Production of specific-molecular-weight hyaluronan by metabolically engineered Bacillus subtilis 168

Low-molecular-weight hyaluronan (LMW-HA) has attracted much attention because of its many potential applications. Here, we efficiently produced specific LMW-HAs from sucrose in Bacillus subtilis. By coexpressing the identified committed genes (tuaD, gtaB, glmU, glmM, and glmS) and downregulating the glycolytic pathway, HA production was significantly increased from 1.01 g L⁻¹ to 3.16 g L⁻¹, with a molecular weight range of 1.40×10⁶–1.83×10⁶ Da. When leech hyaluronidase was actively expressed after N-terminal engineering (1.62×10⁶ U mL⁻¹), the production of HA was substantially increased from 5.96 g L⁻¹ to 19.38 g L⁻¹. The level of hyaluronidase was rationally regulated with a ribosome-binding site engineering strategy, allowing the production of LMW-HAs with a molecular weight range of 2.20×10³–1.42×10⁶ Da. Our results confirm that this strategy for the controllable expression of hyaluronidase, together with the optimization of the HA synthetic pathway, effectively produces specific LMW-HAs, and could also be used to produce other LMW polysaccharides.     

Bench-scale fermentation of Trichoderma viride on wastewater sludge: Rheology,lytic enzymes and biocontrol activity

Conidiation and lytic enzyme production by Trichoderma viride at different solids concentration of pre-treated municipal wastewater sludge was examined in a 15-L fermenter. The maximum conidia concentration (5.94 × 10⁷ CFU mL⁻¹ at 96 h) was obtained at 30 g L⁻¹ suspended solids. The maximum lytic enzyme activities were achieved around 12–30 h of fermentation. Bioassay against a fungal phytopathogen, Fusarium sp. showed maximum activity in the sample drawn around 96 h of fermentation at 30 g L⁻¹ suspended solids concentration. Entomotoxicity against spruce budworm larvae showed maximum value ≈17290 SBU μL⁻¹ at 30 g L⁻¹ suspended solids concentration at the end of fermentation (96 h). Plant bioassay showed dual action of T. viride, i.e., disease prevention and growth promotion. The rheological analyses of fermentation sludges showed the pseudoplastic behaviour. In order to maintain required dissolved oxygen concentration ≥30%, the agitation and aeration requirements significantly increased at 35 g L⁻¹ compared to 30 and 25 g L⁻¹. The oxygen uptake rate and volumetric oxygen mass transfer coefficient, k_La at 35 g L⁻¹ did not increase in comparison to 30 g L⁻¹ due to rheological complexity of the broth during fermentation. Thus, the successful fermentation operation of the biocontrol fungus T. viride is a rational indication of its potential for mass-scale production for agriculture and forest sector as a biocontrol agent.     

Forming nanoparticles of α-amylase and embedding them into solid surfaces

In the current work nanoparticles (NPs) of α-amylase were generated in an aqueous solution using high-intensity ultrasound, and were subsequently immobilized on polyethylene (PE) films, or polycarbonate (PC) plates, or on microscope glass slides. The α-amylase NPs coated on the solid surfaces have been characterized by ESEM, TEM, FTIR, XPS and AFM. The substrates immobilized with α-amylase were used for hydrolyzing soluble potato starch to maltose. The amount of enzyme introduced in the substrates, leaching properties, and the catalytic activity of the immobilized enzyme were compared. The catalytic activity of the amylase deposited on the three solid surfaces was compared to that of the same amount of free enzyme at different pHs and temperatures. α-Amylase coated on PE showed the best catalytic activity in all the examined parameters when compared to native amylase, especially at high temperatures. When immobilized on glass, α-amylase showed better activity than the native enzyme over all pH and temperature values studied. However, the immobilization on PC did not improve the enzyme activity at any pH and any temperature compared to the free amylase. The kinetic parameters, K_m and V_max were also calculated. The amylase coated PE showed the most favorable kinetic parameters (K_m = 5 g L⁻¹ and V_max = 5E−07 mol mL⁻¹ min⁻¹). In contrast, the anchored enzyme-PC exhibited unfavorable kinetic parameters (K_m = 16 g L⁻¹, V_max = 4.2E−07 mol mL⁻¹ min⁻¹). The corresponding values for amylase-glass were K_m = 7 g L⁻¹, V_max = 1.8E−07 mol mL⁻¹ min⁻¹, relative to those obtained for the free enzyme (K_m = 6.6 g L⁻¹, V_max = 3.3E−07 mol mL⁻¹ min⁻¹).     

