Early detection of spoilage moulds in bread using volatile production patterns and quantitative enzyme assays

AIMS: Early detection of spoilage fungi (two Eurotium spp., a Penicillium chrysogenum species) in bread analogues over periods of 72 h at 25 degrees C and 0.95 water activity was evaluated using volatile production patterns, hydrolytic enzyme production, and changes in fungal populations. METHODS AND RESULTS: Using an electronic nose system it was possible to differentiate between uninoculated controls and samples contaminated with P. chrysogenum within 28 h. After 40-48 h it was possible to differentiate between the Eurotium spp., P. chrysogenum and the control using Principal Component Analysis (PCA). Cluster analyses could differentiate between the control, P. chrysogenum and the Eurotium spp. after 40 h. Of seven hydrolytic enzymes examined after 48 h, the specific activities of three were significantly different from uninoculated control bread. There were also differences between the mould species in production of three enzymes. Penicillium chrysogenum populations increased fastest, from about 10(3) cfu g(-1) to 10(6)-10(7) cfu g(-1) after 72 h. For the Eurotium spp. this increase was slower. CONCLUSIONS: Overall, this study suggests, for the first time, that an electronic nose system using a surface polymer sensor array is able to detect qualitative changes in volatile production patterns for the early detection of the activity of spoilage moulds in bakery products. SIGNIFICANCE AND IMPACT OF THE STUDY: Potential exists for application of such systems for microbial quality assurance in intermediate moisture food products.     

A simple and easy method for the monitoring of environmental pollutants using oligotrophic bacteria

Sphingomonas paucimobilis KPS01, an oligotrophic bacterium isolated from soil, may be a useful tool for monitoring heavy metals. Previous methods relying on counting of viable cells require a relatively long time and some skill; we have developed a method based on optical density (O.D.) measurements which is significantly faster and does not require skilled personnel. The results of the O.D. and viable count methods were consistent; both methods detected heavy metals at concentrations ranging from 10-3 to 10-5 mmol l-1 and identified heavy metal contamination in 13 of 18 river water samples. Our results demonstrate that biological detection using this O.D. method and S. paucimobilis KPS01 may be useful for routine environmental monitoring of heavy metals, particularly in water sources.     

Effect of high concentration of Co (II) on Enterobacter liquefaciens strain C-1: a bacterium highly resistant to heavy metals with an unknown genome

Heavy metals are required as nutrients for essential functions in microorganisms. However, higher concentrations of these cations are generally toxic and may produce contrasting effects on living organisms. Enterobacter liquefaciens strain C-1, a bacterium isolated from the Moa mine in Cuba, is able to survive in the presence of high concentrations of heavy metals. The proteomes of Enterobacter liquefaciens strain C-1, grown under aerobic conditions in the presence and absence of Co (II) were compared using two-dimensional gel electrophoresis analysis in the isoelectric point range of 4-7 and the mass range of 15-120 kDa. Significant changes in the expression level (> two-fold) were detected for 13 spots: seven and six were up- and down-regulated, respectively. Because the genome of this bacterium is unknown, identification by peptide mass fingerprinting only succeeded in four cases and most of the cross-species identifications were supported by de novo sequencing of tryptic peptides followed by sequence alignment using the MS BLAST program. Twelve different proteins were identified, ten are involved in cellular antioxidant defence probably induced by the presence of Co (II). This is the first step towards understanding the role of proteins participating in the mechanism of resistance to heavy metals in this bacterium.     

Heavy metals (Cd, Pb, Zn, Ni, Cu and Cr(III)) removal from water in Malaysia: post treatment by high quality limestone

This paper presents the results of research on heavy metals removal from water by filtration using low cost coarse media which could be used as an alternative approach to remove heavy metals from water or selected wastewater. A series of batch studies were conducted using different particle media (particle size 2.36-4.75 mm) shaken with different heavy metal solutions at various pH values to see the removal behaviour for each metal. Each solution of cadmium (Cd), lead (Pb), zinc (Zn), nickel (Ni), copper (Cu) and chromium (Cr(III)) with a concentration of 2 mg/L was shaken with the media. At a final pH of 8.5, limestone has significantly removed more than 90% of most metals followed by 80% and 65% removals using crushed bricks and gravel, respectively. The removal by aeration and settlement methods without solid media was less than 30%. Results indicated that the removal of heavy metals was influenced by the media and not directly by the pH. Investigations on the removal behaviour of these metals indicated that rough solid media with the presence of carbonate were beneficial for the removal process. Adsorption and precipitation as metals oxide and probably as metals carbonate were among the two mechanisms that contributed to the removal of metals from solution.     

