Improved detection of soil microorganisms using fluorescence in situ hybridization (FISH) and catalyzed reporter deposition (CARD-FISH)

In recent years, methods of molecular microbiology have been used for the investigation of soil microbial diversity. Fluorescence in situ hybridization (FISH) represents a method which allows a specific staining and enumeration of soil microorganisms by using fluorescent-labelled oligonucleotide probes. However, the detection of FISH-stained cells is often affected by strong autofluorescence of the background, especially in samples of the top soils.In this study a more efficient FISH-approach coupled with catalyzed reporter deposition (CARD) was adapted to soils. Due to tyramide signal amplification (TSA) the fluorescence intensity has been considerably increased at the target binding site of a probe.Six different soils were investigated to evaluate the effect of sample preparation and pre-treatments, TSA, and the procedure of detection. The results show that both cell permeabilization and TSA are two important factors which improve in situ hybridization of soil microorganisms. Soils with higher clay contents have shown better results when prepared on polycarbonate filters rather than on glass slides.Using specific fluorescence filter systems and dye combinations the detection of hybridized cells was extensively increased compared with the application of monolabelled oligonucleotide probes in regular FISH-analysis. As a result, CARD-FISH-stained cells were suitable for automated counting using digital image analysis. Nevertheless, the counterstain with DAPI had to be analyzed manually as it was strongly affected by autofluorescence.     

多粘芽孢杆菌芽孢荧光原位杂交检测技术研究及其在有机肥发酵中的应用

采用化学和萌发两种预处理方法,对多粘芽孢杆菌SQR21芽孢进行荧光原位杂交(FISH),结合扫描电镜比较不同预处理芽孢表面结构和形态变化。结果表明,两种预处理均可在2 h内完成SQR21芽孢的荧光原位杂交,且萌发预处理比化学预处理杂交效率高,荧光信号强。扫描电镜结果显示,萌发预处理芽孢无裂解,而化学预处理芽孢有不同程度的裂解。将萌发预处理荧光原位杂交用于检测猪粪有机肥中二次发酵的SQR21芽孢,发现在37℃,含水量50%的条件下,2 h后芽孢开始生长,4 h后营养体细胞出现,12 h后营养体细胞有显著优势。本研究建立的芽孢荧光原位杂交检测技术,比平板计数更能准确反映多粘芽孢杆菌芽孢的数量和生长动态,在生物有机肥中芽孢杆菌的检测方面有良好的应用前景。     

Localization of bacteria in lichens from Alpine soil crusts by fluorescence in situ hybridization

Lichens are prominent components of many biological soil crusts. Owing to their persistence, lichen thalli create microhabitats for other microbes. Here, the structure of bacterial communities at the thallus–soil interface in lichen soil crusts was studied by using fluorescence in situ hybridization (FISH), confocal laser scanning microscopy (CLSM) and 3D image reconstruction. Terricolous lichen thalli above the tree-line in open habitats of the Austrian Alps were sampled. We selected six lichen species associated with green algal photobionts: Arthrorhaphis citrinella, Baeomyces placophyllus, B. rufus, Icmadophila ericetorum, Psora decipiens and Trapeliopsis granulosa. Alphaproteobacteria and Acidobacteria are predominant in these soil crust lichens, where the latter are frequently present in the lower part of lichen thalli and in the hypothallosphere. In the inconspicuous thallus structures of Arthrorhaphis citrinella, Baeomyces rufus, Icmadophila ericetorum and Trapeliopsis granulosa we observed association of bacteria with algal cells in soil particles and on the outer surface of the mycobiont–photobiont aggregates. We found bacterial cells intermixed with photobiont cells in the lower part of the lichen thalli and as small colonies on the surface of the squamules of Baeomyces placophyllus and Psora decipiens. Moreover, technical issues of performing FISH and confocal microscopy with biological soil crusts are discussed.     

