WC), pond‐raised healthy and diseased crabs (DC).

Enhancement of the immune response and protection against Vibrio parahaemolyticus by indigenous probiotic Bacillus strains in mud crab (Scylla paramamosain)

In a previous study, bacterial communities of the intestine in three populations of crabs (wild crabs, pond-raised healthy crabs and diseased crabs) were probed by culture-independent methods. In this study, we examined the intestinal communities of the crabs by bacterial cultivation with a variety of media. A total of 135 bacterial strains were isolated from three populations of mud crabs. The strains were screened for antagonistic activity against Vibrio parahaemolyticus using an agar spot assay. Antagonistic strains were then identified by 16S rRNA gene sequence analysis. Three strains (Bacillus subtilis DCU, Bacillus pumilus BP, Bacillus cereus HL7) with the strongest antagonistic activity were further evaluated for their probiotic characteristics. The results showed that two (BP and DCU) of them were able to survive low pH and high bile concentrations, showed good adherence characteristics and a broad spectrum of antibiotic resistance. The probiotic effects were then tested by feeding juvenile mud crabs (Scylla paramamosain) with foods supplemented with 10(5) CFU/g of BP or DCU for 30 days before being subjected to an immersion challenge with V. parahaemolyticus for 48 h. The treated crabs showed significantly higher expression levels of immune related genes (CAT, proPO and SOD) and activities of respiratory burst than that in controlled groups. Crabs treated with BP and DCU supplemented diets exhibited survival rates of 76.67% and 78.33%, respectively, whereas survival rate was 54.88% in crabs not treated with the probiotics. The data showed that indigenous mud-associated microbiota, such as DCU and BP, have potential application in controlling pathogenic Vibriosis in mud crab aquaculture.

The outer membrane protein, LamB (maltoporin), is a versatile vaccine candidate among the Vibrio species

Maltoporin (LamB) is a family of outer membrane proteins. There has been no report of immunological characteristics of LamB in the Vibrio species so far. In this study, lamB genes from eight Vibrio strains were cloned and sequenced. The bioinformatics analysis indicated that sequence similarities of LamB proteins were ranged from 46.7% to 81.1%. Further, the result showed that their antigenic epitopes were highly conserved implying that LamB might be a shared antigen among Vibrios. The Western blot of rabbit sera against recombinant LamB from V. alginolyticus ATCC 33787 with cell lysate of 18 Vibrio strains showed cross-recognition. Bands observed on cell lysate of Vibrio strains immunoblotted with the anti-LamB sera ranged between 40 and 49 kDa. The Whole-cell ELISA assay further confirmed that the antisera of recombinant LamB recognized the tested Vibrio strains indicating the surface-exposed of LamB. Finally, the cross-protective property of recombinant LamB was evaluated through vaccination and subsequent challenge with heterogeneous virulent Vibrio strains in zebrafish. Recorded relative percent survival (RPS) of the vaccinated group varied from 54.1% to 77.8%, showing that zebrafish were protected from Vibrio infection after immunization with LamB protein. The cumulative evidences in this study suggested that LamB was a conserved antigen among tested Vibrio species and might be a potentially versatile vaccine candidate for the prevention of Vibriosis.

Cellulase production in a new mutant strain of Penicillium decumbens ML-017 by solid state fermentation with rice bran

To produce cellulolytic enzyme efficiently, Penicillium decumbens strain L-06 was used to prepare mutants with ethyl methane sulfonate (EMS) and UV-irradiation. A mutant strain ML-017 is shown to have a higher cellulase activity than others. Box-Behnkens design (BBD) and response surface methodology (RSM) were adopted to optimize the conditions of cellulase (filter paper activity, FPA) production in strain ML-017 by solid-state fermentation (SSF) with rice bran as the substrate. And the result shows that the initial pH, moisture content and culture temperature all have significant effect on the production of cellulase. The optimized condition shall be initial pH 5.7, moisture content 72% and culture temperature 30°C. The maximum cellulase (FPA) production was obtained under the optimized condition, which is 5.76 IU g(-1), increased by 44.12% to its original strain. It corresponded well with the calculated results (5.15 IU g(-1)) by model prediction. The result shows that both BBD and RSM are the cellulase optimization methods with good prospects.

Contamination and Ecotoxicology risks of Polycyclic Aromatic Hydrocarbons in Shantou Coastal Waters, China

Nine locations in Shantou coastal waters were chosen for the study on contamination and ecotoxicology risks posed by polycyclic aromatic hydrocarbons (PAHs). Sediment samples were collected to investigate PAH distribution behaviour, sources and understand their origin, which is fundamental in predicting their subsequent behaviour. Many approaches and methods were applied to accomplish these objectives and study purpose. The results found revealed the critical importance of improving our understanding of PAH equilibrium relationships. The serious environmental and health concern, imposed by the high concentrations of PAHs in the area, were widely discussed. Furthermore, the location of Shantou within the town and vicinity of Guiyu, which is a booming E-waste processing centre in China, might explain the significance of atmospheric transportation source of PAHs and enhance the occurrence of air–water exchange.

Cellulase production by solid state fermentation using bagasse with Penicillium decumbens L-06

The cellulase production byPenicillium decumbens L-06 in solid state fermentation (SSF) was investigated using bagasse as the substrate in this paper. The optimum conditions for cellulase production achieved by single factor testing were: the ratio of bagasse to wheat bran 1∶1 (w/w), the ratio of water to material 3∶1 (v/w), culture temperature 30 °C, initial pH 5.0, ammonium sulphate as nitrogen source with the concentration of 1%, 6 day’s fermentation period. BoxuBehnken factorial design (BBD) and response surface methodology (RSM) were further used to optimize conditions for cellulase (Filter paper activity) production. The maximal cellulase (Filter paper activity) production (3.89 FPu g−1) was obtained under the optimized conditions (ratio of water to material 2.38∶1, initial pH 5.28, cultivation time 150.5 h). It was well corresponded to the calculated results (3.97 FPu g−1) by model prediction.

Synergistic enzymatic saccharification and fermentation of agar for biohydrogen production

Nowadays, marine biomass is gradually considered as another utilizable material for the sustainable bioenergy development. In the present study, galactose, the main component of agar polysaccharide, was utilized for the biohydrogen production by Enterobacter sp. CN1. The highest hydrogen yield of 303.2mL/g was obtained in the cultivation media containing 5.87g/L of galactose, together with initial pH of 7.3 and incubation temperature of 36°C, after the response surface methodology (RSM) analysis. After the saccharification process by the agarase (AgaXa) and neoagarobiose hydrolase (NH852), the agar hydrolysate obtained was further applied to generate biohydrogen by strain CN1. Under the synergistic enzymatic saccharification and fermentation process, the production of biohydrogen was obtained to be 5047±228mL/L from 50g/L of agar, resulting in 3.86-fold higher than the control without enzymatic pretreatment.