Merja H. Kontro

Gas chromatographic - mass spectrometric detection of 2- and 3-hydroxy fatty acids as methyl esters from soil, sediment and biofilm

Hydroxy fatty acids (OH-FAs) can be used in the characterization of microbial communities, especially Gram-negative bacteria. We prepared methyl esters of 2- and 3-OH-FAs from the lipid extraction residue of soil, sediment, and biofilm samples without further purification or derivatization of hydroxyl groups. OH-FA methyl esters were analyzed using a gas chromatograph equipped with a mass selective detector (GC-MS). The ions followed in MS were m/z 103 for 3-OH-FAs and m/z 90 and M-59 for 2-OH-FAs. The rapid determination of 3- and 2-OH-FAs concomitantly with phospholipid fatty acids provided more detailed information on the microbial communities present in soil, sediment, and drinking water biofilm.

Wood ash for application in municipal biowaste composting.

This study aimed to clarify the impacts of pH control by wood ash amendment on biowaste composting processes. To achieve this, fresh source separated municipal biowaste was mixed with low doses (2-8% wt/vol) of wood ash and processed in a pilot and large-scale composting systems. The results indicated a correlation between a low initial pH and delay in the early rise of the process temperature. Wood ash elevated the composting temperatures and pH, and stimulated the mineralisation both in the pilot scale and the industrial large-scale processes. According to the results addition of amounts of 4-8% wood ash is sufficient for efficient biowaste composting process and yields a safe end product. However, to minimise the environmental risk for heavy metal contamination, and meet the criteria for the limit values of the impurities in wood ash, strict quality control of the applied wood ashes should be implemented.

Atrazine and terbutryn degradation in deposits from groundwater environment within the boreal region in Lahti, Finland.

The degradation of pesticides atrazine and terbutryn was investigated under aerobic and anaerobic conditions in the northern boreal region subsurface deposits and sterilized controls from the depths of 6.3-21.0 m below the surface and 1.2-16.9 m below the groundwater table. During 1.3-1.7 years of laboratory incubation, atrazine degradation under aerobic conditions varied from rapid (half-live 38 days) to no degradation. Anaerobically, atrazine half-lives were 430-829 days. Organic matter, nitrogen, and lead in deposits correlated positively with the atrazine concentration in groundwater. Aerobic and anaerobic terbutryn half-lives were 193-644 and 266-400 days, respectively. Microbial aerobic atrazine and terbutryn degradation was confirmed in the deep deposits near the water table. Under aerobic conditions, the high amounts of Cr, Mn, Ni, and Zn in deposits decreased the chemical degradation of terbutryn.

Evolution of clostridia and streptomycetes in full-scale composting facilities and pilot drums equipped with on-line temperature monitoring and aeration.

The evolution of sporulating bacteria in full-scale composting facilities with online temperature monitoring has been poorly studied, although organic matter recycling increases. We analysed Clostridium perfringens and sulphite-reducing clostridia (SRC) by cultivation, and streptomycetes by real-time PCR in five full-scale, temperature-monitored and aerated composting processes, and two pilot-scale drum composters. Facilities composted woodchips, sawdust, peat, or bark amended sludge or source-separated biowaste. Streptomycetes genes of 0.21-110×10(7)copies/g feed increased fast to 0.019-33×10(9)copies/g, and then were equal or decreased. SRC of 0.06-2.2×10(7)cfu/g feed decreased to 0-600 cfu/g, with re-growth in two facilities. End products were clean of C. perfringens, detected in sludge composts. Although processes contained large quantities of spore-forming bacteria, in the best facilities end products had the high quality. Temperature (>55°C,>2d) was not related to the end compost quality, but relations between waste and bulking agent qualities, aeration, and processing time should be better controlled.