Deconjugation and degradation of flavonol glycosides by pig cecal microbiota characterized by Fluorescence in situ hybridization (FISH)

As the bioavailability of flavonoids is influenced by intestinal metabolism, we have investigated the microbial deconjugation and degradation of several flavonols and flavonol glycosides using the pig cecum in vitro model system developed in our group. For this model system the microbiota was directly isolated from the cecal lumen of freshly slaughtered pigs. The characterization of the cecal microbiota by fluorescence in situ hybridization (FISH) with 16S rRNA-based oligonucleotide probes confirmed the suitability of the model system for studying intestinal metabolism by the human microbiota. We have investigated the microbial degradation of quercetin-3-O-beta-d-rutinoside 1, quercetin-3-O-beta-d-glucopyranoside 2, quercetin-4'-O-beta-d-glucopyranoside 3, quercetin-3-O-beta-d-galactopyranoside 4, quercetin-3- O-beta-d-rhamnopyranoside 5, quercetin-3- O-[alpha-l-dirhamnopyranosyl-(1-->2)-(1-->6)-beta-d-glucopyranoside 6, kaempferol-3-O-[alpha-l-dirhamnopyranosyl-(1-->2)-(1-->6)-beta-d-glucopyranoside 7, apigenin 8, apigenin-8- C-glucoside (vitexin) 9, and feruloyl-O-beta-d-glucopyranoside 10 (100 microM), representing flavonoids with different aglycones, sugar moieties, and types of glycosidic bonds. The degradation rate was monitored using HPLC-DAD. The flavonol O-glycosides under study were almost completely metabolized by the intestinal microbiota within 20 min and 4 h depending on the sugar moiety and the type of glycosidic bond. The degradation rates of the quercetin monoglycosides showed a clear dependency on the hydroxyl pattern of the sugar moiety. The degradation of 2 with all hydroxyl groups of the glucose in the equatorial position was the fastest. The intestinal metabolism of di- and trisaccharides was much slower compared to the monoglycosides. The structure of the aglycone has not much influence on the intestinal metabolism; however, the type of glycosidic bond ( C- or O-glycoside) has substantial influence on the degradation rate. The liberated aglycones were completely metabolized within 8 h. Phenolic compounds such as 3,4-dihydroxyphenylacetic acid 12, 4-hydroxyphenylacetic acid 13, and phloroglucinol 18 were detected by GC-MS as main degradation products.     

Detection of soil water in macropores of undisturbed soil using microfocus X-ray tube computerized tomography (μCT)

Minimal information has been garnered regarding the spatial distribution of soil water in relation to pores and the soil matrix. Destructive layer-by-layer reconstructions derived from polished section methodology exclude any data of water in the soil. In contrast, microfocus X-ray tube computerized tomography generates images of the internal structure of the soil with a resolution down to 1 μm, at the same time creating a visual image of the spatial distribution of water in undisturbed soils.As X-rays pass through the soil, some radiation is absorbed, some is scattered, and some is transmitted. Using advanced microfocus computerized tomography (μCT) which ensures controlled and stable output intensity for X-ray emissions and thus a constant focal spot size and spatial resolution, the resulting pattern of radiation detects to a 0.5% contrast difference. While 2D X-ray imaging is sufficient in many cases, 3D images derived from X-ray irradiation of a soil sample can reveal complex inner structures in more comprehensive format, providing information on the causal connection of water and soil structure.Using the X-ray Feinfocus Y.FOX System and related programmes, two- and three-dimensional images of two different soils (Haplic Luvisol and Stagnic Anthrosol) at field capacity (pF 1.8) have been produced which show films of water which are associated with the pore surfaces. The mean thickness of the water films was 10.6 μm in the Stagnic Anthrosol and 3.0 μm in the Haplic Luvisol. These results were unexpected in pores >50 μm since at field capacity only the adhesive water should be present which would create water films in the range of nanometres. Myriads of colloidal dispersed nanoparticles, detected with dark field microscopy and SEM, seem to be the source of the adhesion and cohesion, causing micro-rheological effects which lead to water films of up to 30 μm in pores. Additionally, nanoparticles correlated to the clay content (fine clay) appear to conglomerate in the water films, presumably forming surface protuberances on the films of varying extent.     

Reductive dissolution of crystalline and amorphous Fe(III) oxides by microorganisms in submerged soil

Summary Reduction of Fe(III) of amorphous and crystalline Fe(III) oxides to Fe(II) in flooded soils was studied using ⁵⁹Fe(OH)₃ and ⁵⁹Fe₂O₃. The results indicated that Fe(III) in the amorphous oxide was readily amenable to microbial reduction in anaerobic soil condition whereas Fe(III) in the crystalline oxide was not. Following soil submergence, the native as well as the applied crystalline Fe(III) oxides were rapidly converted into the amorphous form. The transformation of the crystalline oxides to the amorphous form appears to be a prerequisite for the reduction of Fe(III) of the oxide. This transformation, probably through hydration, is also mediated by microorganisms.     

