Rising temperatures may increase growth rates and microcystin production in tropical Microcystis species

Rising temperatures (1.4–6 °C) due to climate change have been predicted to increase cyanobacterial bloom occurrences in temperate water bodies; however, the impacts of warming on tropical cyanobacterial blooms are unknown. We examined the effects of four different temperatures on the growth rates and microcystin (MC) production of five tropical Microcystis isolates (M. ichthyoblabe (two strains), M. viridis, M. flos-aquae, and M. aeruginosa). The temperature treatments are based on current temperature range in Singapore's reservoirs (27 °C and 30 °C), as well as projected mean (33 °C) and maximum temperatures (36 °C) based on tropical climate change estimates of +6 °C in air temperature. Increasing temperatures did not significantly affect the maximum growth rates of most Microcystis strains. Higher growth rates were only observed in one M. ichthyoblabe strain at 33 °C and M. flos-aquae at 30 °C where both were isolated from the same reservoir. MC-RR and MC-LR were produced in varying amounts by all four species of Microcystis. Raised temperatures of 33 °C were found to boost total MC cell quota for three Microcystis strains although further increase to 36 °C led to a sharp decrease in total MC cell quota for all five Microcystis strains. Increasing temperature also led to higher MC-LR:MC-RR cell quota ratios in M. ichthyoblabe. Our study suggests that higher mean water temperatures resulting from climate change will generally not influence growth rates of Microcystis spp. in Singapore except for increases in M. ichthyoblabe strains. However, toxin cell quota may increase under moderate warming scenarios depending on the species.     

Cyanotoxin diversity and food web bioaccumulation in a reservoir with decreasing phosphorus concentrations and perennial cyanobacterial blooms

In a shallow multifunction dam reservoir, perennial water blooms formed by several toxin-producing cyanobacteria (Anabaena spp., Aphanizomenon spp., Planktothrix agardhii and Microcystis spp.) were observed. Over a seven-year period, concomitantly with a gradual decrease in phosphate and total phosphorus concentrations in the water and an increase in the DIN to DIP ratio, a reduced biomass of cyanobacteria was noted. Simultaneously, a twofold increase in cyanobacterial species richness was found. The concentration of intracellular anatoxin-a was positively correlated with the total cyanobacterial biomass, but the concentration of intracellular microcystins was significantly negatively correlated with the level of phosphorus in the water. Therefore, in a period with a very low (2.3–3.6) DIN:DIP ratio, intracellular ANTX prevailed in the reservoir, while in the following years (at DIN:DIP = 23–36) much higher MC levels were noted. The highest total concentrations (22.2 μg L⁻¹) of intracellular MCs (MC-LF > -LY > -LR > -LA = -LW) and ANTX (14.4 μg L⁻¹) were found in 2010. In the following year, eight MC iso-forms were detected (MC-LF > -LY > -LA > -LR > -LW > -WR > -YR > -RR). The number of MC variants was positively correlated with the increased contribution of Anabaena planctonica/A. affinis and Microcystis spp. to cyanobacteria biomass. The indigenous bentho-pelagic fish Abramis brama L. accumulated in their tissues relatively high amounts of both ANTX (e.g. 6.2–18.4 μg g⁻¹ FW of liver) and different variants of MCs (up to 4.4 μg g⁻¹ FW of liver). Cyanotoxin tissue contents decreased in the following order: gills > liver > muscles. These observed strong changes in the species structure of cyanobacteria assemblages, even at their considerably smaller biomass, appeared to be an undesirable phenomenon due to the predominance of the efficient MC and ANTX producers, such as Anabaena spp., which is easily digested by fish. The variability of the profile of cyanobacterial blooms that depends on nutrient fluctuations and may account for the diverse toxin accumulation and tissue distribution in freshwater ichthyofauna is noteworthy, especially in water bodies used for fishery.     

