Rapid detection of meat spoilage by measuring volatile organic compounds by using proton transfer reaction mass spectrometry.

The evolution of the microbial spoilage population for air- and vacuum-packaged meat (beef and pork) stored at 4 degrees C was investigated over 11 days. We monitored the viable counts (mesophilic total aerobic bacteria, Pseudomonas spp., Enterobacteriaceae, lactic acid bacteria, and Enterococcus spp.) by the microbiological standard technique and by measuring the emission of volatile organic compounds (VOCs) with the recently developed proton transfer reaction mass spectrometry system. Storage time, packaging type, and meat type had statistically significant (P < 0.05) effects on the development of the bacterial numbers. The concentrations of many of the measured VOCs, e.g., sulfur compounds, largely increased over the storage time. We also observed a large difference in the emissions between vacuum- and air-packaged meat. We found statistically significant strong correlations (up to 99%) between some of the VOCs and the bacterial contamination. The concentrations of these VOCs increased linearly with the bacterial numbers. This study is a first step toward replacing the time-consuming plate counting by fast headspace air measurements, where the bacterial spoilage can be determined within minutes instead of days.

Characterization of hydrocarbon-degrading microbial populations in contaminated and pristine Alpine soils.

Biodegradation of petroleum hydrocarbons in cold environments, including Alpine soils, is a result of indigenous cold-adapted microorganisms able to degrade these contaminants. In the present study, the prevalence of seven genotypes involved in the degradation of n-alkanes (Pseudomonas putida GPo1 alkB; Acinetobacter spp. alkM; Rhodococcus spp. alkB1, and Rhodococcus spp. alkB2), aromatic hydrocarbons (P. putida xylE), and polycyclic aromatic hydrocarbons (P. putida ndoB and Mycobacterium sp. strain PYR-1 nidA) was determined in 12 oil-contaminated (428 to 30,644 mg of total petroleum hydrocarbons [TPH]/kg of soil) and 8 pristine Alpine soils from Tyrol (Austria) by PCR hybridization analyses of total soil community DNA, using oligonucleotide primers and DNA probes specific for each genotype. The soils investigated were also analyzed for various physical, chemical, and microbiological parameters, and statistical correlations between all parameters were determined. Genotypes containing genes from gram-negative bacteria (P. putida alkB, xylE, and ndoB and Acinetobacter alkM) were detected to a significantly higher percentage in the contaminated (50 to 75%) than in the pristine (0 to 12.5%) soils, indicating that these organisms had been enriched in soils following contamination. There was a highly significant positive correlation (P < 0.001) between the level of contamination and the number of genotypes containing genes from P. putida and Acinetobacter sp. but no significant correlation between the TPH content and the number of genotypes containing genes from gram-positive bacteria (Rhodococcus alkB1 and alkB2 and Mycobacterium nidA). These genotypes were detected at a high frequency in both contaminated (41.7 to 75%) and pristine (37.5 to 50%) soils, indicating that they are already present in substantial numbers before a contamination event. No correlation was found between the prevalence of hydrocarbon-degradative genotypes and biological activities (respiration, fluorescein diacetate hydrolysis, lipase activity) or numbers of culturable hydrocarbon-degrading soil microorganisms; there also was no correlation between the numbers of hydrocarbon degraders and the contamination level. The measured biological activities showed significant positive correlation with each other, with the organic matter content, and partially with the TPH content and a significant negative correlation with the soil dry-mass content (P < 0.05 to 0.001).

Biodegradation and bioremediation of hydrocarbons in extreme environments.

Many hydrocarbon-contaminated environments are characterized by low or elevated temperatures, acidic or alkaline pH, high salt concentrations, or high pressure, Hydrocarbon-degrading microorganisms, adapted to grow and thrive in these environments, play an important role in the biological treatment of polluted extreme habitats. The biodegradation (transformation or mineralization) of a wide range of hydrocarbons, including aliphatic, aromatic, halogenated and nitrated compounds, has been shown to occur in various extreme habitats. The biodegradation of many components of petroleum hydrocarbons has been reported in a variety of terrestrial and marine cold ecosystems. Cold-adapted hydrocarbon degraders are also useful for wastewater treatment. The use of thermophiles for biodegradation of hydrocarbons with low water solubility is of interest, as solubility and thus bioavailability, are enhanced at elevated temperatures. Thermophiles, predominantly bacilli, possess a substantial potential for the degradation of environmental pollutants, including all major classes. Indigenous thermophilic hydrocarbon degraders are of special significance for the bioremediation of oil-polluted desert soil. Some studies have investigated composting as a bioremediation process. Hydrocarbon biodegradation in the presence of high salt concentrations is of interest for the bioremediation of oil-polluted salt marshes and industrial wastewaters, contaminated with aromatic hydrocarbons or with chlorinated hydrocarbons. Our knowledge of the biodegradation potential of acidophilic, alkaliphilic, or barophilic microorganisms is limited.