Effect of light Sphagnum peat on odour formation in the early stages of biowaste composting

In the present study, we investigated the effects of two bulking materials, Sphagnum peat and pine wood chips, on the early stages of biowaste composting in two pilot-scale processes. Emphasis was placed on studying the formation conditions of malodorous compost gases in the initial phases of the processes. The results showed that gas emission leaving an open windrow and a closed drum composting system contained elevated concentrations of fermentative microbial metabolites when acid Sphagnum peat (pH 3.2) was used as a bulking material. Moreover, the gas emission of the peat amended drum composter contained a high concentration of odour (up to 450,000oum(-3) of air). The highest odour values in the outlet gas of peat amended composts coincided with the elevated concentrations of volatile organic compounds such as acetoin and buthanedion. We conclude that the acidifying qualities of composting substrates or bulking material may intensify odour emission from biowaste composts and prolong the early stages of the composting process.

Utility of Molecular Tools in Monitoring Large Scale Composting

Composting is an aerobic biological process in which solid organic matter is degraded by microorganisms. The microbiology of composting has been of interest for decades, and microbes in composting have been characterized in many types of composing processes using traditional culture-based methods. In recent years, an immense diversity of bacteria, archaea, and fungi has been found to occupy many different habitats using culture-independent molecular biological methods. Molecular methods which can detect both the culturable and non-culturable fractions of the microbial community are under constant development. In this chapter, several new molecular tools for characterising the microbes present in different composting processes are described, and the advantages and limitations of the application of these methods in studying composting microbiology are discussed.

Silylation can be omitted in gas-liquid chromatographic/mass spectrometric analysis of ergosterol

