Low molecular weight metabolites produced by various Penicillium species

Low-molecular weight volatile metabolites produced by Penicillium farinosum, P. citrinum, P. camemberti and P. chrysogenum were investigated. During first 40 days of cultivation the fungi produced mainly C-8 compounds, and later mainly 2-hexenal was synthesized. Addition of 0.1% linoleic acid significantly stimulated the secretion of volatile metabolites. P. citrinum and P. farenosum produced large quantities of geosmin.     

Microbial cleaning of waste gas containing volatile organic compounds in a bioreactor system with a closed gas circuit

The cleaning of the exhaust gases of a bioreactor containing volatile hydrocarbons in a bioreactor system with a closed gas circuit is described. The bioreactor system consisted of three different reactor types: a stirred tank which was filled with hydrocarbon-containing waste water to simulate the exhaust gases of a remediation process; a trickle-bed reactor for aerobic treatment of the exhaust gas from the stirred tank; and a photoreactor containing an algae culture which assimilated CO₂ from the trickle-bed reactor and also produced O₂. With this bioreactor system, it was possible to efficiently remove volatile organic compounds (VOC) from the waste gases. Depending on the type of waste water investigated, elimination rates of 41% to 93% of BTEX (benzene, ethylbenzene, toluene, xylene) and 29% to 53% of VCH (volatile chlorinated hydrocarbons) were obtained. Due to the photosynthesis of the algae in the system's photoreactor, oxygen concentrations between 12% and 18% [v/v], equivalent to about 57% to 83% DOT, were obtained. This concentration permitted the aerobic degradation to be carried out without having to add fresh air. The trickle-bed reactor and the photoreactor worked continuously, whereas the waste water in the stirred bioreactor was replaced in different batches. The accumulation of toxic compounds in the nutrient solutions of the trickle-bed (EC-50 > 30 g/l) and of the photoreactor (EC-50 > 35 g/l) was low. Carbon dioxide concentrations in the gas flow were higher than in fresh air (1% to 3% [vol/vol]), but no long-term accumulation of CO₂ occurred. This means that the algae in the photoreactor were active enough to assimilate the CO₂ which had been produced. They were also able to produce sufficient oxygen for aerobic hydrocarbon degradation. The system described is a first step towards treating waste gases which results from the bioremediation of hydrocarbon-contaminated media in a closed gas circuit without any emission (e.g. VOC, CO₂, germs).     

Implementation of proton transfer reaction‐mass spectrometry (PTR‐MS) for advanced bioprocess monitoring

We report on the implementation of proton transfer reaction‐mass spectrometry (PTR‐MS) technology for on‐line monitoring of volatile organic compounds (VOCs) in the off‐gas of bioreactors. The main part of the work was focused on the development of an interface between the bioreactor and an analyzer suitable for continuous sampling of VOCs emanating from the bioprocess. The permanently heated sampling line with an inert surface avoids condensation and interaction of volatiles during transfer to the PTR‐MS. The interface is equipped with a sterile sinter filter unit directly connected to the bioreactor headspace, a condensate trap, and a series of valves allowing for dilution of the headspace gas, in‐process calibration, and multiport operation. To assess the aptitude of the entire system, a case study was conducted comprising three identical cultivations with a recombinant E. coli strain, and the volatiles produced in the course of the experiments were monitored with the PTR‐MS. The high reproducibility of the measurements proved that the established sampling interface allows for reproducible transfer of volatiles from the headspace to the PTR‐MS analyzer. The set of volatile compounds monitored comprises metabolites of different pathways with diverse functions in cell physiology but also volatiles from the process matrix. The trends of individual compounds showed diverse patterns. The recorded signal levels covered a dynamic range of more than five orders of magnitude. It was possible to assign specific volatile compounds to distinctive events in the bioprocess. The presented results clearly show that PTR‐MS was successfully implemented as a powerful bioprocess‐monitoring tool and that access to volatiles emitted by the cells opens promising perspectives in terms of advanced process control. Biotechnol. Bioeng. 2012; 109: 3059–3069. © 2012 Wiley Periodicals, Inc.     