Sustainable sources of biomass for bioremediation of heavy metals in waste water derived from coal-fired power generation

Biosorption of heavy metals using dried algal biomass has been extensively described but rarely implemented. We contend this is because available algal biomass is a valuable product with a ready market. Therefore, we considered an alternative and practical approach to algal bioremediation in which algae were cultured directly in the waste water stream. We cultured three species of algae with and without nutrient addition in water that was contaminated with heavy metals from an Ash Dam associated with coal-fired power generation and tested metal uptake and bioremediation potential. All species achieved high concentrations of heavy metals (to 8% dry mass). Two key elements, V and As, reached concentrations in the biomass of 1543 mg.kg(-1) DW and 137 mg.kg(-1) DW. Growth rates were reduced by more than half in neat Ash Dam water than when nutrients were supplied in excess. Growth rate and bioconcentration were positively correlated for most elements, but some elements (e.g. Cd, Zn) were concentrated more when growth rates were lower, indicating the potential to tailor bioremediation depending on the pollutant. The cosmopolitan nature of the macroalgae studied, and their ability to grow and concentrate a suite of heavy metals from industrial wastes, highlights a clear benefit in the practical application of waste water bioremediation.     

Development of urease and glutamic dehydrogenase amperometric assay for heavy metals screening in polluted samples

An amperometric assay based on urease inactivation has been developed for the screening of heavy metals in environmental samples. The enzyme urease catalyses the hydrolysis of urea and the formation of NH(4)(+) is determined using a NADH-glutamate dehydrogenase coupled reaction system. NADH consumption is monitored amperometrically using screen-printed three electrode configuration and its oxidation current is then correlated to urease activity. The presence of heavy metals in the samples inhibits the urease activity, resulting in a lower NH(4)(+) production and therefore a decrease in NADH oxidation. The use of metallised carbon electrodes gave a decrease in NADH oxidation potential from +300 mV versus Ag/AgCl compared with > +600 mV for bare carbon electrodes, and thus minimised interferences from oxidizable species present in the samples. Electrodes fouling and possible contamination after reuse and cleaning was also eliminated by using screen-printed disposable electrodes. The linear range obtained for Hg(II) and Cu(II) was 10-100 microgl(-1) with a detection limit of 7.2 microgl(-1) and 8.5 microgl(-1), respectively. Cd(II) and Zn(II) produced enzyme inhibition in the range 1-30 mgl(-1), with limits of detection of 0.3 mgl(-1) for Cd(II) and 0.2 mgl(-1) for Zn(II). Pb(II) did not inactivate the urease enzyme significantly at the studied range (up to 50 mgl(-1)). Coefficients of variation (CV) values were 6-9% in all cases. Application of the assay system to leachate samples gave reliable and accurate toxicity assessments when compared to atomic absorption spectrometry (AAS) and inductively coupled plasma atomic emission spectroscopy (ICP-MS) analysis. This approach provides to be a simple and rapid (15 min, including enzyme inhibition time) method for metal ions detection.     

Heavy metals alter the electrokinetic properties of bacteria, yeasts, and clay minerals.