Ergosterol (5,7,22-ergostatrien-3b-ol) is a cell membrane sterol compound characteristic of fungi, and absent or a minor compound in other living organisms, including plants, animals, and bacteria. Ergosterol extracted from soil largely results from living cellular material, due to a quite rapid mineralisation in soil. Therefore, ergosterol can be used as a chemical marker for measuring fungal biomass, and coefficients have been calculated for the conversion of ergosterol content into biomass. Ergosterol has been used to analyse fungal communities in a wide range of environments including leaves, soil, house dust, building material, indoor air and activated sludge. In the analysis of ergosterol, lipidbound fatty acids are commonly hydrolysed and ergosterol released, e.g. by the direct saponification in alcoholic KOH. Ergosterol is then extracted with a solvent such as hexane. For analysis by gas-liquid chromatography/mass spectrometry (GC/MS), the hydroxyl group of ergosterol is then converted, for example, into a trimethylsilyl (TMS) derivative. The conversion of ergosterol into its TMS derivative gives a sharper GC peak, whereas without derivatisation ergosterol may be adsorbed onto the column in the free form, resulting in peak tailing. We present data on ergosterol GC/MS analysis, which support the possibility of omitting the resource-consuming derivatisation procedure. Leaves collected in September 2002 from five silver birches (Betula pendula) in Helsinki (Finland) were dried at 22 28C for 14 days. Approximately 1 g of leaves in separate nylon litter bags (10 15 cm, pore size 1mm) was decomposed in soil at the depth of 5 cm for 8 months. The leaves were lyophilised, pulverised in liquid nitrogen, and 75.3–90.0mg (dry weight) were weighed in duplicate for the ergosterol analysis. Leaves were supplemented with 10% KOH in 3mL of methanol, heated at 808C for 90min, and cooled. The internal standard, 7-dehydrocholesterol, and 1mL of water were added, and samples were extracted twice with 2mL of hexane. After evaporation under a stream of nitrogen at 21 28C, the solid residue was dissolved in 1mL of dichloromethane/hexane (1:1, v/v). The sample was applied to a silica solid-phase extraction (SPE) column (100mg of silica, Varian, Palo Alto, CA, USA) which has been preconditioned with 1mL of diethyl ether and 1mL of dichloromethane/hexane (1:1, v/v), and washed twice with 1mL of dichloromethane/hexane. The sterols were eluted twice with 1mL of diethyl ether, and the solvent was evaporated under a stream of nitrogen. Hexane (0.5mL) was added or, alternatively, the trimethylsilyl (TMS) derivative of the alcohol group in ergosterol was prepared by adding 55mL of N,Obis(trimethylsilyl)trifluoroacetamide/ pyridine/trimethylchlorosilane (5:5:1, v/v/v) and heating at 708C for 15min. Pyridine was evaporated, and 0.5mL of hexane was added. The sample was washed twice with 0.5mL of distilled water, and dried with anhydrous Na2SO4. 17 The amount of egosterol at the end of the sample preparation was calculated using an external calibration curve, and in leaves using an internal standard. The ratio of ergosterol after sample preparation to that in leaves, that is the ergosterol recovery, was without silylation 69.0 10.4% and with silylation 51.7 8.4% (average standard deviation, n1⁄4 8). Methanol, KOH and Na2SO4 were from Merck (Darmstadt, Germany), hexane and dichloromethane from Mallinckrodt (Deventer, The Netherlands), diethyl ether from Riedel de-Häen (Seelze, Germany), silylation reagents from Pierce (Rockford, IL, USA), and 7dehydrocholesterol from Sigma (St. Louis, MO, USA). The sterols were analysed with a model GC-17A gas chromatograph terscience.wiley.com) DOI: 10.1002/rcm.3893 (Shimadzu, Kyoto, Japan) equipped with a model GCMC-QP5000 massselective detector and an model AOC17, long automatic sampler. The gasliquid chromatography conditions were as follows: Zebron ZB-5 capillary column (30m 0.25mm 0.25mm) coated with 5% phenyl–95% dimethylpolysiloxane (Phenomonex, Torrance, CA, USA); carrier gas, helium at a flow rate of 1mLmin ; injection of 1mL, splitless; injector temperature, 3108C; and detector interface temperature, 3008C. The oven temperature was programmed to hold at 1708C for 1min, and then increased by 208C min 1 to 290, 300, 310, or 3208C. The electron ionisation (EI) mass spectra were recorded at an electron energy of 70 eV and emission current of 75mA. The ion source temperature was 2158C. In the GC/MS analysis using selected ion monitoring (SIM) and a final oven and column temperature of 2908C, peaks were produced in positions where ergosterol and 7-dehydrocholesterol and their TMS derivatives were assumed to elute according to the retention times of standards (Fig. 1). In leaf samples without TMS derivatisation, two additional unknown peaks observed could be plant sterols. Saraf et al. could not analyse accurately the TMS derivative of egosterol from 4 of 31 house dust preparations to quantify fungi, due to an unknown compound with the same retention time and the same ion in SIM as the ergosterol TMS derivative. The interference of an unknown substance co-eluting with ergosterol or 7-dehydrocholesterol was not observed in this study. The TMS derivative of ergosterol was detected from ions atm/z 337 [M–131]þ, 363 [M– 105]þ and 468 [M]þ, and the TMS derivative of 7-dehydrocholesterol from ions at m/z 325 [M–131]þ, 351 [M–105]þ and 456 [M]þ. The ions in the mass spectra of the TMS-derivatised ergosterol at m/z 337 [M-131]þ, and the TMSderivatised 7-dehydrocholesterol at 325 [M–131]þ, result from the loss of trimethylsilanol and the C1-C3 fragment. The ions in the TMS-derivatised ergosterol at m/z 363 [M–105]þ, and the TMS-derivatised 7-dehydrocholesterol at m/z 351 [M–105]þ, are due to the loss of trimethylsilanol and a methyl group. The direct analysis of ergos-

Influence of organic matter, nutrients, and cyclodextrin on microbial and chemical herbicide and degradate dissipation in subsurface sediment slurries