Bioremediation (natural attenuation and biostimulation) of diesel-oil-contaminated soil in an alpine glacier skiing area.

We investigated the feasibility of bioremediation as a treatment option for a chronically diesel-oil-polluted soil in an alpine glacier area at an altitude of 2,875 m above sea level. To examine the efficiencies of natural attenuation and biostimulation, we used field-incubated lysimeters (mesocosms) with unfertilized and fertilized (N-P-K) soil. For three summer seasons (July 1997 to September 1999), we monitored changes in hydrocarbon concentrations in soil and soil leachate and the accompanying changes in soil microbial counts and activity. A significant reduction in the diesel oil level could be achieved. At the end of the third summer season (after 780 days), the initial level of contamination (2,612 +/- 70 microg of hydrocarbons g [dry weight] of soil(-1)) was reduced by (50 +/- 4)% and (70 +/- 2)% in the unfertilized and fertilized soil, respectively. Nonetheless, the residual levels of contamination (1,296 +/- 110 and 774 +/- 52 microg of hydrocarbons g [dry weight] of soil(-1) in the unfertilized and fertilized soil, respectively) were still high. Most of the hydrocarbon loss occurred during the first summer season ([42 +/- 6]% loss) in the fertilized soil and during the second summer season ([41 +/- 4]% loss) in the unfertilized soil. In the fertilized soil, all biological parameters (microbial numbers, soil respiration, catalase and lipase activities) were significantly enhanced and correlated significantly with each other, as well as with the residual hydrocarbon concentration, pointing to the importance of biodegradation. The effect of biostimulation of the indigenous soil microorganisms declined with time. The microbial activities in the unfertilized soil fluctuated around background levels during the whole study.

Potential of halotolerant and halophilic microorganisms for biotechnology.

Halotolerant or halophilic microorganisms, able to live in saline environments, offer a multitude of actual or potential applications in various fields of biotechnology. The technical applications of bacteriorhodopsin comprise holography, spatial light modulators, optical computing, and optical memories. Compatible solutes are useful as stabilizers of biomolecules and whole cells, salt antagonists, or stress-protective agents. Biopolymers, such as biosurfactants and exopolysaccharides, are of interest for microbially enhanced oil recovery. Other useful biosubstances are enzymes, such as new isomerases and hydrolases, that are active and stable at high salt contents. Halotolerant microorganisms play an essential role in food biotechnology for the production of fermented food and food supplements. The degradation or transformation of a range of organic pollutants and the production of alternative energy are other fields of applications of these groups of extremophiles.

Changes of cell size distribution during the batch culture of Arthrobacter strain PI/1-95.

The size distributions of an arthrobacter, approximately 1 microm in diameter, were analysed using a Coulter Multisizer II instrument thereby making it possible to distinguish between the different stages in the morphological cycle. The results indicated that at the beginning of exponential phase a shift occurred from large to smaller cells, the cell size distributions in both categories were asymmetric, skewed towards higher values than the means. During the course of the batch culture the cells in the larger class decreased in size and the two classes coalesced. The size distribution of the smaller class exhibited an additional shift towards smaller cells. After which neither the number of classes nor the size distribution changed. It was concluded that the Coulter Multisizer II instrument provides a feasible method to extract information not only about mean cell size but also about cell size distributions.

Characterization and immobilization of the laccase from Pleurotus ostreatus and its use for the continuous elimination of phenolic pollutants.