Volatile fatty acids accumulation and rhamnolipid generation in situ from waste activated sludge fermentation stimulated by external rhamnolipid addition

The solubilization and acidification of waste activated sludge (WAS) were apparently enhanced by external rhamnolipid (RL) addition. The maximum solute carbohydrate concentrations increased linearly from 48 ± 5 mg COD L⁻¹ in the un-pretreated WAS (blank) to 566 ± 19 mg COD L⁻¹, and protein increased from 1050 ± 8 to 3493 ± 16 mg COD L⁻¹ at RL dosage of 0.10 g g⁻¹ TSS. The highest VFAs concentration peaked at 3840 mg COD L⁻¹ at RL dosage of 0.04 g g⁻¹ TSS, which was 4.24-fold higher than the blank test. RL was generated in situ during WAS fermentation when external RL was added. It was detected that RL concentration was increased from initial 880 ± 92 mg L⁻¹ to 1312 ± 7 mg L⁻¹ at the end of 96 h with RL dosage of 0.04 g g⁻¹ TSS, which was increased to 1.49-fold. Meanwhile, methane production was notably reduced to a quite low level of 2.0 mL CH₄ g⁻¹ VSS, showing effective inhibition of methanogens by RL (58.8 mL CH₄ g⁻¹ VSS in the blank). In addition, the activity of hydrolytic enzymes (protease and α-glucosidase) was enhanced accordingly. VFAs accumulation and RL generation in situ demonstrated that the additional RL substantially performed enhanced biological effects for waste activated sludge fermentation.     

Urine nitrification with a synthetic microbial community

During long-term extra-terrestrial missions, food is limited and waste is generated. By recycling valuable nutrients from this waste via regenerative life support systems, food can be produced in space. Astronauts’ urine can, for instance, be nitrified by micro-organisms into a liquid nitrate fertilizer for plant growth in space. Due to stringent conditions in space, microbial communities need to be be defined (gnotobiotic); therefore, synthetic rather than mixed microbial communities are preferred. For urine nitrification, synthetic communities face challenges, such as from salinity, ureolysis, and organics.In this study, a synthetic microbial community containing an AOB (Nitrosomonas europaea), NOB (Nitrobacter winogradskyi), and three ureolytic heterotrophs (Pseudomonas fluorescens, Acidovorax delafieldii, and Delftia acidovorans) was compiled and evaluated for these challenges. In reactor 1, salt adaptation of the ammonium-fed AOB and NOB co-culture was possible up to 45 mS cm⁻¹, which resembled undiluted nitrified urine, while maintaining a 44 ± 10 mg NH₄⁺–N L⁻¹ d⁻¹ removal rate. In reactor 2, the nitrifiers and ureolytic heterotrophs were fed with urine and achieved a 15 ± 6 mg NO₃⁻–N L⁻¹ d⁻¹ production rate for 1% and 10% synthetic and fresh real urine, respectively. Batch activity tests with this community using fresh real urine even reached 29 ± 3 mg N L⁻¹ d⁻¹. Organics removal in the reactor (69 ± 15%) should be optimized to generate a nitrate fertilizer for future space applications.     

d- and l-lactic acid production from fresh sweet potato through simultaneous saccharification and fermentation