Growth rates of Aporrectodea caliginosa (Oligochaetae: Lumbricidae) as influenced by soil temperature and moisture in disturbed and undisturbed soil columns

Earthworm growth is affected by fluctuations in soil temperature and moisture and hence, may be used as an indicator of earthworm activity under field conditions. There is no standard methodology for measuring earthworm growth and results obtained in the laboratory with a variety of food sources, soil quantities and container shapes cannot easily be compared or used to estimate earthworm growth in the field. The objective of this experiment was to determine growth rates of the endogeic earthworm Aporrectodea caliginosa (Savigny) over a range of temperatures (5–20 °C) and soil water potentials (−5 to−54 kPa) in disturbed and undisturbed soil columns in the laboratory. We used PVC cores (6 cm diameter, 15 cm height) containing undisturbed and disturbed soil, and 1 l cylindrical pots (11 cm diameter, 14 cm height) with disturbed soil. All containers contained about 500 g of moist soil. The growth rates of juvenile A. caliginosa were determined after 14–28 days. The instantaneous growth rate (IGR) was affected significantly by soil moisture, temperature, and the temperature×moisture interaction, ranging from −0.092 to 0.037 d⁻¹. Optimum growth conditions for A. caliginosa were at 20 °C and −5 kPa water potential, and they lost weight when the soil water potential was −54 kPa for all temperatures and also when the temperature was 5 °C for all water potentials. Growth rates were significantly greater in pots than in cores, but the growth rates of earthworms in cores with undisturbed or disturbed soil did not differ significantly. The feeding and burrowing habits of earthworms should be considered when choosing the container for growth experiments in order to improve our ability to extrapolate earthworm growth rates from the laboratory to the field.     

Identification of in situ 2,4-dichlorophenoxyacetic acid-degrading soil microorganisms using DNA-stable isotope probing

Stable isotope probing (SIP) was used to investigate the microorganisms responsible for degradation of the herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) in soil samples. Soils were unamended or amended with either unlabeled 2,4-D or UL(ring) ¹³C-2,4-D. Degradation of 2,4-D was complete after 17 days, whereas little removal (11±3%) was observed in the sterile controls. Terminal restriction fragment length polymorphism (TRFLP) on soil DNA after 17 days indicated a consistent increase in the relative abundance of one fragment (217 bp in Hae III digests) in soils spiked with 2,4-D (both unlabeled and labeled samples) compared to the unamended soils. DNA extracts from labeled and unlabeled 2,4-D amended soils were subject to ultracentrifugation, fractionation of centrifuged samples, followed by TRFLP on each fraction. TRFLP profiles from ultracentrifugation fractions illustrated that the same fragment experienced an increase in buoyant density (BD) in samples spiked with ¹³C-labeled 2,4-D. This increase in DNA BD indicates the organisms represented by this fragment were responsible for uptake and degradation of the herbicide. 16S rRNA sequencing of the heavy, ¹³C-enriched fraction suggests the organisms belong to the β subdivision of Proteobacteria. Herein, SIP facilitated the identification of unique organisms degrading 2,4-D in soil without the need for isolation and provided more direct evidence for a functional role of these organisms than would have been possible with the molecular-based methods alone.     

Degradation of Sevin by soil microorganisms

Microorganisms capable of transforming the pesticide 1-naphthyl N-methyl-carbamate (Sevin) were isolated from soil. Three isolates were able to accelerate the hydrolysis of Sevin to 1-naphthol. Several unidentified intermediates were separated by thin-layer chromatography and also by following the decomposition of Sevin-methyl-¹⁴C. Since 1-naphthol is a biological as well as a chemical decomposition product of Sevin, its transformation by the isolated microbes was also studied. A fungus, identified as Fusarium solani, altered 1-naphthol rapidly. Whereas one strain of bacterium degraded the hydrolysis product gradually, another strain accumulated it under certain conditions. Mixed cultures of the investigated microbes were more effective in transforming Sevin than pure cultures, and this phenomenon was also observed with 1-naphthol as substrate with the exception of one bacterial strain.     

