Bacterial community structure and variation in a full-scale seawater desalination plant for drinking water production.

Microbial processes inevitably play a role in membrane-based desalination plants, mainly recognized as membrane biofouling. We assessed the bacterial community structure and diversity during different treatment steps in a full-scale seawater desalination plant producing 40,000 m(3)/d of drinking water. Water samples were taken over the full treatment train consisting of chlorination, spruce media and cartridge filters, de-chlorination, first and second pass reverse osmosis (RO) membranes and final chlorine dosage for drinking water distribution. The water samples were analyzed for water quality parameters (total bacterial cell number, total organic carbon, conductivity, pH, etc.) and microbial community composition by 16S rRNA gene pyrosequencing. The planktonic microbial community was dominated by Proteobacteria (48.6%) followed by Bacteroidetes (15%), Firmicutes (9.3%) and Cyanobacteria (4.9%). During the pretreatment step, the spruce media filter did not impact the bacterial community composition dominated by Proteobacteria. In contrast, the RO and final chlorination treatment steps reduced the Proteobacterial relative abundance in the produced water where Firmicutes constituted the most dominant bacterial group. Shannon and Chao1 diversity indices showed that bacterial species richness and diversity decreased during the seawater desalination process. The two-stage RO filtration strongly reduced the water conductivity (>99%), TOC concentration (98.5%) and total bacterial cell number (>99%), albeit some bacterial DNA was found in the water after RO filtration. About 0.25% of the total bacterial operational taxonomic units (OTUs) were present in all stages of the desalination plant: the seawater, the RO permeates and the chlorinated drinking water, suggesting that these bacterial strains can survive in different environments such as high/low salt concentration and with/without residual disinfectant. These bacterial strains were not caused by contamination during water sample filtration or from DNA extraction protocols. Control measurements for sample contamination are important for clean water studies.

Morphology, mineralogy, and solid-liquid phase separation characteristics of Cu and Zn precipitates produced with biogenic sulfide.

The morphology, mineralogy, and solid-liquid phase separation of the Cu and Zn precipitates formed with sulfide produced in a sulfate-reducing bioreactor were studied at pH 3, 5, and 7. The precipitates formed at pH 7 display faster settling rates, better dewaterability, and higher concentrations of settleable solids as compared to the precipitates formed at pH 3 and 5. These differences were linked to the agglomeration of the sulfidic precipitates and coprecipitation of the phosphate added to the bioreactor influent. The Cu and Zn quenched the intensity of the dissolved organic matter peaks identified by fluorescence-excitation emission matrix spectroscopy, suggesting a binding mechanism that decreases supersaturation, especially at pH 5. X-ray absorption fine structure spectroscopy analyses confirmed the precipitation of Zn-S as sphalerite and Cu-S as covellite in all samples, but also revealed the presence of Zn sorbed on hydroxyapatite. These analyses further showed that CuS structures remained amorphous regardless of the pH, whereas the ZnS structure was more organized at pH 5 as compared to the ZnS formed at pH 3 and 7, in agreement with the cubic sphalerite-type structures observed through scanning electron microscopy at pH 5.

Leaching and accumulation of trace elements in sulfate reducing granular sludge under concomitant thermophilic and low pH conditions.

The leaching and/or accumulation of trace elements in sulfate reducing granular sludge systems was investigated. Two thermophilic up-flow anaerobic sludge bed (UASB) reactors operated at pH 5 were fed with sucrose (4 gCOD l(reactor)(-1)d(-1)) and sulfate at different COD/SO(4)(2-) ratios. During the start-up of such acidogenic systems, an initial leaching of trace elements from the inoculum sludge occurred regardless of trace elements supplementation in the reactor influent. The granular sludge maintained the physical structure despite high Fe leaching. After start-up and nonetheless the acidic conditions, Co, Ni, Cu, Zn, Mo and Se were retained or accumulated by the sludge when added. Particularly, Ni and Co accumulated in the carbonates and exchangeable fractions ensuring potential bioavailability. Otherwise, the initial stock in the inoculum sludge sufficed to operate the process for nearly 1 year without supplementation of trace elements and no significant sludge wash-out occurred.