Microextraction and Gas Chromatography/Mass Spectrometry for improved analysis of geosmin and other fungal “off” volatiles in grape juice

Geosmin is a volatile fungal metabolite with an earthy aroma produced in grape products from rotten grapes. The accumulation of geosmin in grapes is caused by the interaction of Botrytis cinerea and Penicillium expansum. Solid Phase Microextraction (SPME) has great utility for collecting volatile compounds in wine. However, contamination with earthy odours may have occurred previously in the must and novel methods are required for this commodity. In the present report, several parameters of the SPME were evaluated to optimize geosmin extraction. The method permitted quantification of geosmin and other fungal volatiles by Gas Chromatography-Mass Spectrometer (GC-MS) at very low concentrations. Limits of detection and quantification (L_D and L_Q) for geosmin were 4.7 ng L⁻¹ and 15.6 ng L⁻¹ respectively. The RSD was 4.1% and the recovery rates ranged from 115% to 134%. Uniquely, haloanisoles were analyzed by using only one internal standard (2,3,6-trichloroanisole) thus avoiding the synthesis of deuterated anisole analogues that are used as internal standard in other methods. The method was used for the analysis of grape juice samples inoculated with B. cinerea and P. expansum. Geosmin and methylisoborneol were the compounds that appeared to contribute most to earthy odours, although other fungal compounds which are claimed to cause earthy or mouldy off-odours were detected (e.g. 1-octen-3-ol and fenchol).     

Biodegradation of the Water‐Soluble Gasoline Components in a Novel Hybrid Bioreactor

A novel hybrid bioreactor was designed to remove volatile organic compounds from water contaminated with water‐soluble gasoline components, and the performance of this new bioreactor was investigated. It was composed of two biotrickling filter sections and one biofilter section. The liquid phase pollutants were removed by a mixed culture in the biotrickling filter sections and the gas phase pollutants stripped by air injection in the biofilter section. The specific rates of chemical oxygen demand (COD) removal obtained in the reactor were directly proportional to the pollutant‐loading rate. A stable operation of the hybrid bioreactor was attained for long periods of time. The bioreactor had the potential to simultaneously treat a complex mixture of volatile organic compounds, e.g., those present in the water‐soluble fraction of gasoline, as well as the capacity to readily adapt to changing operational conditions, such as an increased contaminant loading, and variations in the airflow rate.     

Origin of (−)-Geosmin on Grapes: On the Complementary Action of Two Fungi, <Emphasis Type="Italic">Botrytis Cinerea</Emphasis> and <Emphasis Type="Italic">Penicillium Expansum</Emphasis>

One of the consequences of rot on grapes is the development of volatile compounds giving fungal, mouldy or earthy odours. Among these compounds, (−)-geosmin (trans-1,10-dimethyl-trans-9-decalol), a powerful aromatic compound with an earthy smell is a persistent defect in grape juice and wines made with at least partially rotten grapes. A microbiota analysis of rotten grapes containing (−)-geosmin was carried out on sites from four French regions from 1999 to 2002, to clarify the involvement in geosmin appearance of Streptomyces spp. and Penicillium spp., two types of microorganisms present on grape, that are known for their ability to produce geosmin. In earthy grapes, Botrytis cinerea was largely present. Different species of Streptomyces were also isolated, but their pH sensitivity was an extremely limiting parameter for their development on grape juice, grapes or stem, and consequently for their potentiality to generate geosmin in the vineyard. Penicillium expansum, producing geosmin on a model medium, was omnipresent. Penicillium carneum, which is also a geosmin producer, was represented by a single colony during the 4 years of this study. P. expansum alone was able to produce geosmin on a model medium but not on grapes. However, after 7 days’ pre-culture of some B. cinerea strains on grape juice, this juice became favourable to geosmin production by P. expansum. We demonstrated the necessary and complementary action of B. cinerea and P. expansum in geosmin production in grape juice and in crushed grape berries.     