Evidence of trophic transfer of microcystins from the gastropod Lymnaea stagnalis to the fish Gasterosteus aculeatus

According to our previous results the gastropod Lymnaea stagnalis exposed to MC-producing cyanobacteria accumulates microcystins (MCs) both as free and covalently bound forms in its tissues, therefore representing a potential risk of MC transfer through the food web. This study demonstrates in a laboratory experiment the transfer of free and bound MCs from L. stagnalis intoxicated by MC-producing Planktothrix agardhii ingestion to the fish Gasterosteus aculeatus. Fish were fed during five days with digestive glands of L. stagnalis containing various concentrations of free and bound MCs, then with toxin-free digestive glands during a 5-day depuration period. MC accumulation was measured in gastropod digestive gland and in various fish organs (liver, muscle, kidney, and gills). The impact on fish was evaluated through detoxification enzyme (glutathion-S-transferase, glutathion peroxydase and superoxyde dismutase) activities, hepatic histopathology, and modifications in gill ventilation, feeding and locomotion. G. aculeatus ingestion rate was similar with intoxicated and toxin-free diet. Fish accumulated MCs (up to 3.96 ± 0.14 μg g⁻¹ DW) in all organs and in decreasing order in liver, muscle, kidney and gills. Hepatic histopathology was moderate. Glutathion peroxydase was activated in gills during intoxication suggesting a slight reactive oxygen species production, but without any impact on gill ventilation. Intoxication via ingestion of MC-intoxicated snails impacted fish locomotion. Intoxicated fish remained significantly less mobile than controls during the intoxication period possibly due to a lower health condition, whereas they showed a greater mobility during the depuration period that might be related to an acute foraging for food. During depuration, MC elimination was total in gills and kidney, but partial in liver and muscle. Our results assess the MC transfer from gastropods to fish and the potential risk induced by bound MCs in the food web.     

Mechanism of photosynthetic response in Microcystis aeruginosa PCC7806 to low inorganic phosphorus

Photosynthetic response of Microcystis aeruginosa PCC7806 to different concentrations of phosphorus supply was studied so as to elucidate if the declining process of Microcystis bloom under freshwater ecosystem is related to soluble reactive phosphorus (SRP) decrease in water volume. Growth rate of M. aeruginosa PCC7806 was significantly reduced under P-deficient conditions, and its photosynthetic activity in terms of rETR_max (maximum electron transport rate) decreased significantly after 48 h growth, while it kept elevating and reached to a relative stable value when supplied with rich phosphorus of 0.6 mg/L. With the increasing actinic irradiance along the rapid light curves of M. aeruginosa PCC7806 cultured under low-phosphorus level, qP (photochemical quenching) and rETR (relative electron transport rate) decreased greatly, and the increase in qN (non-photochemical quenching) and Φ_PS (actual photochemical efficiency of PSII) was obviously inhibited. The affinity of M. aeruginosa PCC7806 to inorganic carbon was reduced evidently in 0.02 mg/L P compared with in 0.6 mg/L P. When P was reduced from 0.6 to 0.02 mg/L, the decreasing rate of rETR_max (77%) was significantly greater than that of photosynthetic carbon assimilation (22%), which indicated that down-regulation of CO₂ affinity caused by P-deficiency was, but not the only reason that resulted in the decline of photosynthetic efficiency. Instantaneous low-temperature significantly limited rETR_max under rich-P condition but had no effect on it when P was insufficient, and 1% ethanol could enhance rETR_max at low-P level but did not influence it at rich-P level. These two results proved that the decrease in thylakoid membrane fluidity caused by P-deficiency was another important reason that results in the decline of photosynthetic efficiency of M. aruginosa PCC7806.     