A laccase, the only ligninolytic enzyme produced by the basidiomycete Pleurotus ostreatus strain RK 36 was purified to homogeneity and characterized. The enzyme is a monomeric protein with a molecular weight of 67 000 Da and an isoelectric point of 3.6. Type I and type III Cu(2+) centers were identified by spectrophotometry. With syringaldazine as substrate laccase showed the highest oxidation rates at pH 5.8, 50 degrees C, and in 40 mM phosphate buffer. Among the tested stabilization parameters laccase retained most of its activity in high ionic buffer, pH 10, -20 degrees C, in the presence of 10 mM benzoic acid and with 35% ethylene glycol respectively. Crude laccase was covalently immobilized to Eupergit((R))C. Benzoate was found to stabilize the enzyme during the immobilization process. The activity loss of laccase during 10 days at 25 degrees C storage was 2% on average. Continuous elimination of 2,6-dimethoxyphenol by immobilized laccase was carried out in a packed bed reactor followed by filtration of the formed precipitate. The solubility of the polymerisates of oxidized syringaldazine, o-dianisidine, and 2,6-dimethoxyphenol with respect to temperature, pH-value and organic solvents were examined. The precipitates were found to be insoluble under non-extreme environmental conditions.

Monitoring of bioremediation by soil biological activities.

An evaluation of soil biological activities as a monitoring instrument for the decontamination process of a mineral-oil-contaminated soil was made using measurements of microbial counts, soil respiration, soil biomass and several enzyme activities. The correlations between these parameters and with the levels of hydrocarbon residues were investigated; the effects of different N- and P-sources on hydrocarbon decontamination and soil biological activities were determined. Inorganic nutrients stimulated hydrocarbon biodegradation but not all biological activities to a significant extent. Biodegradation could be monitored well by soil biological parameters: the residual hydrocarbon content correlated positively with soil respiration, biomass-C (substrate-induced respiration), and with activities of soil dehydrogenase, urease and catalase. Soil lipase activity and the number of hydrocarbon utilizers correlated negatively (P < 0.0001) with the remaining hydrocarbon content.

A practicable and accurate method to differentiate between intra- and extracellular water of microbial cells.

A thermographimetric method which allows for a quick and accurate estimation of intra- and extracellular water of microbial cells is reviewed and improved. Knowledge of these fractions is important for physiological as well as for toxicological investigations. Results of the study indicate that besides the species, nutrient availability and growth conditions affect the intracellular water content. Intra- and extracellular water, dry matter, volume and density of a single cell of Arthrobacter sp. are calculated. There are indications that intracellular compartments of eukaryotes could also be investigated with this method.

Responses of the soil microbiota to elevated CO2 in an artificial tropical ecosystem.

Plants in artificial tropical ecosystems were grown under ambient (340 microl l(-1)) and elevated (610 microl l(-1)) atmospheric CO2 for 530 d under low-nutrient conditions on a substrate free of organic C. At the end of the experiment a number of soil chemical and microbiological variables were determined. Although we found no changes in total soil organic matter under elevated CO2, we did find that after physical fractionation the amount of organic C in the supernatant (< 0.2 microm) and the amount of water extractable organic C (WEOC) was lower under elevated CO2. The extractable optical density (OD) indicated a higher degree of humification for the elevated than for the ambient CO2 samples (P = 0.032). Microbial biomass C was not significantly altered under high CO2, but total bacterial counts were significantly higher. The microbial biomass C-to-N ratio was also higher at elevated (15.0) than at ambient CO2 (10.0). The number of mycorrhizal spores was lower at high CO2, but ergosterol contents and fungal hyphal lengths were not significantly affected. Changes were found neither in community level physiological profiles (CLPPs) nor in the structural attributes (phospholipid fatty acids, PLFAs) of the microbial community. Overall, the effects on the soil microbiota were small, perhaps as a result of the low nutrient supply and low organic matter content of the soil used in our study. The few significant results showing changes in specific, though relatively minor, organic matter pools may point to possible long-term changes of the more major pools. Furthermore, the data suggest increased competition between plants and microbes for N at high CO2.

Biodegradation of diesel oil by cold-adapted microorganisms in presence of sodium dodecyl sulfate.

The effect of different concentrations of the anionic surfactant sodium dodecyl sulfate (SDS) on biodegradation of diesel oil was assessed during 32 days at 10 degrees C, under simulated environmental conditions, in liquid culture and in an alpine soil. Low SDS concentrations (50-100 mg l-1) significantly enhanced oil biodegradation by a psychrotrophic inoculum in liquid culture, whereas higher SDS concentrations (500-1000 mg l-1) inhibited hydrocarbon biodegradation. Oil biodegradation by the indigenous microorganisms in soil was inhibited at all SDS concentrations tested. The surfactant itself was rapidly biodegraded both in liquid culture and in soil.

A microbial biosensor based on Yarrowia lipolytica for the off-line determination of middle-chain alkanes.