Potential of on-line CIMS for bioprocess monitoring

Chemical-ionization mass spectrometry (CIMS) using flow reactors is an emerging method for on-line monitoring of trace concentrations of organic compounds in the gas phase. In this study, a flow-reactor CIMS instrument, employing the H(3)O(+) cation as the ionizing reagent, was used to simultaneously monitor several volatile metabolic products as they are released into the headspace during bacterial growth in a bioreactor. Production of acetaldehyde, ethanol, acetone, butanol, acetoin, diacetyl, and isoprene by Bacillus subtilis is reported. Ion signal intensities were related to solution-phase concentrations using empirical calibrations and, in the case of isoprene, were compared with simultaneous gas chromatography measurements. Identification of volatile and semivolatile metabolites is discussed. Flow-reactor CIMS techniques should be useful for bioprocess monitoring applications because of their ability to sensitively and simultaneously monitor many volatile metabolites on-line.     

Identification of odorous compounds from nine fermentor-cultivated Streptomyces strains

The chemical composition was determined of odors produced by nine strains of streptomycetes (Streptomyces aureofaciens, S. avermitilis, S. cinnamonensis, S. coelicolor, S. griseus, S. lividans, S. rimosus, S. spectabilis, S. virginiae) cultivated in a fermentor under similar cultivation conditions. GC-MS analysis identified more than twenty noteworthy volatile chemical individuals. The main components of the odor spectrum were geosmin and unique homologues of oxolones (dihydrofuranones), minor compounds included, e.g., pyrazine derivatives, acetoin and its homologues, aromatic esters, furan derivatives.     

Fungal volatiles as indicators of food and feeds spoilage.

Fungal growth leads to spoilage of food and animal feeds and to formation of mycotoxins and potentially allergenic spores. Fungi produce volatile compounds, during both primary and secondary metabolism, which can be used for detection and identification. Fungal volatiles from mainly Aspergillus, Fusarium, and Penicillium have been characterized with gas chromatography, mass spectrometry, and sensory analysis. Common volatiles are 2-methyl-1-propanol, 3-methyl-1-butanol, 1-octen-3-ol, 3-octanone, 3-methylfuran, ethyl acetate, and the malodorous 2-methyl-isoborneol and geosmin. Volatile sesquiterpenes can be used for taxonomic classification and species identification in Penicillium, as well as to indicate mycotoxin formation in Fusarium and Aspergillus. Developments in sensor technology have led to the construction of "electronic noses" (volatile compound mappers). Exposure of different nonspecific sensors to volatile compounds produces characteristic electrical signals. These are collected by a computer and processed by multivariate statistical methods or in an artificial neural network (ANN). Such systems can grade cereal grain with regard to presence of molds as efficiently as sensory panels evaluating grain odor. Volatile compound mapping can also be used to predict levels of ergosterol and fungal colony-forming units in grain. Further developments should make it possible to detect individual fungal species as well as the degree of mycotoxin contamination of food and animal feeds.     

Causative microorganisms for musty odor occurrence in the eutrophic Lake Kasumigaura

The musty odor phenomenon in Lake Kasumigaura, Japan (a eutrophic lake that is used for various purposes, including water supply source) was examined. The causative microorganisms responsible for the production of the odor compounds, geosmin and/or 2-methylisoborneol (MIB), and the odor release mechanism were studied in vitro and in situ. The numbers of the filamentous P. tenue in the lake water column were closely correlated with the concentration of MIB, but not geosmin. The isolated monoxenic P. tenue in culture produced only MIB. Geosmin concentration was well correlated with the number of actinomycetes in the sediment. Forty isolates of actinomycetes from the sediment around the water supply intake tower produced both geosmin and MIB in culture. Furthermore, the average ratio of production of geosmin to MIB was 1.40–1.0. Actinomycetes in the sediment played an important role in geosmin production. We concluded that geosmin and MIB from actinomycetes in the sediment, and MIB from P. tenue in the water column were jointly responsible for the severe musty odor occurrence of the waters supply source at Lake Kasumigaura.     