Estimating spatial variation in the abundance of potential microcystin-producing Microcystis spp. using real-time PCR during summer bloom in Lake Taihu

Lake Taihu, which is the third largest freshwater lakes in China, is a hypertrophic shallow lake in eastern China that has experienced lake-wide cyanobacterial blooms annually during the last few decades. In this study, quantitative real-time PCR assays targeting on phycocyanin intergenic spacer (PC-IGS) and a microcystin synthetase gene mcyD were established, respectively. Water samples collected from eight sampling sites (including Zhushan Bay (N5), Meiliang Bay (N2), Gonghu Bay (N4), West lake areas (W2 and W4), south-middle lake areas (S2, S4 and S5)) in August of 2009 and 2010 were analyzed using real time PCR for the distribution and abundance of toxic and total Microcystis populations. The results showed that Microcystis exists as a mixed population of potential toxic and non-toxic genotypes, and there was significant spatial changes in the abundance of potential toxic Microcystis on the basis of quantification by quantitative real-time PCR analysis: the abundance of toxic Microcystis population in 2009 and 2010 varied from 4.08 × 10⁴ to 8.28 × 10⁶ copies mL^?1, from 4.45 × 10⁵ to 5.22 × 10⁷ copies mL^?1, respectively. Meanwhile the ratio of the mcyD subpopulation to the total Microcystis varied considerably, from 5.7% to 41.1% in 2009 and from 10.3% to 65.8% in 2010 in all sampling sites, and the value is high in Zhushan Bay and Meiliang Bay with the high level of eutrophication. Correlation analysis showed the abundance of toxic and total Microcystis being strongly related (P < 0.01). However, there is different effects of environmental factors on the abundance of toxic and non-toxic Microcystis populations. The abundance of toxic and total Microcystis populations were positively correlated with chlorophyll-a (Chl-a) concentration (P < 0.01) suggesting that Microcystis is dominated genera of cyanobacterial bloom in Lake Taihu. It was also found that the abundance of toxic Microcystis and the proportion of toxic subpopulation to the total Microcystis were positively correlated with total phosphorus and orthophosphate concentrations (P < 0.01), whereas there was no significant correlation with total nitrogen and nitrate concentration (P > 0.05). All data suggest that phosphorus concentration is a critical factor for determining the abundance of toxic Microcystis population.     

Combined effect of probiotics and specific immunoglobulin Y directed against Escherichia coli on growth performance,diarrhea incidence,and immune system in calves

Enterotoxigenic Escherichia coli (ETEC) K99 is one of the major pathogens associated with calf diarrhea. The induction of passive immunity in animals by immunoglobulin Y and using probiotics are inexpensive alternatives to antibiotics for the prevention and treatment of a number of bacterial infections, including diarrhea. Hence, the aim of this research was to evaluate the impact of dietary probiotics and ETEC K99-specific egg yolk antibody supplements, alone and in combination with each other, on health and growth parameters, diarrhea incidence and immune stimulation in newborn Holstein calves. One hundred and twenty neonatal calves were allocated randomly into 4 dietary groups (n = 30 per group) received colostrum/milk without any additives (control group), or supplemented with egg yolk powder contained E. coli K99-specific antibody (Ab group; 1 g/day), a commercial probiotic, Hypro-calves (Pro group; 3 g/day), and their combination (Ab + Pro group), from day (d) 1 to d28 of age. Analyses of the growth parameters, feed efficiency, fecal score, and microbiota and immune function were carried out on d0, 14, 21, and 28 of the experiment. Calves in Ab or Ab + Pro group had higher (P < 0.05) average daily gain compared to control and Pro groups during 0–14d. Feed efficiency of calves in Ab and Ab + Pro groups was significantly higher than that in control group during the period of 0–14d; however, no significant differences were observed in 0–28d period. Diarrhea prevalence and fecal score in Ab + Pro group were lower than control group (P < 0.05). Calves in Ab + Pro group had the lowest number of fecal E. coli in comparison to other groups on d28 (P < 0.05). Feeding Ab + Pro supplement increased (P < 0.05) concentrations of blood IgA and serum CD4 compared to the control group. Likewise, calves in Pro group had higher CD4 levels as compared to the control calves (P < 0.05). Serum concentration of interferon-gamma in control group was lower than other groups (P < 0.05). Overall, these data suggest that feeding a combination of probiotic and specific antibody against ETEC to neonate Holstein calves enhances feed efficiency, boosts immunity, and reduces diarrhea prevalence.     

Distribution,occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009–2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.     