Decomposition of atmospheric hydrogen by soil microorganisms and soil enzymes

The decomposition of atmospheric hydrogen in different types of soil was measured. The decomposition of H₂ was apparently a first-order reaction. H₂ decomposition activity was proportional to the amount of soil with maximum activities at soil water contents of approx. 6–11% (w/w). The activity was lower under anaerobic conditions, but was constant between 1–20% O₂. It was destroyed by autoclaving and was partially inactivated by fumigation with NH₃, CHC1₃ or acetone, by u.v. irradiation and by treatment with NaCN or NaN₃, indicating that biological processes in the soil were responsible for the observed H₂ decomposition. Treatment of soil with toluene or CHCl₃ caused only a partial inactivation. Incubation of soil in the presence of streptomycin or actidione reduced H₂ decomposition by less than 50%, whereas CO consumption was abolished. The H₂ decomposition rates showed H₂ saturation curves with apparent Michaelis-Menten kinetics. Cooperative effects were not observed. V_max was reached at approx. 200 μl1^?1. The K_m values for H₂ were in the range of 30μl 1^?1, but increased to higher values, when the soil had been pretreated with high H₂ mixing ratios. Apparently, the observed H₂ decomposition by soil is not only due to the activity of viable microorganisms, but soil enzymes as well.     

Metabolism of fluorodifen by soil microorganisms

The metabolism of fluorodifen (p-nitrophenyl α,α,α,-trifluoro-2-nitro-p-tolyl ether) by soil microorganisms in the presence or absence of other carbon and nitrogen sources was studied. The degradation of this herbicide continued for 5 days, when benzoate or acetate and ammonium sulphate were included in the cultures, and for more than 5 weeks when fluorodifen was used as a sole source of carbon and nitrogen.Under all conditions nitrite ions were produced at concentrations ranging between 5 and 80 per cent of the nitro-nitrogen of the fluorodifen present. The highest concentration of nitrite was obtained when added carbon sources were used with fluorodifen. The lowest nitrite concentration accumulated when the fluorodifen was used as sole source of carbon and nitrogen. The nitrite reached a maximum value after a few days of incubation, followed by rapid disappearance.p-Nitrophenol and quinol were identified in the acid-ether extract of cultures. It is suggested that the first step in the degradation of fluorodifen is the hydrolysis of the ether linkage followed by the direct elimination of the nitro-groups as nitrite ions.     

Nondestructive evaluation of anthocyanins in olive (Olea europaea) fruits by in situ chlorophyll fluorescence spectroscopy

Anthocyanins (Anths) in olive (Olea europaea L.) fruits at different degrees of pigmentation were assessed nondestructively by measuring chlorophyll fluorescence (ChlF). The method is based on the comparison of the ChlF excitation spectra from olives with different pigmentation from green to green-red, reddish-purple, and purple. The logarithm of the ratio between the fluorescence excitation spectra (logFER) from two different colored zones gave the difference in the absorption spectrum between them. The absorbance spectrum derived from the logFER between a red olive and the same olive devoid of the skin showed the typical Anth green band (at 550 nm). It matched that recorded by microspectrophotometry on a single pulp cell and the in vitro absorbance spectrum of the olive skin extract. As expected, the in vivo Anths absorption maximum increased in intensity going from less to more mature olives and was higher in the sun-exposed olive side with respect to the sun-shaded side. Absolute quantitative nondestructive determination of Anths for each olive sample was obtained by the logFER calculated for two excitation wavelengths, 550 and 625 nm, of ChlF at 740 nm. Going from green to purple skin colors, the Log[ChlF(625)/ChlF(550)] was fairly well-correlated to the extract Anths concentration. Finally, the relationship between the Anths and the other main phenolics present in the olives analyzed by high-performance liquid chromatography was evaluated. The main result was a net increase of verbascoside with increasing Anths content. On the basis of our results, the development of a new rapid and noninvasive method for the monitoring of olive development and ripening can be envisaged.     