A microbial biosensor based on immobilised psychrotrophic yeast Yarrowia lipolytica integrated to FIA for the determination of middle chain alkanes was developed. The system responded very well to middle chain alkanes even at low operational temperatures down to +5 degrees C. The maximum sensitivity was obtained at 15 degrees C. A linear relationship was observed between the sensor response and dodecane concentration up to 100 microM.

Low-temperature bioremediation of a waste water contaminated with anionic surfactants and fuel oil.

We conducted a laboratory study at 10 degrees C on the biological decontamination of the waste water from a garage and car-wash that was contaminated with anionic surfactants (57 mg 1(-1)) and fuel oil (184 mg hydrocarbons 1(-1)). The indigenous microorganisms degraded both contaminants efficiently after biostimulation by an inorganic nutrient supply. After 7 days at 10 degrees C, the residual contaminations were 11 mg anionic surfactants 1(-1) and 26 mg hydrocarbons 1(-1). After 35 days, only the anionic surfactants had been further reduced to 3 mg 1(-1). Bioaugmentation of the unfertilized waste water with a cold-adapted inoculum, able to degrade both hydrocarbons (diesel oil) and anionic surfactants (sodium dodecyl sulphate), resulted in a significant increase of the hydrocarbon biodegradation during the first 3 days of decontamination, whereas biodegradation of anionic surfactants was inhibited during the first 21 days following inoculation. Bioaugmentation of the nutrient-amended waste water was without any effect.

Efficiency of indigenous and inoculated cold-adapted soil microorganisms for biodegradation of diesel oil in alpine soils.

Biodegradation of diesel oil (5 g(middot)kg [soil dry weight](sup-1)) was investigated in five alpine subsoils, differing in soil type and bedrock, in laboratory experiments during 20 days at 10(deg)C. The biodegradation activities of the indigenous soil microorganisms and of a psychrotrophic diesel oil-degrading inoculum and the effect of biostimulation by inorganic fertilization (C/N/P ratio = 100:10:2) were determined. Fertilization significantly enhanced diesel oil biodegradation activity of the indigenous soil microorganisms. Biostimulation by fertilization enhanced diesel oil biodegradation to a significantly greater degree than bioaugmentation with the psychrotrophic inoculum. In none of the five soils did fertilization plus inoculation result in a higher decontamination than fertilization alone. A total of 16 to 23% of the added diesel oil contamination was lost by abiotic processes. Total decontamination without and with fertilization was in the range of 16 to 31 and 27 to 53%, respectively.

Heavy metal resistant Arthrobacter sp.--a tool for studying conjugational plasmid transfer between gram-negative and gram-positive bacteria.

The role of two heavy metal-resistant strains of the Gram-positive genus Arthrobacter sp. as a tool in studying conjugational plasmid transfer between Gram-positive and Gram-negative bacteria is described. The high nickel resistance and the cobalt resistance of Arthrobacter sp. strain RM1/6 could be transferred to Arthrobacter sp. strain WS14. IncQ plasmids (pKT240, pKT240::czc, pML10) could be mobilized from E. coli into Arthrobacter spp. strains; antibiotic (Km, Ap, Tc) and heavy metal (Co) resistance genes were expressed in the recipient stains. IncQ plasmid pKT240 could be mobilized between Arthrobacter spp. strains. IncP plasmid RP4::Tn4371 was transferred from A. eutrophus to Arthrobacter sp., RP4-mediated antibiotic resistance to Km was expressed in the recipient strain.

High-yield production of oxalic acid for metal leaching processes by Aspergillus niger.

The complex-forming compound oxalic acid can effectively solubilise metals such as aluminium, iron, lithium and manganese. In order to produce high amounts of oxalic acid for biohydrometallurgical processes, it was the aim of this work to optimise oxalic acid production by Aspergillus niger, a fungus well known for its ability to produce oxalic acid. A. niger excreted 427 mmol oxalic acid l-1 if it was cultivated in a pH-controlled (pH 6.0) fed-batch run in a 2-l stirred tank reactor. Sucrose and lactose permeate were suitable carbon sources for oxalic acid production. In sucrose medium, A. niger produced high amounts of gluconic and oxalic acids, whereas in lactose permeate medium only oxalic acid was produced. Cultivation in green syrup and molasses media lead to high yields of biomass, but low oxalic acid production (< 20 mmol l-1).

Buffer-stimulated citrate efflux in Penicillium simplicissimum: an alternative charge balancing ion flow in case of reduced proton backflow?