Uptake and inhibition kinetics of nitrogen in Microcystis aeruginosa: Results from cultures and field assemblages collected in the San Francisco Bay Delta,CA

The nitrogen (N) uptake kinetic parameters for Microcystis field assemblages collected from the San Francisco Bay Delta (Delta) in 2012 and non-toxic and toxic laboratory culture strains of M. aeruginosa were assessed. The ¹⁵N tracer technique was used to investigate uptake of ammonium (NH₄⁺), nitrate (NO₃⁻), urea and glutamic acid over short-term incubations (0.5–1 h), and to study inhibition of NO₃⁻, NH₄⁺ and urea uptake by NH₄⁺, NO₃⁻ and NH₄⁺, respectively. This study demonstrates that Delta Microcystis can utilize different forms of inorganic and organic N, with the greatest capacity for NH₄⁺ uptake and the least for glutamic acid uptake, although N uptake did not always follow the classic Michaelis–Menten hyperbolic relationship at substrate concentrations up to 67 μmol N L⁻¹. Current ambient N concentrations in the Delta may be at sub-saturating levels for N uptake, indicating that if N loading (especially NH₄⁺) were to increase, Delta Microcystis assemblages have the potential for increased N uptake rates. Delta Microcystis had the highest specific affinity, α, for NH₄⁺ and the lowest for NO₃⁻. In culture, N uptake by non-toxic and toxic M. aeruginosa strains was much higher than from the field, but followed similar N utilization trends to those in the field. Neither strain showed severe inhibition of NO₃⁻ uptake by NH₄⁺ or inhibition of NH₄⁺ uptake on NO₃⁻, but both strains showed some inhibition of urea uptake by NH₄⁺.     

Myricetin and quercetin are naturally occurring co-substrates of cyclooxygenases in vivo

Bioflavonoids are ubiquitously present in the plant kingdom, and some of them are presently being sold as healthy dietary supplements around the world. Recently, it was shown that some of the dietary polyphenols were strong stimulators of the catalytic activity of cyclooxygenase I and II, resulting in increased formation of certain prostaglandin (PG) products in vitro and also in intact cells in culture. In the present study, we investigated the effect of two representative dietary compounds, quercetin and myricetin, on plasma and tissue levels of several PG products in normal Sprague-Dawley rats. We found that these two dietary bioflavonoids could strongly stimulate the formation of PG products in vivo in a time-dependent manner, and the stimulatory effect of these two bioflavonoids was dose-dependent with a unique biphasic pattern. At lower doses (<0.3 mg/kg b.w.), they strongly stimulated the formation of PGE₂, but at higher doses (>0.3 mg/kg b.w.), there was a dose-dependent reduction of the stimulatory effect. These results provide support for the hypothesis that some of the bioflavonoids are naturally occurring physiological co-substrates for the cyclooxygenases in vivo.     

LOCALIZATION OF MICROCYSTIN SYNTHETASE GENES IN COLONIES OF THE CYANOBACTERIUM MICROCYSTIS USING FLUORESCENCE IN SITU HYBRIDIZATION1

To better understand the production of microcystins (MCs) in Microcystis colonies, fluorescence in situ hybridization (FISH) methods were developed to detect DNA involved in the synthesis of these cyanobacterial hepatotoxins. Using colonies of Microcystis aeruginosa (Kütz.) Kütz. isolated from environmental blooms of cyanobacteria and from a colony‐forming, MC‐producing laboratory strain of Microcystis, amplified PCR products were observed, coincident with positive controls. The total MC content of individual colonies of Microcystis, determined by ELISA, showed a positive correlation with colony cross‐sectional area. FISH analysis of Microcystis colonies gave high fluorescence in comparison to negative controls, indicating the presence of MC synthetase DNA (mcyA) in situ. FISH analysis for MC synthetase genes has the potential to be developed into an effective early warning tool for drinking and recreational water management.     

