Prokaryote communities in post-glacial profundal freshwater sediments of Windermere, representing 10-12 000 years of deposition, were examined for culturability, viability and community structure. The potential for active geochemical cycles was inferred from the presence of specific groups of bacteria. Direct count procedures revealed 10l2 cells (g dry wt sediment)-l in the surface sediments, which declined to approximately lo9 cells (g dry wt sediment)" a t 6 m depth of core (representing approximately 10000 years of deposition). The majority of the cells in the upper sediments were metabolically active when challenged with viability probes and responded to the direct viable count method. Below 250 cm, viability shown by 5-cyano-2,3-diotyl tetrazolium chloride (CTC) dye was not significantly different from the direct count; however, counts obtained with 5-carboxyfluorescein diacetate (CFDA) and the direct viable count both declined significantly from the direct count below 250 cm and 1 m, respectively. Culture was achieved from samples throughout the core, although the numbers of culturable bacteria decreased significantly with depth, from lo7 c.f.u. (g dry wt sediment)" to lo1-lo2 c.f.u.(g dry wt sediment)'l below 3 m depth. Among culturable isolates, Grampositives and Gram-negatives were found at all levels of the core, and sporeforming heterotrophs dominated. Although sulphate-reducing bacteria were not detected below 20 cm, isolates demonstrating denitrifying activity were detected a t all depths. PCR performed on samples taken below 3 m (deposited more than 7000 years ago) using eubacterial and archaeal primers revealed sequences similar to those found in deep sediments of the Pacific Ocean and the presence of methanogenic archaea. These observations indicate that bacteria and archaea are capable of long-term persistence and activity in deep, aged freshwater sediments.

The effects of past applications of farmyard manure (FYM, applied from 1942(FYM, applied from to 1967, metalcontaminated sewage sludge (applied from 1942 to 1961) and mineral fertilizer (NPK, applied from 1942 until now) on the microbial biomass and community structure in a sandy loam, arable soil from the Woburn Market Garden Experiment, UK, were investigated in 1998. Concentrations of Cu, Ni and Zn in soils which previously received sewage sludge were less than current European Union (EU) limits, but the soil Cd concentration was more than twice the permitted limit. Organic-C concentration in the FYMtreated soil and contaminated soils was about twice that of NPK-treated soil. The initial microbial biomass-C and estimates of total bacterial numbers by acridine orange direct count were significantly (P < 0.05) greater in the FYM-treated soil compared with the NPK-treated and the most contaminated soils. Total phospholipid fatty acid (PLFA) concentration (another measure of biomass) was significantly greater in the FYM-treated soil compared with either the low or high metal-contaminated soils, both of which contained similar PLFA concentrations. In the metal-contaminated soils, in contrast, fluorescent Pseudomonas counts, as a percentage of total plate counts, were at least 1.5 times greater than in the uncontaminated soils. The concentrations of these microbial parameters were significantly (P < 0.05) less in the NPK soil than in all the other treatments. Biomass-C as a percentage of organic-C was also significantly (P < 0.05) greater in the uncontaminated soils compared with the metal-contaminated soils. Biomass specific respiration rates in the metal-contaminated soils were c. 1.5 times those in the FYMtreated soil. In the metal-contaminated soils, the concentration of mono-unsaturated and hydroxy-fatty acids (derived from phospholipids), and lipopolysaccharide hydroxy-fatty acids (all indicative of Gramnegative bacteria) were significantly (P < 0.05) greater than branched fatty acids (indicative of Grampositive bacteria). Furthermore, Gram-negative counts were 62-68% greater than Gram-positive counts in the metal-contaminated soils. Branched fatty acid concentration was significantly (P < 0.05) greater in the FYM-treated soil than in the metal-contaminated soils. Gram-positive counts were also 63% greater than Gram-negative counts in the FYM-treated soil. We found that effects of the relatively small heavy metal concentration caused measurable decreases in soil microbial biomass-C concentrations, acridine orange direct counts and Gram-positive counts. There were also increases in biomass specific respiration rates, and the microbial community had changed substantially, nearly 40 years after the metal inputs ceased. We conclude that, at the very least, the current EU permitted limits for heavy metals in agricultural soils should not be relaxed.