The aim of this study was to develop a bioprocess for l- and d-lactic acid production from raw sweet potato through simultaneous saccharification and fermentation by Lactobacillus paracasei and Lactobacillus coryniformis, respectively. The effects of enzyme and nitrogen source concentrations as well as of the ratio of raw material to medium were investigated. At dried material concentrations of 136.36–219.51 g L⁻¹, yields of 90.13–91.17% (w/w) and productivities of 3.41–3.83 g L⁻¹ h⁻¹ were obtained with lactic acid concentrations as high as 198.32 g L⁻¹ for l-lactic acid production. In addition, d-lactic acid was produced with yields of 90.11–84.92% (w/w) and productivities of 2.55–3.11 g L⁻¹ h⁻¹ with a maximum concentration of 186.40 g L⁻¹ at the same concentrations of dried material. The simple and efficient process described in this study will benefit the tuber and root-based lactic acid industries without requiring alterations in plant equipment.     

Lethal effects of ichthyotoxic raphidophytes,Chattonella marina,C. antiqua,and Heterosigma akashiwo,on post-embryonic stages of the Japanese pearl oyster,Pinctada fucata martensii

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5 × 10² cells ml⁻¹), C. antiqua (10³ cells ml⁻¹), and H. akashiwo (5 × 10³ cells ml⁻¹) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5 × 10³ cells ml⁻¹ and C. marina at 8 × 10³ cells ml⁻¹. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 10⁵ cells ml⁻¹ more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8 × 10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (10⁴ cells ml⁻¹, 24 h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (5 × 10⁴ cells ml⁻¹, 24 h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5 × 10²–8 × 10³ cells ml⁻¹, 48–86 h) and C. antiqua (10³–8 × 10³ cells ml⁻¹, 72–86 h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.     

The effects of light intensity on the growth of Japanese Gambierdiscus spp. (Dinophyceae)

Marine toxic dinoflagellates of the genus Gambierdiscus are the causative agents of ciguatera fish poisoning (CFP), a form of seafood poisoning that is widespread in tropical, subtropical and temperate regions worldwide. The distributions of Gambierdiscus australes, Gambierdiscus scabrosus and two phylotypes of Gambierdiscus spp. type 2 and type 3 have been reported for the waters surrounding the main island of Japan. To explore the bloom dynamics and the vertical distribution of these Japanese species and phylotypes of Gambierdiscus, the effects of light intensity on their growth were tested, using a photoirradiation-culture system. The relationship between the observed growth rates and light intensity conditions for the four species/phylotypes were formulated at R > 0.92 (p < 0.01) using regression analysis and photosynthesis-light intensity (P-L) model. Based on this equation, the optimum light intensity (L_max) and the semi-optimum light intensity range (L_s-opt) that resulted in the maximum growth rate (μ_max) and ≥80% μ _max values of the four species/phylotypes, respectively, were as follows: (1) the L_max and L_s-opt of G. australes were 208 μmol photons m⁻² s⁻¹ and 91–422 μmol photons m⁻² s⁻¹, respectively; (2) those of G. scabrosus were 252 and 120–421 μmol photons m⁻² s⁻¹, respectively; (3) those of Gambierdiscus sp. type 2 were 192 and 75–430 μmol photons m⁻² s⁻¹, respectively; and (4) those of Gambierdiscus sp. type 3 were ≥427 and 73–427 μmol photons m⁻² s⁻¹, respectively. All four Gambierdiscus species/phylotypes required approximately 10 μmol photons m⁻² s⁻¹ to maintain growth. The light intensities in coastal waters at a site in Tosa Bay were measured vertically at 1 m intervals once per season. The relationships between the observed light intensity and depth were formulated using Beer’s Law. Based on these equations, the range of the attenuation coefficients at Tosa Bay site was determined to be 0.058–0.119 m⁻¹. The values 1700 μmol photons m⁻² s⁻¹, 500 μmol photons m⁻² s⁻¹, and 200 μmol photons m⁻² s⁻¹ were substituted into the equations to estimate the vertical profiles of light intensity at sunny midday, cloudy midday and rainy midday, respectively. Based on the regression equations coupled with the empirically determined attenuation coefficients for each of the four seasons, the ranges of the projected depths of L_max and L_s-opt for the four Gambierdiscus species/phylotypes under sunny midday conditions, cloudy midday conditions, and rainy midday conditions were 12–38 m and 12–54 m, 1–16 m and 1–33 m, and 0 m and 0–16 m, respectively. These results suggest that light intensity plays an important role in the bloom dynamics and vertical distribution of Gambierdiscus species/phylotypes in Japanese coastal waters.     