Anti-quorum sensing and anti-biofilm activity of Amomum tsaoko (Amommum tsao-ko Crevost et Lemarie) on foodborne pathogens

Cell-to-cell communication or quorum sensing (QS) leads to biofilm formation and causing other virulence factors which are extreme problems for food safety, biofilm related infectious diseases etc. This study evaluated the anti-QS activity of the Amomum tsaoko extract (0.5–4 mg/ml) by using Chromobacterium violaceum a biosensor strain and biofilm formation by crystal violate assay. Experimental results demonstrated that the overall yield of Amomum tsao-ko extract was 11.33 ± 0.3% (w/w). MIC for Staphylococcus aureus (Gram positive), Salmonella Typhimurium and Pseudomonas aeruginosa (Gram negative) was 1, 2 and 2 mg/ml, respectively. A concentration of 4 mg/ml extract showed highest biofilm inhibition 51.96% on S. Typhimurium when 47.06%, 45.28% were shown by S. aureus, P. aeruginosa respectively. The damage of biofilm architecture was observed by Confocal Laser Scanning Microscopy (CLSM). A level of 44.59% inhibition of violacein production was demonstrated when the dose was 4 mg/ml. Swarming motility inhibition was observed in a dose dependent manner. Taken together, the treatment of A. tsaoko extract can deliver value to food product and medicine by controlling pathogenesis.     

Comparison of ELISA and SPR biosensor technology for the detection of paralytic shellfish poisoning toxins

An enzyme labeled immunosorbent assay (ELISA) and surface plasmon resonance (SPR) biosensor assay for the detection of paralytic shellfish poisoning (PSP) toxins were developed and a comparative evaluation was performed. A polyclonal antibody (BC67) used in both assay formats was raised to saxitoxin–jeffamine–BSA in New Zealand white rabbits. Each assay format was designed as an inhibition assay. Shellfish samples (n = 54) were evaluated by each method using two simple rapid extraction procedures and compared to the AOAC high performance liquid chromatography (HPLC) and the mouse bioassay (MBA). The results of each assay format were comparable with the HPLC and MBA methods and demonstrate that an antibody with high sensitivity and broad specificity to PSP toxins can be applied to different immunological techniques. The method of choice will depend on the end-users needs. The reduced manual labor and simplicity of operation of the SPR biosensor compared to ELISA, ease of sample extraction and superior real time semi-quantitative analysis are key features that could make this technology applicable in a high-throughput monitoring unit.     

Environmental factors controlling colony formation in blooms of the cyanobacteria Microcystis spp. in Lake Taihu,China

Nitrogen (N) and phosphorus (P) over-enrichment has accelerated eutrophication and promoted cyanobacterial blooms worldwide. The colonial bloom-forming cyanobacterial genus Microcystis is covered by sheaths which can protect cells from zooplankton grazing, viral or bacterial attack and other potential negative environmental factors. This provides a competitive advantage over other phytoplankton species. However, the mechanism of Microcystis colony formation is not clear. Here we report the influence of N, P and pH on Microcystis growth and colony formation in field simulation experiments in Lake Taihu (China). N addition to lake water maintained Microcystis colony size, promoted growth of total phytoplankton, and increased Microcystis proportion as part of total phytoplankton biomass. Increases in P did not promote growth but led to smaller colonies, and had no significant impact on the proportion of Microcystis in the community. N and P addition together promoted phytoplankton growth much more than only adding N. TN and TP concentrations lower than about TN 7.75–13.95 mg L⁻¹ and TP 0.41–0.74 mg L⁻¹ mainly promoted the growth of large Microcystis colonies, but higher concentrations than this promoted the formation of single cells. There was a strong inverse relationship between pH and colony size in the N&P treatments suggesting CO₂ limitation may have induced colonies to become smaller. It appears that Microcystis colony formation is an adaptation to provide the organisms adverse conditions such as nutrient deficiencies or CO₂ limitation induced by increased pH level associated with rapidly proliferating blooms.     