Above- and below-ground methane fluxes and methanotrophic activity in a landfill-cover soil.

Landfills are a major anthropogenic source of the greenhouse gas methane (CH(4)). However, much of the CH(4) produced during the anaerobic degradation of organic waste is consumed by methanotrophic microorganisms during passage through the landfill-cover soil. On a section of a closed landfill near Liestal, Switzerland, we performed experiments to compare CH(4) fluxes obtained by different methods at or above the cover-soil surface with below-ground fluxes, and to link methanotrophic activity to estimates of CH(4) ingress (loading) from the waste body at selected locations. Fluxes of CH(4) into or out of the cover soil were quantified by eddy-covariance and static flux-chamber measurements. In addition, CH(4) concentrations at the soil surface were monitored using a field-portable FID detector. Near-surface CH(4) fluxes and CH(4) loading were estimated from soil-gas concentration profiles in conjunction with radon measurements, and gas push-pull tests (GPPTs) were performed to quantify rates of microbial CH(4) oxidation. Eddy-covariance measurements yielded by far the largest and probably most representative estimates of overall CH(4) emissions from the test section (daily mean up to ∼91,500µmolm(-2)d(-1)), whereas flux-chamber measurements and CH(4) concentration profiles indicated that at the majority of locations the cover soil was a net sink for atmospheric CH(4) (uptake up to -380µmolm(-2)d(-1)) during the experimental period. Methane concentration profiles also indicated strong variability in CH(4) loading over short distances in the cover soil, while potential methanotrophic activity derived from GPPTs was high (v(max)∼13mmolL(-1)(soil air)h(-1)) at a location with substantial CH(4) loading. Our results provide a basis to assess spatial and temporal variability of CH(4) dynamics in the complex terrain of a landfill-cover soil.

Quantifying methane oxidation in a landfill-cover soil by gas push-pull tests.

Methane (CH(4)) oxidation by aerobic methanotrophs in landfill-cover soils decreases emissions of landfill-produced CH(4) to the atmosphere. To quantify in situ rates of CH(4) oxidation we performed five gas push-pull tests (GPPTs) at each of two locations in the cover soil of the Lindenstock landfill (Liestal, Switzerland) over a 4 week period. GPPTs consist of the injection of a gas mixture containing CH(4), O(2) and noble gas tracers followed by extraction from the same location. Quantification of first-order rate constants was based upon comparison of breakthrough curves of CH(4) with either Ar or CH(4) itself from a subsequent inactive GPPT containing acetylene as an inhibitor of CH(4) oxidation. The maximum calculated first-order rate constant was 24.8+/-0.8 h(-1) at location 1 and 18.9+/-0.6 h(-1) at location 2. In general, location 2 had higher background CH(4) concentrations in vertical profile samples than location 1. High background CH(4) concentrations in the cover soil during some experiments adversely affected GPPT breakthrough curves and data interpretation. Real-time PCR verified the presence of a large population of methanotrophs at the two GPPT locations and comparison of stable carbon isotope fractionation of CH(4) in an active GPPT and a subsequent inactive GPPT confirmed that microbial activity was responsible for the CH(4) oxidation. The GPPT was shown to be a useful tool to reproducibly estimate in situ rates of CH(4) oxidation in a landfill-cover soil when background CH(4) concentrations were low.

Effect of yeast extract on speciation and bioavailability of nickel and cobalt in anaerobic bioreactors.

The speciation of metals plays an important role in their bioavailability. In the case of anaerobic reactors for the treatment of wastewaters, the ubiquitous presence of sulfide leads to extensive precipitation of metals like nickel and cobalt, which are essential for the metabolism of the anaerobic microorganisms that carry out the mineralization of the pollutants present in the wastewater. In practice, nickel, cobalt, and iron are added in excessive amounts to full-scale installations. This study is concerned with the complexation of nickel and cobalt with yeast extract and its effect on the biogas production by methanogenic biomass. Adsorptive stripping voltammetry (AdSV) was used to get information about the stability and complexing capacity of the metal-yeast extract complexes formed. Nickel and cobalt form relatively strong organic complexes with yeast extract. The bioavailability of these essential metals in anaerobic batch reactors was dramatically increased by the addition of yeast extract. This is due to the formation of dissolved bioavailable complexes, which favors the dissolution of metals from their sulfides. Trace doses of yeast extract may be effective in keeping additions of essential metals to anaerobic reactors at a minimum.