Growth of the fungus Paecilomyces lilacinus with n-hexadecane in submerged and solid-state cultures and recovery of hydrophobin proteins

The filamentous fungus Paecilomyces lilacinus was grown on n-hexadecane in submerged (SmC) and solid-state (SSC) cultures. The maximum CO₂ production rate in SmC (V_max = 11.7 mg CO₂ L_g⁻¹ day⁻¹) was three times lower than in SSC (V_max = 40.4 mg CO₂ L_g⁻¹ day⁻¹). The P. lilacinus hydrophobin (PLHYD) yield from the SSC was 1.3 mg PLHYD g protein⁻¹, but in SmC, this protein was not detected. The PLHYD showed a critical micelle concentration of 0.45 mg mL⁻¹. In addition, the PLHYD modified the hydrophobicity of Teflon from 130.1 ± 2° to 47 ± 2°, forming porous structures with some filaments <1 μm and globular aggregates <0.25 μm diameter. The interfacial studies of this PLHYD could be the basis for the use of the protein to modify surfaces and to stabilize compounds in emulsions.     

Biochemical and biophysical combined study of bicarinalin,an ant venom antimicrobial peptide

We have recently characterized bicarinalin as the most abundant peptide from the venom of the ant Tetramorium bicarinatum. This antimicrobial peptide is active against Staphylococcus and Enterobacteriaceae. To further investigate the antimicrobial properties of this cationic and cysteine-free peptide, we have studied its antibacterial, antifungal and antiparasitic activities on a large array of microorganisms. Bicarinalin was active against fifteen microorganisms with minimal inhibitory concentrations ranging from 2 and 25 μmol L⁻¹. Cronobacter sakazakii, Salmonella enterica, Candida albicans, Aspergilus niger and Saccharomyces cerevisiae were particularly susceptible to this novel antimicrobial peptide. Resistant strains of Staphylococcus aureus, Pseudomonas aeruginosa and C. albicans were as susceptible as the canonical strains. Interestingly, bicarinalin was also active against the parasite Leishmania infantum with a minimal inhibitory concentrations of 2 μmol L⁻¹. The bicarinalin pre-propeptide cDNA sequence has been determined using a combination of degenerated primers with RACE PCR strategy. Interestingly, the N-terminal domain of bicarinalin pre-propeptide exhibited sequence similarity with the pilosulin antimicrobial peptide family previously described in the Myrmecia venoms. Moreover, using SYTOX green uptake assay, we showed that, for all the tested microorganisms, bicarinalin acted through a membrane permeabilization mechanism. Two dimensional-NMR experiments showed that bicarinalin displayed a 10 residue-long α-helical structure flanked by two N- and C-terminal disordered regions. This partially amphipathic helix may explain the membrane permeabilization mechanism of bicarinalin observed in this study. Finally, therapeutic value of bicarinalin was highlighted by its low cytotoxicity against human lymphocytes at bactericidal concentrations and its long half-life in human serum which was around 15 h.     