The electrokinetic patterns of four bacterial species (Bacillus subtilis, Bacillus megaterium, Pseudomonas aeruginosa, and Agrobacterium radiobacter), two yeasts (Saccharomyces cerevisiae and Candida albicans), and two clay minerals (montmorillonite and kaolinite) in the presence of the chloride salts of the heavy metals, Cd, Cr, Cu, Hg, Ni, Pb, and Zn, and of Na and Mg were determined by microelectrophoresis. The cells and kaolinite were net negatively charged at pH values above their isoelectric points (pI) in the presence of Na, Mg, Hg, and Pb at an ionic strength (mu) of 3 x 10(-4); montmorillonite has no pI and was net negatively charged at all pH values in the presence of these metals. However, the charge of some bacteria, S. cerevisiae, and kaolinite changed to a net positive charge (charge reversal) in the presence of Cd, Cr, Cu, Ni, and Zn at pH values above 5.0 (the pH at which charge reversal occurred differed with the metal) and then, at higher pH values, again became negative. The charge of the bacteria and S. cerevisiae also reversed in solutions of Cu and Ni with a mu of greater than 3 x 10(-4), whereas there was no reversal in solutions with a mu of less than 3 x 10(-4). The clays became net positively charged when the mu of Cu was greater than 3 x 10(-4) and that of Ni was greater than 1.5 x 10(-4). The charge of the cells and clays also reversed in solutions containing both Mg and Ni or both Cu and Ni (except montmorillonite) but not in solutions containing both Mg and Cu (except kaolinite) (mu = 3 x 10(-4)). The pIs of the cells in the presence of the heavy metals were at either higher or lower pH values than in the presence of Na and Mg. Exposure of the cells to the various metals at pH values from 2 to 9 for the short times (ca. 10 min) required to measure the electrophoretic mobility did not affect their viability. The specific adsorption on the cells and clays of the hydrolyzed species of some of the heavy metals that formed at higher pH values was probably responsible for the charge reversal. These results suggest that the toxicity of some heavy metals to microorganisms varies with pH because the hydrolyzed speciation forms of these metals, which occur at higher pH values, bind on the cell surface and alter the net charge of the cell.(ABSTRACT TRUNCATED AT 400 WORDS)     

Heavy metals alter the electrokinetic properties of bacteria, yeasts, and clay minerals.

The electrokinetic patterns of four bacterial species (Bacillus subtilis, Bacillus megaterium, Pseudomonas aeruginosa, and Agrobacterium radiobacter), two yeasts (Saccharomyces cerevisiae and Candida albicans), and two clay minerals (montmorillonite and kaolinite) in the presence of the chloride salts of the heavy metals, Cd, Cr, Cu, Hg, Ni, Pb, and Zn, and of Na and Mg were determined by microelectrophoresis. The cells and kaolinite were net negatively charged at pH values above their isoelectric points (pI) in the presence of Na, Mg, Hg, and Pb at an ionic strength (mu) of 3 x 10(-4); montmorillonite has no pI and was net negatively charged at all pH values in the presence of these metals. However, the charge of some bacteria, S. cerevisiae, and kaolinite changed to a net positive charge (charge reversal) in the presence of Cd, Cr, Cu, Ni, and Zn at pH values above 5.0 (the pH at which charge reversal occurred differed with the metal) and then, at higher pH values, again became negative. The charge of the bacteria and S. cerevisiae also reversed in solutions of Cu and Ni with a mu of greater than 3 x 10(-4), whereas there was no reversal in solutions with a mu of less than 3 x 10(-4). The clays became net positively charged when the mu of Cu was greater than 3 x 10(-4) and that of Ni was greater than 1.5 x 10(-4). The charge of the cells and clays also reversed in solutions containing both Mg and Ni or both Cu and Ni (except montmorillonite) but not in solutions containing both Mg and Cu (except kaolinite) (mu = 3 x 10(-4)). The pIs of the cells in the presence of the heavy metals were at either higher or lower pH values than in the presence of Na and Mg. Exposure of the cells to the various metals at pH values from 2 to 9 for the short times (ca. 10 min) required to measure the electrophoretic mobility did not affect their viability. The specific adsorption on the cells and clays of the hydrolyzed species of some of the heavy metals that formed at higher pH values was probably responsible for the charge reversal. These results suggest that the toxicity of some heavy metals to microorganisms varies with pH because the hydrolyzed speciation forms of these metals, which occur at higher pH values, bind on the cell surface and alter the net charge of the cell.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection and identification of fungal species by electronic nose technology: A systematic review

A rapid and effective identification of fungal species is essential for numerous applications, and electronic nose systems are being proposed as suitable alternatives to currently available fungi identification techniques. Hence, the present review aims to unveil all published information concerning fungi identification by electronic nose systems.A systematic review of the literature was conducted according to the PRISMA guidelines. A total of 16 articles met the inclusion criteria and were included in the analysis. The results of the reviewed studies demonstrated that effective detection of fungi was possible through sensor-based electronic nose systems, which may actually function as a mycotoxin screening tool for several applications.The obtained results suggest that the sensor-based electronic nose systems may not only screen different fungi genera, but also identify the associated species. This technology has already been experimented in several fields, from food industry to clinical practice.By summarizing these results, the present review may accelerate the standardization of electronic noses in fungi detection and discrimination, allowing a faster and more efficient screening of samples.     