Conversion and toxicity characteristics of formaldehyde in acetoclastic methanogenic sludge.

An unadapted mixed methanogenic sludge transformed formaldehyde into methanol and formate. The methanol to formate ratio obtained was 1:1. Formaldehyde conversion proceeded without any lag phase, suggesting the constitutive character of the formaldehyde conversion enzymes involved. Because the rate of formaldehyde conversion declined at increased formaldehyde additions, we hypothesized that some enzymes and/or cofactors might become denatured as a result of the excess of formaldehyde. Furthermore, formaldehyde was found to be toxic to acetoclastic methanogenesis in a dual character. Formaldehyde toxicity was partly reversible because once the formaldehyde concentration was extremely low or virtually removed from the system, the methane production rate was partially recovered. Because the degree of this recovery was not complete, we conclude that formaldehyde toxicity was partly irreversible as well. The irreversible toxicity likely can be attributed to biomass formaldehyde-related decay. Independent of the mode of formaldehyde addition (i.e., slug or continuous), the irreversible toxicity was dependent on the total amount of formaldehyde added to the system. This finding suggests that to treat formaldehyde-containing waste streams, a balance between formaldehyde-related decay and biomass growth should be attained.

Assessment of metabolic properties and kinetic parameters of methanogenic sludge by on-line methane production rate measurements.

This report presents a new approach to studying the metabolic and kinetic properties of anaerobic sludge from single batch experiments. The two main features of the method are that the methane production is measured on-line with a relatively cheap system, and that the methane production data can be plotted as rate vs time curves. The case studies of specific methanogenic activity, biodegradability and toxicity tests here presented show that very accurate kinetic data can be obtained. The method is specifically useful in experiments in which strong changes in methane production occur, and it is proposed as a powerful tool to study methanogenic systems. Furthermore, the method is simple and could be implemented by industry in the routine analysis of sludge.

Cluster structure of anaerobic aggregates of an expanded granular sludge bed reactor.

The metabolic properties and ultrastructure of mesophilic aggregates from a full-scale expanded granular sludge bed reactor treating brewery wastewater are described. The aggregates had a very high methanogenic activity on acetate (17.19 mmol of CH(4)/g of volatile suspended solids [VSS].day or 1.1 g of CH(4) chemical oxygen demand/g of VSS.day). Fluorescent in situ hybridization using 16S rRNA probes of crushed granules showed that 70 and 30% of the cells belonged to the archaebacterial and eubacterial domains, respectively. The spherical aggregates were black but contained numerous whitish spots on their surfaces. Cross-sectioning these aggregates revealed that the white spots appeared to be white clusters embedded in a black matrix. The white clusters were found to develop simultaneously with the increase in diameter. Energy-dispersed X-ray analysis and back-scattered electron microscopy showed that the whitish clusters contained mainly organic matter and no inorganic calcium precipitates. The white clusters had a higher density than the black matrix, as evidenced by the denser cell arrangement observed by high-magnification electron microscopy and the significantly higher effective diffusion coefficient determined by nuclear magnetic resonance imaging. High-magnification electron microscopy indicated a segregation of acetate-utilizing methanogens (Methanosaeta spp.) in the white clusters from syntrophic species and hydrogenotrophic methanogens (Methanobacterium-like and Methanospirillum-like organisms) in the black matrix. A number of physical and microbial ecology reasons for the observed structure are proposed, including the advantage of segregation for high-rate degradation of syntrophic substrates.

Kinetics and mass-transfer phenomena in anaerobic granular sludge.