An amperometric biosensor based on horseradish peroxidase immobilized onto maize tassel-multi-walled carbon nanotubes modified glassy carbon electrode for determination of heavy metal ions in aqueous solution

A biosensor for trace metal ions based on horseradish peroxidase (HRP) immobilized on maize tassel-multiwalled carbon nanotube (MT-MWCNT) through electrostatic interactions is described herein. The biosensor was characterized using Fourier transform infrared (FTIR), UV–vis spectrometry, voltammetric and amperometric methods. The FTIR and UV–vis results inferred that HRP was not denatured during its immobilization on MT-MWCNT composite. The biosensing principle was based on the determination of the cathodic responses of the immobilized HRP to H₂O₂, before and after incubation in trace metal standard solutions. Under optimum conditions, the inhibition rates of trace metals were proportional to their concentrations in the range of 0.092–0.55 mg L⁻¹, 0.068–2 mg L⁻¹ for Pb²⁺ and Cu²⁺ respectively. The limits of detection were 2.5 μg L⁻¹ for Pb²⁺ and 4.2 μg L⁻¹ for Cu²⁺. Representative Dixon and Cornish-Bowden plots were used to deduce the mode of inhibition induced by the trace metal ions. The inhibition was reversible and mixed for both metal ions. Furthermore, the biosensor showed good stability, selectivity, repeatability and reproducibility.     

A new bioprocess for the production of prebiotic lactosucrose by an immobilized β-galactosidase

A new bioprocess for the synthesis of lactosucrose was studied using a covalently immobilized β-galactosidase on macrospheres of chitosan. The effects of temperature and pH on the production of lactosucrose and other oligosaccharides were evaluated. At 30 °C and pH 7.0, the maximum concentration of lactosucrose reached to 79 g L⁻¹. The change of the reaction conditions allowed to modify the qualitative profile of the final products without quantitative change in the total of oligosaccharides produced. At pH 7 and 30 °C, products profile was 79 g L⁻¹ of lactosucrose, 37 g L⁻¹ of galactooligosaccharides and 250 g L⁻¹ of total oligosaccharides, while at pH 5 and 64 °C the concentrations for the same compounds were 40, 62 and 250 g L⁻¹, respectively. The immobilization increased the thermal stability up to 260-fold. Using 300 g L⁻¹ of sucrose and 300 g L⁻¹ of lactose, and 8.5 mg of chitosan mL⁻¹, 30 cycles of reuse were performed and the biocatalyst kept the maximal lactosucrose synthesis. These results fulfill some important aspects for the enzyme immobilization and oligosaccharides synthesis: the simplicity of the protocols, the high operational stability of the enzyme and the possibility of driving the final products.     

CO2 fixation for succinic acid production by engineered Escherichia coli co-expressing pyruvate carboxylase and nicotinic acid phosphoribosyltransferase

In wild-type Escherichia coli, 1 mol of CO₂ was fixated in 1 mol of succinic acid generation anaerobically. The key reaction in this sequence, catalyzed by phosphoenolpyruvate carboxylase (PPC), is carboxylation of phosphoenolpyruvate to oxaloacetate. Although inactivation of pyruvate formate-lyase and lactate dehydrogenase is found to enhance the PPC pathway for succinic acid production, it results in excessive pyruvic acid accumulation and limits regeneration of NAD⁺ from NADH formed in glycolysis. In other organisms, oxaloacetate is synthesized by carboxylation of pyruvic acid by pyruvate carboxylase (PYC) during glucose metabolism, and in E. coli, nicotinic acid phosphoribosyltransferase (NAPRTase) is a rate-limiting enzyme of the NAD(H) synthesis system. To achieve the NADH/NAD⁺ ratio decrease as well as carbon flux redistribution, co-expression of NAPRTase and PYC in a pflB, ldhA, and ppc deletion strain resulted in a significant increase in cell mass and succinic acid production under anaerobic conditions. After 72 h, 14.5 g L⁻¹ of glucose was consumed to generate 12.08 g L⁻¹ of succinic acid. Furthermore, under optimized condition of CO₂ supply, the succinic acid productivity and the CO₂ fixation rate reached 223.88 mg L⁻¹ h⁻¹ and 83.48 mg L⁻¹ h⁻¹, respectively.     