土壤重金属生物毒性研究进展

世界范围内土壤重金属污染不断加重,由污染所带来的问题以及如何治理污染已经受到人们越来越多的关注.土壤重金属将对土壤生物产生影响,而土壤生物在重金属的胁迫下也会产生不同的响应.综述了国内外近年来土壤重金属生物毒性的研究进展,介绍了土壤重金属污染对陆地生态系统中植物、动物和微生物生长的影响;土壤重金属生物毒性的影响因素;土壤重金属生物毒性的研究方法;土壤重金属生物毒性的预测模型,最后提出了问题和展望.     

蛋白酶分泌菌对重金属胁迫的反应

本文研究了Pb、Zn、Cu和Cd对3种蛋白酶产生菌：碱性地衣芽孢杆菌2709(Bacillus licheniformis 2709)、中性枯草杆菌1.398(Bacillus subtilis1.398)和酸性宇佐美曲霉537(Aspergillus usamii 537)生长及其酶活性的影响, 并采用生长抑制皿分析(GIPA)方法针对3种菌株在Pb、Zn、Cu、Cd不同胁迫浓度下的生长量进行打分, 分析其耐性与抗性指标(MTC与MIC)。结果表明4种重金属在超过一定临界浓度后, 对3种菌株的生长和蛋白酶活性均产生了较大的抑制作用。碱性蛋白酶对4种重金属均具有较大的适应性, 其次是中性蛋白酶对Zn和Cd具有一定的适应性, 而酸性蛋白酶对4种重金属均表现出被抑制状态。3种菌株对Pb和Zn耐性与抗性最高(2.0 mmol/L~6.0 mmol/L), 其次是对Cd也具有一定的抗性(0.5 mmol/L~0.75 mmol/L)。     

Role of Iron-Oxidizing Microorganism in Bioleaching of Heavy Metals From Sewage Sludge Contaminated with Textile Dyes

This article reports the role of indigenous iron-oxidizing microorganisms in bioleaching of heavy metals from anaerobically digested sewage sludge in presence of toxic dyes namely, methylene blue (MB), Remazol black B (RBB) and mixture of both the dyes (DM). To achieve this goal, different concentrations of dyes (0, 500, 10,000, 15,000 and 25,000 mg/L) were added to the sewage sludge (initial pH ～ 7) and subsequently, the solubilization of heavy metals (Cu, Ni, Zn and Cr) was measured at time intervals of 48 h for 16 days. The results showed that an increase in dye concentration adversely affected the population of indigenous iron-oxidizing microorganisms, thereby decreased the bioleaching of metals. The metal solubilization from sludge is more adversely affected in presence of RBB and DM than MB. In majority of cases, the maximum metal solubilization was recorded at dye concentration of 5,000 mg/L. Two factor analyses (ANOVA) suggests the impact of both factors: dye concentration and dye type on metal leaching rate in sludge system.     

Impact of industrial waste water effluents on mycoflora of the shore sediments of the 3rd oxidation pond,with reference to biosorption of heavy metals

The third oxidation pond at 10th of Ramadan desert receives a number of industrial waste water effluents contaminated with the heavy metal ions Zn, Cd, Cu and Ni. The species diversity and fungal community structure of seven different sites at the onshore sediments and offshore were studied. Mycological analysis resulted in isolation of 3912 fungal colonies, 11.7% of this count were recovered from the onshore sediment sites (4 sites) whereas 88.3% were from the offshore sites (3 sites), in the desert. Fungal counts and species diversity at the onshore sites tend to increase with increasing distance far from the waste water input. A complete accordance was observed among the total fungal counts and species variabilities with organic matter content at each sampling site. This relationship was reversed in case of heavy metal contents with both counts and diversity. Seventeen fungal species belonging to seven genera were isolated from all sites under study. Aspergillus spp. constituted the majority of the isolates (51.7% of the total isolates), followed by Curvularia, Cephalosporium, and Humicola. Of nine isolated Aspergillus spp., A. humicola was the most dominant (37.4% of the total catch) and appeared at all polluted sites. Therefore, A. humicola was chosen to investigate its potential for heavy metals sorption from the contaminated waste water effluent. Four days old biomass pellets could sorb a large amount of heavy metals according to the following sequence: Zn>Cd>Cu>Ni ions. Agitation significantly increased Zn and Cd sorption, but not Cu and Ni. Heavy metals sorption took place at a wide pH range and particularly increased at alkaline pH (8-9).     