Kinetics of glucose uptake by soil microorganisms

The kinetics of glucose uptake by soil microorganisms was investigated. Soil amended with an inorganic nutrient solution containing C glucose at concentrations of 2.5, 5.0, 10.0 or 20.0 mmol 1⁻¹ was maintained at 4, 12 or 25°C for varying times. The soil was analyzed for glucose, soluble ^14C, total organic ¹⁴C and evolved ¹⁴CO₂ to develop a carbon balance for the system and to define Michaelis-Menten kinetic parameters (K_m and V_max) for glucose uptake at each temperature.Glucose uptake rates, as measured by the depletion of glucose or soluble ¹⁴C from solution, were similar in soils maintained at 12 or 25°C. Based on the depletion of soluble ¹⁴C, values for K_m were 2.25 and 2.43 mmol I⁻¹ at 12 and 25°C, respectively, while V_max values were 0.25 and 1.61 h¹⁴', respectively. Glucose depletion at 4°C was faster than at 12C, while soluble ¹⁴C was removed at a significantly slower rate, suggesting soluble-C intermediates were produced in the 4°C system. Based on Chromatographie techniques and GC-MS, a soluble ¹⁴C-compound accumulating in the 4°C system was identified as maltose. The conversion of glucose to maltose resulted in K_m and V_max values of 17.29 mmol I⁻¹ and 0.12h⁻¹, respectively, for soluble ¹⁴C depletion and 4.96mmol1⁻¹ and 0.43 h⁻, respectively, for glucose depletion at 4δC. These results demonstrate the need to differentiate uptake rates for the parent compound as well as for transitory intermediates excreted into the growth medium. Evolution of CO₂ was shown to be a poor indicator of the rapid disappearance of glucose in soils.     

Biomass and respiration activity of soil microorganisms in anthropogenically transformed ecosystems (Moscow region)

In the forest, meadow, arable, and urban ecosystems (recreational, residential, and industrial zones) of Sergiev Posad, Shatura, Serpukhov, and Serebryanye Prudy districts of Moscow region, spatially separated sites (3–5 points per site) have been randomly selected and soil samples have been taken from the 0–10 (plant litter excluded) and 10- to 150-cm layers (a total of 201 samples have been taken). In the samples, the microbial biomass carbon (C_mic), the rate of the basal (microbial) respiration (BR), and the physical parameters (the particle size distribution (PSD), organic carbon (C_org), pH, heavy metals, and nutrients (NPK)) have been determined. High spatial variability has been revealed for C_mic and BR in all the ecosystems and the functional zones of the studied districts, and a clear tendency of a decrease in these parameters has been shown in the arable soils (by 1.4–3.2 times) and the industrial zone (by 1.7–3.3 times) compared to the natural analogues and other corresponding functional zones. It has been shown that the spatial distribution of the microbiological parameters is significantly (p ≤ 0.05) affected by the physicochemical properties of the soil (C_mic by the PSD and PSD × C_org; BR by the pH and pH × NPK; contributions of 40 and 63%, respectively), as well as by the type of ecosystem and the region of study (the contribution of the sum of these factors to the C_mic and BR was 56 and 67%, respectively). A tendency toward the deterioration of the functioning of the microbial community under the anthropogenic transformation of the soil has been shown. The contribution of the urban soils as a potential source of CO₂ emission to the atmosphere has been calculated and discussed.     

Fertilizer and temperature effects on urea hydrolysis in undisturbed soil

Summary Few published studies have examined the effects of a continuous fertilizer application on urea hydrolysis. In the present study we investigated the effects of 9 years of continuous application of urea and P fertilizers on urea hydrolysis in undisturbed soil samples as affected by temperature (5–45°C). Undisturbed soil samples of surface horizons (0–7 cm) were obtained with cutting rings (50 cm³) from different fertilizer-treatment plots and inserted in polyethylene bottles (with cutting rings). Each soil sample (in the cutting ring) was treated with 10 ml urea solution (0.5 mmol urea N g^-1 soil) and then broght to 90% field capacity. The samples were left to equilibrate for 30 min at a temperature of 4°C, then placed in an incubator at 37°C for 6 h. The results indicated that 9 years of continuous application of urea but not P had a significant effect on urea hydrolysis in soil. There was a good correlation between temperature and urea hydrolysis in soil. Q ₁₀ was between 1.97 and 2.08 in the temperature range 5–45°C.     

荧光原位杂交技术检测土壤中博德特氏菌探针的设计与应用

根据博德特氏菌(Bordetellasp.)的16S rRNA基因序列,设计荧光原位杂交(FISH)检测博德特氏菌的寡核苷酸探针FW_iso_62和FW_iso_761,在20%～60%甲酰胺均有很强的荧光信号。采用探针FW_iso_62及其竞争探针,结合Nycodenz和DAPI技术,建立定量检测土壤中博德特氏菌的DAPI-FISH方法。该方法可排除土壤颗粒的自动荧光对细菌信号的掩盖,保证图片中有大量微生物供统计分析,还能有效保存微生物的原位信息。应用该方法分析土壤中1,2,4-三氯苯降解菌-博德特氏菌,结果未受氯苯污染的农田土壤中没有检测到博德特氏菌,而氯苯污染土壤中检测到大量的博德特氏菌,每克土壤含3.78×106个。将该污染土壤中分离的博德特氏降解菌及其降解菌群接种至农田土壤中,降解菌的数量均随培养而增加,一个月后分别占DAPI计数的1.7%和3.8%。本研究设计的探针可有效用于复杂环境样品中博德特氏菌的定性与定量检测。     