Effect of Zinc nanoparticles on oxidative stress-related genes and antioxidant enzymes activity in the brain of Oreochromis niloticus and Tilapia zillii

This study was carried out to determine the median lethal concentrations (LC₅₀) of Zinc nanoparticles (ZnNPs) on Oreochromis niloticus and Tilapia zillii. The biochemical and molecular potential effects of ZnNPs (500 and 2000 μg L⁻¹) on the antioxidant system in the brain tissue of O. niloticus and T. zillii were investigated. Four hundred fish were used for acute and sub-acute studies. ZnNP LC₅₀ concentrations were investigated in O. niloticus and T. zillii. The effect of 500 and 2000 μg L⁻¹ ZnNPs on brain antioxidants of O. niloticus and T. zillii was investigated. The result indicated that 69 h LC₅₀ was 5.5 ± 0.6 and 5.6 ± 0.4 for O. nilotica and T. zillii, respectively. Fish exposed to 500 μg L⁻¹ ZnNPs showed a significant increase in reduced glutathione (GSH), total glutathione (tGSH) levels, superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) activity and gene expression. On the contrary, malondialdehyde (MDA) levels significantly decreased. Meanwhile, fish exposed to 2000 μg L⁻¹ ZnNPs showed a significant decrease of GSH, tGSH levels, SOD, CAT, GR, GPx and GST activity and gene expression. On the contrary, MDA levels significantly increased. It was concluded that, the 96 h LC₅₀ of ZnNPs was 5.5 ± 0.6 and 5.6 ± 0.4 for O. nilotica and T. zillii, respectively. ZnNPs in exposure concentrations of 2000 μg/L induced a deleterious effect on the brain antioxidant system of O. nilotica and T. zillii. In contrast, ZnNPs in exposure concentrations of 500 μg L⁻¹ produced an inductive effect on the brain antioxidant system of O. nilotica and T. zillii.     

Sulfate reduction and microbial community of autotrophic biocathode in response to acidity

Microbial electrolysis cells (MECs) with autotrophic biocathode are a promising technology for removal of pollutants in wastewater. The aim of this study was to investigate the effect of initial acidity of wastewater on performance of sulfate-reducing biocathodes. MECs with biocathodes were operated with initial pH values of catholyte ranged from 3.0 to 7.0. The optimum initial pH value was 6.0 with a maximum sulfate reductive rate and biomass of 57 mg L⁻¹ d⁻¹ and 2.1 ± 0.4 mg g⁻¹, respectively. With initial pH 7.0, the pH value of catholyte increased to 9.8 ± 0.2 after an operation cycle, which resulted in low performance of the biocathode. A considerable sulfate reductive rate of 31 ± 0.85 mg L⁻¹ d⁻¹ was achieved with initial pH 3.0. Desulfovibrio sp. grew dominantly with abundance of 46%–66% in the cathode biofilm with initial pH values from 3.0 to 6.0 and contributed to the sulfate reduction. Clostridium and Parapedobacter also had high abundance in pH 6.0 cathode, indicated that interspecies electron transfer between electrochemical active and sulfate-reducing bacteria could play an important role in sulfate removal. The results suggest that acidity of catholyte is an important factor to be considered to utilize autotrophic biocathode MECs for wastewater treatment.     