Identification of Major High-Boiling Volatile Compounds Produced During Refrigerated Storage of Haddock Fillets

The two major high-boiling volatile compounds produced during refrigerated storage of haddock fillets were found by gas chromatography and mass spectroscopy to be phenethyl alcohol and phenol.     

The influence of different nitrogen sources on the production of volatile compounds by Dipodascus aggregatus

Summary The yeast fungus Dipodascus aggregatus was grown aerobically on 9 different nitrogen sources and the production of volatile compounds determined by a gas chromatographic head-space technique. Excellent growth was supported by glutamine, aspartic acid, asparagine, (NH₄)₂-tartrate and NH₄H₂PO₄. Valine, leucine, and particularly isoleucine were utilized with a somewhat lower growth rate. Lysine was rapidly utilized after a prolonged lag phase.The highest production of volatile compounds was obtained from leucine and isoleucine. At least 20 volatile compounds were formed from each of them and many products were detected in high concentrations. Intermediate amounts of volatile compounds were produced from asparagine, the ammonium salts and valine, and low amounts from lysine, glutamine and aspartic acid.Ethyl acetate was a major product irrespective of the nitrogen source used. Regarding the pattern of volatile compounds produced, leucine, isoleucine and valine had much in common. Most of the volatile products formed from these amino acids contained a branched carbon chain and at least three high-boiling components eluted later than n-amyl acetate from the gas chromatographic column. The other six nitrogen sources could be grouped together. In general the same volatile compounds were formed from these sources, but the quantities of the individual compounds differed. Only one component eluted later than n-amyl acetate. No basic difference in production of volatile compounds was observed between the ammonium salts and -amino compounds like lysine and asparagine.     

Fermentation odor and bioprocess scale-up

During scaleup of Streptomyces avermitilis batch culture based on similarity of geometry, aeration and mixing conditions, the production of avermectins was proportional to the intensity of fermentation odor represented by geosmin. The active stimulatory role of odor component on antibiotic production was detected after addition of volatile compounds separated from the fermentation liquor by steam distillation.     

微波蒸馏-顶空固相微萃取-气质联用检测鱼体中土霉昧化合物

采用微波蒸馏-顶空固相微萃取-气质联用检测鱼体中常见的两种土霉味化合物,即2-甲基异茨醇(2-MIB)和土腥素(Geosmin).研究并优化了微波蒸馏萃取过程的关键参数(微波蒸馏时间、载气流量),结果表明微波蒸馏6min、载气流70 mL/min为土霉味化合物微波蒸馏萃取的最佳条件.在此优化的条件下,土霉味化合物能够充分地从鱼体中蒸馏出来,再采用顶空固相微萃取的方法使馏分中的土霉味化合物吸附于纤维涂层上,将其在250℃高温下解吸,并用GC-MS分析.基于此测定方法,鱼肉中2-甲基异茨醇和土腥素的检测限均达到0.1μg/kg,且其在1-20μg/kg的范围内线性关系良好,相关系数R分别达到0.987、0.995.因此,用该方法分析鱼体中痕量的(ppb级)的土霉味化合物,结果可靠.     

Identification ofStreptomyces odor spectrum

The chemical composition of odors produced by nine strains ofStreptomyces was determined. StrainsStreptomyces aureofaciens, S. avermitilis, S. cinamomensis, S. coelicolor, S. griseus, S. lividans, S. rimosus, S. spectabilis, S. virginiae (as representatives of producers of biologically active compounds) were cultivated at the same time statically in dishes and in shaken flasks at similar cultivation conditions. According to the GC-MS analysis of odor compounds, more than twenty noteworthy volatile chemical individuals were identified. As the main component of odor spectrum geosmin and homologues of oxolones (dihydrofuranones) were found; the other compounds (pyrazine derivatives, acetoin and its homologues, aromatic esters, furan derivatives,etc.) were in minority. An erratum to this article is available at .     

High-resolution gas chromatographic profiles of volatile organic compounds produced by microorganisms at refrigerated temperatures.