Interactions of soil microorganisms on growth and disease incidence of pepper (Capsicum annum)

The effect of interactions between soil borne pathogen, Phytophthora infestans the antagonistic organism Trichoderma viride and Arbuscular mycorrhizal Glomus etunicatum on growth and disease severity of pepper Capsicum annum was investigated in a greenhouse experiment. Pepper seedlings inoculated with the pathogen alone had sever disease symptoms. While those inoculated with mycorrhizal alone had the highest flowering and fruiting values, followed by the pepper seedlings inoculated with Trichoderma viride. But pepper seedlings simultaneously or dually inoculated with the three microorganisms also had good growth parameters, such as early and high flowering incidence, fruit maturity increase in leaf number, height and growth, while the effect of the pathogen was highly suppressed.     

Stability and transportability of biosolid colloids through undisturbed soil monoliths

This study evaluated the stability of water-suspended biosolid colloids fractionated from municipal and agricultural wastes and their transportability through undisturbed soil monoliths. The aim of the study was to assess potential risks posed by dispersed biosolid colloid particles as carriers of contaminants associated with organic waste amendments applied to soils. All biosolid colloids showed remarkable stability over a wide range of pH conditions, with 50–90% remaining in suspension after 24 h. Lime-stabilized biosolid colloids (LSB) were more stable than poultry manure (PMB) and aerobically digested (ADB) biosolid colloids, with pH and organic matter (OM) content being the dominant factors influencing colloid stability. However, increased colloid stability did not always result in greater transportability. In spite of their higher overall stability, LSB colloids showed low transportability potential through undisturbed soil monoliths averaging < 0.1C/C₀, probably due to carbonate dissolution and increased ionic interaction with the soil matrix. The ADB and PMB colloids, with the highest OM content, exhibited increased mobility, particularly through soils with significant macroporosity. Breakthrough curves were mostly irregular, suggesting considerable macropore flow and increased interaction with the soil matrix. Eluent pH and EC fluctuations appeared to mainly affect LSB colloid transport, while the mobility of the remaining biosolid colloids was maintained over a wide range of ionic conditions. The findings of this study suggest that some biosolid colloids derived from soil-applied agricultural or municipal wastes may exhibit considerable mobility through soil macropores with potential to migrate to great distances in subsoil environments. Considering the high surface charge and electronegativity of these colloids, they may be a significant vector for hydrophobic contaminant transport and pollution of groundwater resources.     

土壤和土壤悬液中1,2,4-三氯苯的微生物强化降解

Inoculating soil with an adapted microbial community is a more effective bioaugmentation approach than inoculation with pure strains in bioremediation.However,information on the potential of different inocula from sites with varying contamination levels and pollution histories in soil remediation is lacking.The objective of the study was to investigate the potential of adapted microorganisms in soil inocula,with different contamination levels and pollution histories,to degrade 1,2,4-trichlorobenzene (1,2,4-TCB).Three different soils from chlorobenzene-contaminated sites were inoculated into agricultural soils and soil suspension cultures spiked with 1,2,4-TCB.The results showed that 36.52% of the initially applied 1,2,4-TCB was present in the non-inoculated soil,whereas about 19.00% of 1,2,4-TCB was present in the agricultural soils inoculated with contaminated soils after 28 days of incubation.The soils inoculated with adapted microbial biomass (in the soil inocula) showed higher respiration and lower 1,2,4-TCB volatilization than the non-inoculated soils,suggesting the existence of 1,2,4-TCB adapted degraders in the contaminated soils used for inoculation.It was further confirmed in the contaminated soil suspension cultures that the concentration of inorganic chloride ions increased continuously over the entire experimental period.Higher contamination of the inocula led not only to higher degradation potential but also to higher residue formation.However,even inocula of low-level contamination were effective in enhancing the degradation of 1,2,4-TCB.Therefore,applying adapted microorganisms in the form of soil inocula,especially with lower contamination levels,could be an effective and environment-friendly strategy for soil remediation.