蛋白酶分泌菌对重金属胁迫的反应

本文研究了Pb、zn、cu和Cd对3种蛋白酶产生菌:碱性地衣芽孢杆菌2709(Bacilluslicheniformis 2709)、中性枯草杆菌1.398(Bacillus subtilis1.398)和酸性宇佐美曲霉537(Aspergillususamii 537)生长及其酶活性的影响,并采用生长抑制皿分析(GIPA)方法针对3种菌株在Pb、zn、Cu、Cd不同胁迫浓度下的生长量进行打分,分析其耐性与抗性指标(MTC与MIC).结果表明4种重金属在超过一定临界浓度后,对3种菌株的生长和蛋白酶活性均产生了较大的抑制作用.碱性蛋白酶对4种重金属均具有较大的适应性,其次是中性蛋白酶对Zn和Cd具有一定的适应性.而酸性蛋白酶对4种重金属均表现出被抑制状态.3种菌株对Pb和Zn耐性与抗性最高(2.0 mmol/L～6.0 mmol/L),其次是对Cd也具有一定的抗性(0.5 mmol/L～0.75 mmol/L).     

Bioleaching of Heavy Metals from Textile Sludge by Indigenous Sulfur-and-Iron-Oxidizing Microorganisms Using Elemental Sulfur and Ferrous Sulfate as Energy Sources: A Comparative Study

The present study was undertaken to investigate the potential of enriched indigenous sulfur-and-iron-oxidizing microorganisms in the bioleaching of Cu, Ni, Zn and Fe from textile sludges by using elemental sulfur and ferrous sulfate (FS), respectively, as an energy source under batch conditions. The experiments were performed with three different textile sludges (S1, S2 and S3) at initial neutral pH of the sludges procured from different parts of the country i.e., UP, Haryana and Punjab. The three sludges used were not only procured from different parts of the country but also differ in physiochemical characteristics. The extent of heavy metals solubilization in each sludge was found to be different using sulfur- and iron-oxidizing microorganisms. The results of the study indicate that sulfur-oxidizing microorganisms were found more efficient in the bioleaching process, irrespective of any sludge. The use of sulfur-oxidizing microorganisms led to higher solubilization of heavy metals and after 7 days of bioleaching about 84–96% Cu, 64–78% Ni, 81–92% Zn and 74–88% Fe were removed compared to 62–73% Cu, 62–66% Ni, 74–78% Zn and 70–78% Fe using iron-oxidizing microorganisms. This study had shown the feasibility of applying the bioleaching process to textile sludge contaminated with heavy metals. The results of the present study indicate that the bioleached sludge would be safer for land application.     

Detection and differentiation between mycotoxigenic and non-mycotoxigenic strains of two Fusarium spp. using volatile production profiles and hydrolytic enzymes

Volatile profiles and hydrolytic enzyme production by one non-mycotoxigenic and three mycotoxigenic strains of Fusarium moniliforme and F. proliferatum, grown in vitro for up to 96 h on a grain medium at 25 degrees C/0.95 water activity, were examined for differentiation of isolates. After spore lawn inoculation, measurements were made after 48, 72 and 96 h by sampling the head space above cultures with an electronic nose system using a 14 sensor surface polymer array, and by extraction and quantification of hydrolytic enzymes. There was good reproducibility of volatile patterns between replicates of the same treatment. Principal component analysis indicated that discrimination could be achieved between the uninoculated controls, the non-mycotoxigenic strain and the mycotoxin-producing strains for both species after 48 h. The total and specific activity of three out of seven enzymes (beta-D-glucosidase, alpha-D-galactosidase and N-acetyl-beta-D-glucosaminidase) were found to increase significantly in the non-mycotoxigenic when compared with the toxigenic strains of both species after 72 h. Activities of the others (beta-D-fucosidase, alpha-D-mannosidase, beta-D-xylosidase and N-acetyl-alpha-D-glucosaminidase) were not significantly different between strains. The study has shown for the first time that it is possible to differentiate between mycotoxigenic and non-mycotoxigenic strains of such spoilage fungi based on their volatile production patterns using an electronic nose system. These results have significance in the development of methods for the early detection of toxin-producing spoilage moulds in the food industry.     