Pesticides leaching from soil to surface and groundwater are a global threat for drinking water safety, as no cleaning methods occur for groundwater environment. We examined whether peat, compost-peat-sand (CPS) mixture, NH4NO3, NH4NO3 with sodium citrate (Na-citrate), and the surfactant methyl-β-cyclodextrin additions enhance atrazine, simazine, hexazinone, dichlobenil, and the degradate 2,6-dichlorobenzamide (BAM) dissipations in sediment slurries under aerobic and anaerobic conditions, with sterilized controls. The vadose zone sediment cores were drilled from a depth of 11.3-14.6m in an herbicide-contaminated groundwater area. The peat and CPS enhanced chemical atrazine and simazine dissipation, and the peat enhanced chemical hexazinone dissipation, all oxygen-independently. Dichlobenil dissipated under all conditions, while BAM dissipation was fairly slow and half-lives could not be calculated. The chemical dissipation rates could be associated with the chemical structures and properties of the herbicides, and additive compositions, not with pH. Microbial atrazine degradation was only observed in the Pseudomonas sp. ADP amended slurries, although the sediment slurries were known to contain atrazine-degrading microorganisms. The bioavailability of atrazine in the water phase seemed to be limited, which could be due to complex formation with organic and inorganic colloids. Atrazine degradation by indigenous microbes could not be stimulated by the surfactant methyl-β-cyclodextrin, or by the additives NH4NO3 and NH4NO3 with Na-citrate, although the nitrogen additives increased microbial growth.

Intrapopulation genotypic variation in leaf litter chemistry does not control microbial abundance and litter mass loss in silver birch, Betula pendula

Background and aimsDifferences among plant genotypes can influence ecosystem functioning such as the rate of litter decomposition. Little is known, however, of the strength of genotypic links between litter quality, microbial abundance and litter decomposition within plant populations, or the likelihood that these processes are driven by natural selection.MethodsWe used 19 Betula pendula genotypes randomly selected from a local population in south-eastern Finland to establish a long-term, 35-month litter decomposition trial on forest ground. We analysed the effect of litter quality (N, phenolics and triterpenoids) of senescent leaves and decomposed litter on microbial abundance and litter mass loss.ResultsWe found that while litter quality and mass loss both had significant genotypic variation, the genotypic variation among silver birch trees in the quantity of bacterial and fungal DNA was marginal. In addition, although the quantity of bacterial DNA at individual tree level was negatively associated with most secondary metabolites of litter and positively with litter N, litter chemistry was not genotypically linked to litter mass loss.ConclusionsContrary to our expectations, these results suggest that natural selection may have limited influence on overall microbial DNA and litter decomposition rate in B. pendula populations by reworking the genetically controlled foliage chemistry of these populations.

A zero-valent iron and organic matter mixture enhances herbicide and herbicide degradation product removal in subsurface waters.

The pesticide atrazine, its degradation products, and 2,6-dichlorobenzamide (BAM) are persistent in groundwater environment. We studied whether their dissipation can be enhanced with a mixture of a complex carbon source and zero-valent iron (ZVI) called EHC®. The application rates were 1.0% and 2.0% (by weight) in subsurface sediments slurries (atrazine 30mg/L), and 2.0% in 1.5m pilot-scale sediment columns with groundwater flowing through (atrazine 0.08, desethylatrazine DEA 0.03, BAM 0.02μg/L). In the slurries under aerobic conditions, atrazine of 0.88±0.14mg/g of EHC® was dissipated chemically, as concentrations did not differ significantly between the slurries and their sterilized controls. No degradation occurred in the slurries under anaerobic conditions. In the pilot-scale columns under water-saturated conditions, atrazine, DEA and BAM were not detected in effluents during 33, 64 and 64days from the beginning of the water flow through EHC® columns, respectively, but thereafter traces of compounds could be detected. No atrazine or degradation products (BAM, DEA, deisopropylatrazine, desethyldeisopropylatrazine) could be extracted from the column sediments at the end of the experiment. As a result, the sum of dissipated pesticides was about 7.6μg/g of EHC® in columns under water-saturated conditions, and 0.88mg/g of EHC® in slurries under aerobic conditions. EHC® can be used to enhance the dissipation of studied pesticides in small quantities, preferentially under aerobic conditions.