Evidence of freshwater algal toxins in marine shellfish: Implications for human and aquatic health

The occurrence of freshwater harmful algal bloom toxins impacting the coastal ocean is an emerging threat, and the potential for invertebrate prey items to concentrate toxin and cause harm to human and wildlife consumers is not yet fully recognized. We examined toxin uptake and release in marine mussels for both particulate and dissolved phases of the hepatotoxin microcystin, produced by the freshwater cyanobacterial genus Microcystis. We also extended our experimental investigation of particulate toxin to include oysters (Crassostrea sp.) grown commercially for aquaculture. California mussels (Mytilus californianus) and oysters were exposed to Microcystis and microcystin toxin for 24 h at varying concentrations, and then were placed in constantly flowing seawater and sampled through time simulating riverine flushing events to the coastal ocean. Mussels exposed to particulate microcystin purged the toxin slowly, with toxin detectable for at least 8 weeks post-exposure and maximum toxin of 39.11 ng/g after exposure to 26.65 μg/L microcystins. Dissolved toxin was also taken up by California mussels, with maximum concentrations of 20.74 ng/g after exposure to 7.74 μg/L microcystin, but was purged more rapidly. Oysters also took up particulate toxin but purged it more quickly than mussels. Additionally, naturally occurring marine mussels collected from San Francisco Bay tested positive for high levels of microcystin toxin. These results suggest that ephemeral discharge of Microcystis or microcystin to estuaries and the coastal ocean accumulate in higher trophic levels for weeks to months following exposure.     

The efficiency of combined coagulant and ballast to remove harmful cyanobacterial blooms in a tropical shallow system

We tested the hypothesis that a combination of coagulant and ballast could be efficient for removal of positively buoyant harmful cyanobacteria in shallow tropical waterbodies, and will not promote the release of cyanotoxins. This laboratory study examined the efficacy of coagulants [polyaluminium chloride (PAC) and chitosan (made of shrimp shells)] alone, and combined with ballast (lanthanum modified bentonite, red soil or gravel) to remove the natural populations of cyanobacteria collected from a shallow eutrophic urban reservoir with alternating blooms of Cylindrospermopsis and Microcystis. PAC combined with ballast was effective in settling blooms dominated by Microcystis or Cylindrospermopsis. Contrary to our expectation, chitosan combined with ballast was only effective in settling Cylindrospermopsis-dominated blooms at low pH, whereas at pH ≥ 8 no effective flocculation and settling could be evoked. Chitosan also had a detrimental effect on Cylindrospermopsis causing the release of saxitoxins. In contrast, no detrimental effect on Microcystis was observed and all coagulant-ballast treatments were effective in not only settling the Microcystis dominated bloom, but also lowering dissolved microcystin concentrations. Our data show that the best procedure for biomass reduction also depends on the dominant species.     

Curcumin and docosahexaenoic acid block insulin-induced colon carcinoma cell proliferation

Diets high in fish and curcumin are associated with a decreased risk of CRC. Insulin resistance and obesity are associated with increased CRC risk and higher reoccurrence rates. We utilized cell culture to determine if dietary compounds could reduce insulin-induced cell proliferation comparing the response in normal and metastatic colon epithelial cells. We treated model normal murine colon epithelial cells (YAMC) and adenocarcinoma cells (MC38) with docosahexaenoic acid (DHA) or curcumin alone and then co-treatments of the diet-derived compound and insulin were combined. Cell proliferation was stimulated with insulin (1 ug/mL) to model insulin resistance in obesity. Despite the presence of insulin, proliferation was reduced in the MC38 cells treated with 10 μM curcumin (p<0.001) and 50 μM DHA (p<0.001). Insulin stimulated MAPK and MEK phosphorylation was inhibited by DHA and curcumin in MC38 cancer cells. Here we show that curcumin and DHA can block insulin-induced colon cancer cell proliferation in vitro via a MEK mediated mechanism.     