Investigation on the properties and kinetics of glucose-fed aerobic granular sludge

Aerobic granulation is a process in which suspended biomass aggregate and form discrete well-defined granules in aerobic systems. To investigate the properties and kinetics of aerobic granular sludge, aerobic granules were cultivated with glucose synthetic wastewater in a series of sequencing batch reactors (SBR). The spherical shaped granules were observed on 8th day with the mean diameter of 0.1 mm. With the organic loading rate (OLR) being increased to 4.0 g COD L⁻¹ d⁻¹, aerobic granules grew matured with spherical shape. The size of granules ranged from 1.2 to 1.8 mm, and the corresponding settling velocity of individual granule was 24.2–36.4 m h⁻¹. The oxygen utilization rate (OUR) of mature granules was 41.90 g O₂ kg MLSS⁻¹ h⁻¹, which was two times higher than that of activated sludge (18.32 g O₂ kg MLSS⁻¹ h⁻¹). The experimental data indicated that the substrate utilization and biomass growth kinetics generally followed Monod's kinetics model. The corresponding kinetic coefficients of k (maximum specific substrate utilization rate), K_s (half velocity coefficient), Y (growth yield coefficient) and K_d (decay coefficient) were determined as follows, k_c = 23.65 d⁻¹, K_c = 3367.05 mg L⁻¹, K_N = 0.038 d⁻¹, K_N = 29.65 mg L⁻¹, Y = 0.1927–0.2022 mg MMLS (mg COD)⁻¹ and K_d = 0.00845–0.0135 d⁻¹, respectively. Those properties of aerobic granules made aerobic granules system had a short setup period, high substrate utilization rate and low sludge production.     

Effect of separate and combined exposure of selenium and diazinon on rat sperm motility by computer assisted semen analysis

Effects of selenium (Se) and diazinon (DZN) on sperm motility parameters in rats were investigated. Male rats received a separate dose of Se (2 mg kg⁻¹ b.w., intraperitoneally, 5 mg L⁻¹, per os in drinking water), diazinon (20 mg kg⁻¹ b.w., intraperitoneally, 40 mg L⁻¹, per os in drinking water), and in combination (Se + DZN) with the same dosage as in the separate administration. 36 h an intraperitoneal (i.p.) and after 90 days of per oral (p.o.) exposure, thirteen parameters of sperm motility were evaluated using a Computer Assisted Sperm Analyzer (CASA). Almost all the evaluated sperm motility parameters significantly decreased in Se p.o. exposed groups. In the Se i.p. group decrease was noted only in beat cross frequency (BCF) and progressive motility. Significant decline in the sperm motility, progressive motility, BCF and increase in amplitude of lateral head displacement (ALH) were recorded after DZN i.p. administration. In DZN p.o. group, significant increase in ALH, velocity average path (VAP) and curvilinear velocity (VCL) but decrease in progressive motility and BCF was detected. Se + DZN i.p. administration caused a significant decrease in motility, progressive motility and BCF. Per oral administration of Se + DZN decreased all motility parameters except LIN, WOB and ALH. Sperm abnormalities increased in all experimental conditions. Se and DZN negatively affected sperm structure and function in separate doses or in combination. No protective effect of Se was observed.     

Galactose-limited fed-batch cultivation of Escherichia coli for the production of lacto-N-tetraose

Lacto-N-tetraose (Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc) is one of the most abundant oligosaccharide structures in human milk. We recently described the synthesis of lacto-N-tetraose by a whole-cell biotransformation with recombinant Escherichia coli cells. However, only about 5% of the lactose was converted into lacto-N-tetraose by this approach. The major product obtained was the intermediate lacto-N-triose II (GlcNAc(β1-3)Gal(β1-4)Glc).In order to improve the bioconversion of lactose to lacto-N-tetraose, we have investigated the influence of the carbon source on the formation of lacto-N-tetraose and on the intracellular availability of the glycosyltransferase substrates, UDP-N-acetylglucosamine and UDP-galactose. By growth of the recombinant E. coli cells on D-galactose, the yield of lacto-N-tetraose (810.8 mg L⁻¹ culture) was 3.6-times higher compared to cultivation on D-glucose.Using fed-batch cultivation with galactose as sole energy and carbon source, a large-scale synthesis of lacto-N-tetraose was demonstrated. During the 26 h feeding phase the growth rate (μ = 0.05) was maintained by an exponential galactose feed. In total, 16 g L⁻¹ lactose were fed and resulted in final yields of 12.72 ± 0.21 g L⁻¹ lacto-N-tetraose and 13.70 ± 0.10 g L⁻¹ lacto-N-triose II. In total, 173 g of lacto-N-tetraose were produced with a space-time yield of 0.37 g L⁻¹ h⁻¹.     