Three different strains of bacteria isolated from spoiled, uncooked chicken were grown in pure culture on Trypticase soy agar supplemented with yeast extract. The volatile organic compounds produced by each culture were concentrated on a porous polymer precolumn and analyzed by high-resolution gas chromatographic mass spectrometry. Twenty different compounds were identified. Both qualitative and quantitative differences in the chromatographic profiles from each culture were found.     

High-resolution gas chromatographic profiles of volatile organic compounds produced by microorganisms at refrigerated temperatures.

Three different strains of bacteria isolated from spoiled, uncooked chicken were grown in pure culture on Trypticase soy agar supplemented with yeast extract. The volatile organic compounds produced by each culture were concentrated on a porous polymer precolumn and analyzed by high-resolution gas chromatographic mass spectrometry. Twenty different compounds were identified. Both qualitative and quantitative differences in the chromatographic profiles from each culture were found.     

Production of volatile organic compounds by the yeast fungus Dipodascus aggregatus

Summary The yeast fungus Dipodascus aggregatus was grown aerobically in a synthetic nutrient solution and the volatile compounds produced were concentrated. Identification of the volatiles was performed by combined gas chromatographymass spectrometry or by one of these methods. The compounds identified were 11 esters, 9 alcohols, 5 acids and 3 carbonyls.The time course production of volatile neutral compounds was followed. During the phase of no apparent growth only a few substances were formed (mostly alcohols). The rapid phase of growth was characterized by an intense synthesis of many compounds in relatively high concentrations and later a sudden decrease in the number and amounts of substances. A slow successive, decline in the number and amounts of volatile components was observed during the phase of no net growth.The volatiles emitted by the fungus were concentrated, when most of the compounds were most abundant and the relative amounts of the major volatile neutral compounds were determined. The main components were ethyl acetate, ethyl propionate and ethanol.     

Metabolism of gamma-hexachlorocyclohexane by Arthrobacter citreus strain BI-100: Identification of metabolites

Growth characteristics of the aerobic bacterial strain Arthrobacter citreus BI-100 in mineral salts medium with gamma-hexachlorocyclohexane (gamma-HCH) as the sole source of carbon and degradation of gamma-HCH by the strain are reported. The highest yield of the bacteria is observed at a gamma-HCH concentration of 100 mg/L. At this concentration, the bacteria entered the exponential phase of growth without any lag. At 8 h of growth, no residual HCH, but its metabolites, was detectable in the medium. The bacterium attained its stationary phase at 48 h and at 72 h; no metabolite of gamma-HCH could be detected by gas chromatography. Six metabolic intermediates of gamma-HCH produced by A. citreus BI-100 at different periods of growth were characterized by using gas chromatography-mass spectrometry and high-performance liquid chromatography, which furnished evidence for the presence of gamma-1,3,4,5,6-pentachlorocyclohexene, tetrachlorocyclohexene, trichlorocyclohexa-diene, 2-chlorophenol, phenol, and catechol, among others.     

Dynamics of the Volatile Organic Substances Associated with Cyanobacteria and Algae in a Eutrophic Shallow Lake

The volatile substances present in a eutrophic shallow lake were determined qualitatively and quantitatively during the growing season, allowing a first-time analysis of the dynamics of these compounds in a water body. The major compounds found were β-cyclocitral, α-cyclocitral, β-ionone, 1,3,3-trimethylcyclohexene, 2,2,6-trimethylcyclohexanone, 2,6,6-trimethylcyclohex-2-en-1-one, eucalyptol, geosmin, two argosmin isomers, pent-1-en-3-ol, pent-1-en-3-one, heptadec-1-ene, heptadec-cis 5-ene, heptadeca-1, cis 8-diene, decanal, and hexanal. Most of the substances have not been detected before in natural waters. The occurrence of geosmin, the argosmin isomers, heptadec-cis 5-ene, and an unknown sesquiterpene could clearly be correlated with the appearance of Aphanizomenon gracile, as could the occurrence of β-cyclocitral with the appearance of Microcystis wesenbergii. The data presented indicate that a rapid metabolism or the elimination of these compounds occurs in lake water.