Proteomic analysis of Arabidopsis halleri shoots in response to the heavy metals cadmium and zinc and rhizosphere microorganisms

Arabidopsis halleri has the rare ability to colonize heavy metal‐polluted sites and is an emerging model for research on adaptation and metal hyperaccumulation. The aim of this study was to analyze the effect of plant–microbe interaction on the accumulation of cadmium (Cd) and zinc (Zn) in shoots of an ecotype of A. halleri grown in heavy metal‐contaminated soil and to compare the shoot proteome of plants grown solely in the presence of Cd and Zn or in the presence of these two metals and the autochthonous soil rhizosphere‐derived microorganisms. The results of this analysis emphasized the role of plant–microbe interaction in shoot metal accumulation. Differences in protein expression pattern, identified by a proteomic approach involving 2‐DE and MS, indicated a general upregulation of photosynthesis‐related proteins in plants exposed to metals and to metals plus microorganisms, suggesting that metal accumulation in shoots is an energy‐demanding process. The analysis also showed that proteins involved in plant defense mechanisms were downregulated indicating that heavy metals accumulation in leaves supplies a protection system and highlights a cross‐talk between heavy metal signaling and defense signaling.     

Effects of organic and heavy metal pollution on chironomids within a pristine upland catchment

Several studies, particularly in the Northern Hemisphere, have demonstrated that chironomids are responsive to pollution (e.g., heavy metals, organic). In Australia, there is limited evidence that chironomid species have differential sensitivities to water pollution, with several studies reporting high tolerance of heavy metal pollution. We investigated the impact of both zinc-rich mine waste and organic effluent on chironomids within an effectively pristine background using the Chironomid Pupal Exuviae Technique (CPET). Chironomid species assemblages were strongly influenced by both mine drainage and organic pollution. Community composition differed between unpolluted streams and locations downstream of the pollution sources, and between the two different sources of pollution. Thirty seven of the most abundant species exhibited strong responses to water pollution varying from greatly increased abundance to complete absence. Ten species had higher abundance at one of the polluted sites compared to unpolluted sites; six in the presence of zinc pollution and five in the presence of organic pollution. One species responded positively to both pollution types. Our results indicate that contrary to the mixed reports of Australian chironomids for being insensitive to pollution, we found that they displayed a strong pollution response to both organic and heavy metal contamination.     

Effects of heavy metals on ultrastructure and HSP70s induction in the aquatic moss Leptodictyum riparium Hedw

The effects of heavy metals, both toxic (Pb, Cd) and essential (Cu, Zn) on the ultrastructure and the induction of Heat Shock Protein 70 (HSP70) have been studied in the aquatic moss Leptodictyum riparium Hedw. In vitro cultured L. riparium was treated with different heavy metals, both toxic, as cadmium or lead; and essential microelements such as Copper or Zinc concentrations ranging from 10(-3) to 10(-6) M to investigate both ultrastructural damage and HSP induction. TEM observations showed that sub-lethal concentrations of heavy metals caused only slight changes, largely localized in the chloroplasts. Among all the heavy metals tested, cadmium caused the most severe modifications. Heavy metals caused the decrease of the soluble protein content and the enhancement of proteins reacting versus HSP70 antibodies, suggesting that molecular chaperons might be involved in the resistance to toxic effects of lead, cadmium, copper and zinc. Therefore, the induction of HSP70 in L. riparium would confer a higher resistance to pollutants under stressful conditions lethal for other mosses and higher plant species. These results suggest that the moss L. riparium can tolerate heavy metals stress without incurring severe cellular/subcellular damage. Therefore it can be used as a useful indicator of heavy metals accumulation.