Investigation on the formation and kinetics of glucose-fed aerobic granular sludge

Aerobic granular sludge was cultivated in a glass sequencing batch reactor (SBR) with glucose synthetic wastewater. The spherical shaped granules were observed on 4th day with the mean diameter of 0.1 mm. With the increase of chemical oxygen demand (COD) concentration of the influent, aerobic granules grew matured, the size of which ranged from 1.2 to 1.9 mm. The aerobic granular sludge could sustain high organic loading rate (about 4.0 g COD L⁻¹ d⁻¹), with good settling ability (settling velocity 36 m/h) and high biomass concentration (MLSS 6.7 ±0.2 g/L). Experimental data indicated that the substrate utilization and biomass growth kinetics followed Monod's kinetics model approximately. The corresponding kinetic coefficients of maximum specific substrate utilization rate (k), half velocity coefficient (K_s), growth yield coefficient (Y) and decay coefficient (K_d) were 13.2 d⁻¹, 275.8 mg/L, 0.183–0.250 mg MLSS/mg COD and 0.023–0.075 d⁻¹, respectively, which made aerobic granules have short setup period, high rate of substrate utilization and little surplus sludge.     

Thalassia testudinum response to the interactive stressors hypersalinity,sulfide and hypoxia

A large-scale mesocosm (sixteen 500 L tanks) experiment was conducted to investigate the effects of hypersalinity (45–65 psu), porewater sulfide (2–6 mM) and nighttime water column hypoxia (5–3 mg L⁻¹) on the tropical seagrass Thalassia testudinum Banks ex König. We examined stressor effects on growth, shoot survival, tissue sulfur (S⁰, TS, δ³⁴S) and leaf quantum efficiencies, as well as, porewater sulfides (∑TS_pw) and mesocosm water column O₂ dynamics. Sulfide was injected into intact seagrass cores of T. testudinum exposing below-ground tissues to 2, 4, and 6 mM S²⁻, but rapid oxidation resulted in ∑TS_pw < 1.5 mM. Hypersalinity at 65 psu lowered sulfide oxidation and significantly affected plant growth rates and quantum efficiencies (F_v/F_m < 0.70). The most depleted rhizome δ³⁴S signatures were also observed at 65 psu, suggesting increased sulfide exposure. Hypoxia did not influence ∑TS_pw and plant growth, but strengthened the hypersalinity response and decreased rhizome S⁰, indicating less efficient oxidation of ∑TS_pw. Following nighttime hypoxia treatments, ecosystem level metabolism responded to salinity treatments. When O₂ levels were reduced to 5 and 4 mg L⁻¹, daytime O₂ levels recovered to approximately 6 mg L⁻¹; however, this recovery was more limited when O₂ levels were lowered to 3 mg L⁻¹. Subsequent to O₂ reductions to 3 mg O₂ L⁻¹, nighttime O₂ levels rose in the 35 and 45 psu tanks, stayed the same in the 55 psu tanks, and declined in the 65 psu tanks. Thus, hypersalinity at 65 psu affects T. testudinum's oxidizing capacity and places subtle demands on the positive O₂ balance at an ecosystem level. This O₂ demand may influence T. testudinum die-off events, particularly after periods of high temperature and salinity. We hypothesize that the interaction between hypersalinity and sulfide toxicity in T. testudinum is their synergistic effect on the critical O₂ balance of the plant.