Co-expression of d-glucose isomerase and d-psicose 3-epimerase: Development of an efficient one-step production of d-psicose

d-Psicose has been attracting attention in recent years because of its alimentary activities and is used as an ingredient in a range of foods and dietary supplements. To develop a one-step enzymatic process of d-psicose production, thermoactive d-glucose isomerase and the d-psicose 3-epimerase obtained from Bacillus sp. and Ruminococcus sp., respectively, were successfully co-expressed in Escherichia coli BL21 strain. The substrate of one-step enzymatic process was d-glucose. The co-expression system exhibited maximum activity at 65 °C and pH 7.0. Mg²⁺ could enhance the output of d-psicose by 2.32 fold to 1.6 g/L from 10 g/L of d-glucose. When using high-fructose corn syrup (HFCS) as substrate, 135 g/L d-psicose was produced under optimum conditions. The mass ratio of d-glucose, d-fructose, and d-psicose was almost 3.0:2.7:1.0, when the reaction reached equilibrium after an 8 h incubation time. This co-expression system approaching to produce d-psicose has potential application in food and beverage products, especially softdrinks.     

A yeast co-culture-based biosensor for determination of waste water contamination levels

Artificial microbial co-cultures were formed to develop the receptor element of a biosensor for assessment of biological oxygen demand (BOD). The co-cultures possessed broad substrate specificities and enabled assays of water and fermentation products within a broad BOD range (2.4–80 mg/dm³) with a high correlation to the standard method (R = 0.9988). The use of the co-cultures of the yeasts Pichia angusta, Arxula adeninivorans and Debaryomyces hansenii immobilized in N-vinylpyrrolidone-modified poly(vinyl alcohol) enabled developing a BOD biosensor possessing the characteristics not inferior to those in the known biosensors. The results are indicative of a potential of using these co-cultures as the receptor element base in prototype models of instruments for broad application.     

Activation of nylon net and its application to a biosensor for determination of glucose in human serum

A glucose biosensor using a glucose oxidase (GO_x)-immobilized nylon net with glutaraldehyde as cross-linking reagent and an oxygen (O₂) electrode for the determination of glucose has been fabricated. The detection scheme was based on the utilization of dissolved O₂ in oxidation of glucose by the membrane bound GO_x. Crucial factors including O-alkylation temperature, reaction times of nylon net with dimethyl sulfate, l-lysine, and glutaraldehyde, and enzyme loading were examined to determine the optimal enzyme immobilization conditions for the best sensitivity of the developed glucose biosensor. In addition, the effects of pH and concentration of phosphate buffer on the response of the biosensor were studied. The glucose biosensor had a linear range of 18 μM to 1.10 mM with the detection limit of 9.0 μM (S/N = 3) and response time of 80 s. The biosensor exhibited both good operational stability with over 200 measurements and long-term storage stability. The results from this biosensor compared well with those of a commercial glucose assay kit in analyzing human serum glucose samples.     

Optimization of biomass production with enhanced glucan and dietary fibres content by Pleurotus ostreatus ATHUM 4438 under submerged culture

This work was aimed at optimizing biomass production by the edible basidiomycete Pleurotus ostreatus ATHUM 4438 in a submerged process with enhanced glucan and dietary fibres content. β-Glucan from Pleurotus sp. (pleuran) has been used as food supplements due to its immunosuppressive activity. Like other dietary fibre components, oyster mushroom polysaccharides can stimulate the growth of colon microorganisms (probiotics), i.e. act as prebiotics. We used the FF MicroPlate for substrate utilization and growth monitoring. The pattern of substrate catabolism forms a substrate assimilation fingerprint which is useful in selecting media components for media optimization of maximum biomass production. Different carbon sources (95) were used and then 8 of them were tested in shake flask cultures. The effect of various organic and complex nitrogen sources on biomass production was also examined and response surface methodology based on central composite design was applied to explore the optimal medium composition. When the optimized culture medium was tested in a 20-L stirred tank bioreactor, using 57 g L⁻¹ xylose and 37 g L⁻¹ corn steep liquor, high yields (39.2 g L⁻¹) of dry biomass was obtained. The yield coefficients for total glucan and dietary fibres on mycelial biomass formed were 140 ± 4 and 625 ± 9 mg g⁻¹ mycelium dry weight, respectively.