Soil pollution with hexachlorocyclohexane (HCH) has caused serious environmental problems. Here we describe the targeted degradation of all HCH isomers by applying the aerobic bacterium Sphingobium indicum B90A. In particular, we examined possibilities for large-scale cultivation of strain B90A, tested immobilization, storage and inoculation procedures, and determined the survival and HCH-degradation activity of inoculated cells in soil. Optimal growth of strain B90A was achieved in glucose-containing mineral medium and up to 65% culturability could be maintained after 60 days storage at 30 degrees C by mixing cells with sterile dry corncob powder. B90A biomass produced in water supplemented with sugarcane molasses and immobilized on corncob powder retained 15-20% culturability after 30 days storage at 30 degrees C, whereas full culturability was maintained when cells were stored frozen at -20 degrees C. On the contrary, cells stored on corncob degraded gamma-HCH faster than those that had been stored frozen, with between 15 and 85% of gamma-HCH disappearance in microcosms within 20 h at 30 degrees C. Soil microcosm tests at 25 degrees C confirmed complete mineralization of [(14)C]-gamma-HCH by corncob-immobilized strain B90A. Experiments conducted in small pits and at an HCH-contaminated agricultural site resulted in between 85 and 95% HCH degradation by strain B90A applied via corncob, depending on the type of HCH isomer and even at residual HCH concentrations. Up to 20% of the inoculated B90A cells survived under field conditions after 8 days and could be traced among other soil microorganisms by a combination of natural antibiotic resistance properties, unique pigmentation and PCR amplification of the linA genes. Neither the addition of corncob nor of corncob immobilized B90A did measurably change the microbial community structure as determined by T-RFLP analysis. Overall, these results indicate that on-site aerobic bioremediation of HCH exploiting the biodegradation activity of S. indicum B90A cells stored on corncob powder is a promising technology.

Sphingomonas paucimobilis B90A is able to degrade the ␣-, ␤-, ␥-, and ␦-isomers of hexachlorocyclohexane (HCH). It contains the genes linA, linB, linC, linD, linE, and linR, which have been implicated in HCH degradation. In this study, dynamic expression of the lin genes was measured in chemostat-grown S. paucimobilis B90A by RNA dot blot hybridization and real-time reverse transcriptase PCR upon exposure to a pulse of different HCH isomers. Irrespective of the addition of HCH, linA, linB, and linC were all expressed constitutively. In contrast, linD and linE were induced with ␣-HCH (2 mg/liter) and ␥-HCH (7 mg/liter). A sharp increase in mRNA levels for linD and linE was observed from 10 to 45 min after the addition of ␣-or ␥-HCH. Induction of linD and linE was not detectable upon the addition of 0.7 mg of ␥-HCH per liter, although the compound was degraded by the cells. The addition of ␤-HCH (5 mg/liter) or ␦-HCH (20 mg/liter) did not lead to linE and linD induction, despite the fact that 50% of the compounds were degraded. This suggests that degradation of ␤-and ␦-HCH proceeds by a different pathway than that of ␣-and ␥-HCH.Hexachlorocyclohexane (HCH) has been extensively used for the control of insect pests on agriculturally important crops, seeds, and vegetables, in forestry, and in vector control (34). Mainly two forms of HCH, lindane (␥-HCH) and a technical mixture of all isomers, have been applied. Technical grade HCH (33) largely consists of ␣-HCH (60 to 70%), with ␤-HCH (5 to 12%), ␥-HCH (10 to 15%), and ␦-HCH (6 to 10%) (16). As a result of the extensive use of lindane dust and technical HCH over the years and despite the recent ban on the use of HCH, several countries are currently faced with two very serious problems: (i) soil contamination with small amounts of HCH and (ii) highly contaminated sites where lindane was produced and purified or disposed of (5, 25).Although HCH is persistent and difficult to biodegrade, a few microorganisms have been isolated which can degrade one or more HCH isomers under aerobic conditions (27,29,31). Most of the strains, such as Sphingomonas paucimobilis UT26, degrade ␣-and ␥-HCH but do not degrade ␤-HCH. Only one strain has been described, S. paucimobilis B90A (9), the parent strain of strain B90 (14, 15), that is able to degrade the four ␣-, ␤-, ␥-, and ␦-HCH isomers, although with different rates and not to completion for the ␤-and ␦-HCH isomers. The degradation pathway for ␥-HCH is well established from work on S. paucimobilis strain UT26 (20-24). Degradation of ␥-HCH is mediated by the products of the so-called lin genes (23). It is assumed that ␣-HCH is degraded through the same pathway, but this has not been proven for ␤-and ␦-HCH. S. paucimobilis strains UT26 and B90A have very similar lin gene sequences and organizations (9,15). In contrast to typical degradation pathways in, for example, Pseudomonas or Ralstonia, where very long polycistronic operons are common (10,17,36), the genes for lindane degradation in S. paucimobilis are not organized within one or...

A mixed culture utilizing EDTA as the sole carbon source was isolated from a mixed inoculum of water from the River Mersey (United Kingdom) and sludge from an industrial effluent treatment plant. Fourteen component organisms were isolated from the culture, including representatives of the genera Methylobacterium,Variovorax, Enterobacter,Aureobacterium, and Bacillus. The mixed culture biodegraded metal-EDTA complexes slowly; the biodegradability was in the order Fe>Cu>Co>Ni>Cd. By incorporation of inorganic phosphate into the medium as a precipitant ligand, heavy metals were removed in parallel to EDTA degradation. The mixed culture also utilized a number of possible EDTA degradation intermediates as carbon sources.