Organic acids excreted by filamentous fungi may be used to win metals from industrial secondary raw materials. For a future commercial use a high production rate of organic acids is necessary. The conditions under which the commercially used fungus Aspergillus niger excretes high amounts of citric acid can not be maintained in metal leaching processes. However, Penicillium simplicissimum showed an enhanced citric acid efflux in the presence of an industrial filter dust containing 50% zinc oxide. Because Good buffers of high molarity were able to mimic the effect of zinc oxide, the high buffering capacity of zinc oxide and not an effect of the zinc ions was held responsible for the enhanced citric acid efflux. The presence of ammonium and trace elements reduced this buffer-stimulated citric acid efflux, whereas the plant hormone auxine canceled this reduction. This citric acid efflux was influenced by a depolarization of the membrane: the freely permeable compound tetraphenylphosphoniumbromide decreased the citric acid efflux, without decreasing intracellular citric acid or consumption of glucose and oxygen. Vanadate, an inhibitor of the plasma membrane H(+)-ATPase also reduced the buffer-stimulated citric acid efllux. The role of the efflux of citrate anions as an alternative charge balancing ion flow in case of impaired backflow of extruded protons because of a high extracellular buffering capacity is discussed.

Influence of medium components and metabolic inhibitors on citric acid production by Penicillium simplicissimum.

Penicillium simplicissimum excreted more than 100 mmol citric acid l-1 [2.9 mmol (g dry wt)-1; 9 d] if an industrial filter dust (> 50% ZnO) providing a high extracellular buffering capacity was present in the medium. A similar specific [2 mmol (g dry wt)-1], but lower absolute (26 mmol l-1), citric acid excretion occurred in the absence of an extracellular buffer and if amino acids or urea were used as nitrogen source. P. simplicissimum excreted no citric acid under conditions where Aspergillus niger produces citric acid (deficiency of trace elements, low pH and reduced biomass formation). Citric acid excretion by P. simplicissimum always paralleled biomass formation and occurred in a pH range between 4 and 7. This indicated that different imbalances of metabolism were responsible for citric acid excretion in A. niger and P. simplicissimum. However, provided a high extracellular buffering capacity was present, the response of the Penicillium system to different carbon and nitrogen sources was similar to the Aspergillus system. In contrast, the metals iron and copper had virtually no effect on citric acid excretion compared with A. niger. Estimation of intracellular citric acid, as well as the effects of the uncoupler 2,4-dinitrophenol, and the H(+)-ATPase inhibitor sodium orthovanadate, led to the conclusion that the buffer-stimulated citric acid efflux was dependent on metabolic energy and an energized plasma membrane, respectively. Despite similarities to the Aspergillus system, a different mechanism for buffer-stimulated citric acid excretion by P. simplicissimum seems probable.

Leaching with Penicillium simplicissimum: Influence of Metals and Buffers on Proton Extrusion and Citric Acid Production.

In the presence of insoluble metal oxides (industrial filter dust, zinc oxide, synthetic mixture of metal oxides), Penicillium simplicissimum developed the ability to excrete considerable amounts of citric acid (>100 mM). Parallel with the increase of citric acid concentration in the culture broth, zinc was solubilized from zinc oxide. The adsorption of filter dust onto the mycelium (the pellets formed were less than 1 mm in diameter) was required for not only the citric acid excretion but also the leaching of zinc. When the filter dust was replaced with a synthetic mixture of metal oxides or with zinc oxide in combination with trace elements, levels of adsorption and citric acid production were observed to be similar to those in experiments where industrial filter dust was used. The two most important properties of the filter dust were its heavy-metal content and its buffering capacity. These properties were simulated by adding heavy metals in soluble form (as chlorides, sulfates, or nitrates) or soluble buffers to the medium. Both heavy metals and buffers were not able to induce a citric acid efflux. As with citric acid production by Aspergillus niger, the addition of manganese lowered citric acid excretion (by 40% with metal oxide-induced citric acid efflux and by 100% with urea-induced citric acid efflux). Copper antagonized the effect of manganese. The mechanism for the bulk of citric acid excretion by P. simplicissimum, however, seemed to be different from that described for citric acid accumulation by A. niger. Because of the inefficiency of metals in solubilized form and of soluble buffers to induce a strong citric acid efflux, adsorption of an insoluble metal compound (zinc oxide) turned out to be essential. Surface phenomena possibly involving the plasma membrane H-ATPase are thought to participate in the induction of citric acid excretion by P. simplicissimum in the presence of industrial filter dust.