The kinetic properties of acetate-degrading methanogenic granular sludge of different mean diameters were assessed at different up-flow velocities (V(up)). Using this approach, the influence of internal and external mass transfer could be estimated. First, the apparent Monod constant (K(S)) for each data set was calculated by means of a curve-fitting procedure. The experimental results revealed that variations in the V(up) did not affect the apparent K(S)-value, indicating that external mass-transport resistance normally can be neglected. With regard to the granule size, a clear increase in K(S) was found at increasing granule diameters. The experimental data were further used to validate a dynamic mathematical biofilm model. The biofilm model was able to describe reaction-diffusion kinetics in anaerobic granules, using a single value for the effective diffusion coefficient in the granules. This suggests that biogas formation did not influence the diffusion-rates in the granular biomass.

Effects of nickel and cobalt on kinetics of methanol conversion by methanogenic sludge as assessed by on-line CH4 monitoring.

When metals were added in a pulse mode to methylotrophic-methanogenic biomass, three methane production rate phases were recognized. Increased concentrations of Ni and Co accelerated the initial exponential and final arithmetic increases in the methane production rate and reduced the temporary decrease in the rate. When Ni and Co were added continuously, the temporary decrease phase was eliminated and the exponential production rate increased. We hypothesize that the temporary decrease in the methane production rate and the final arithmetic increase in the methane production rate were due to micronutrient limitations and that the precipitation-dissolution kinetics of metal sulfides may play a key role in the biovailability of these compounds.

Regulation of crp transcription by oscillation between distinct nucleoprotein complexes.

FIS belongs to the group of small abundant DNA-binding proteins of Escherichia coli. We recently demonstrated that, in vivo, FIS regulates the expression of several genes needed for catabolism of sugars and nucleic acids, a majority of which are also transcriptionally regulated by cAMP-cAMP-receptor protein (CRP) complex. Here we provide evidence that FIS represses transcription of the crp gene both in vivo and in vitro. Employing crp promoter-lacZ fusions, we demonstrate that both FIS and cAMP-CRP are required to keep the crp promoter in a repressed state. We have identified in the crp promoter other transcription initiation sites which are located 73, 79 and 80 bp downstream from the previously mapped start site. Two CRP- and several FIS-binding sites with different affinities are located in the crp promoter region, one of them overlapping the downstream transcription initiation sites. We show that initiation of transcription at the crp promoter is affected by the composition of nucleoprotein complexes resulting from the outcome of competition between proteins for overlapping binding sites. Our results suggest that the control of crp transcription is achieved by oscillation in the composition of these regulatory nucleoprotein complexes in response to the physiological state of the cell.

FIS is a regulator of metabolism in Escherichia coli.

The Escherichia coli DNA-binding protein FIS (factor for inversion stimulation) stimulates site-specific recombination reactions catalysed by DNA invertases and is an activator of stable RNA synthesis. To address the question of whether FIS is involved in other cellular processes we have identified and sequenced proteins whose expression pattern is affected by FIS. This has led to the identification of several E. coli genes whose expression in vivo is either enhanced or repressed by FIS. All of these genes encode enzymes or transport proteins involved in the catabolism of sugars or nucleic acids, and their expression is also dependent on the cAMP-CRP complex. In most cases studied the regulation by FIS is indirect and occurs through effects on the synthesis of the respective repressor proteins. We conclude that FIS is a transcriptional modulator involved in the regulation of metabolism in E. coli.

Analysis of baseline and BrdU-dependent SCEs at different BrdU concentrations.

In the present paper we have used a rationale based on the development of theoretical equations that define sister-chromatid exchange (SCE) frequencies as a function of two variables, namely the baseline (BrdU-independent) and the BrdU-dependent SCE frequencies. The experimental design includes the estimation of SCE frequencies in second division chromosomes when both cycles occurred in the presence of BrdU and when BrdU incubation took place only during the first cycle in a wide range of BrdU concentrations. The final SCE yields in second division chromosomes could be separated into three different components: (1) The BrdU-independent, 'spontaneous' or baseline SCEs, whose low but biologically significant frequency was calculated to be about 0.06 SCEs per pg of DNA; this figure could be similar for most of the cell types; (2) the BrdU-dependent SCEs whose frequency increases with BrdU dose, probably as a result of BrdU substitution for thymidine; (3) the BrdU-dependent SCEs as a consequence of other cellular factors such as disturbance of nucleotide pool sizes. At high BrdU concentrations (300 microM upward) the three components appear to have a significant value in the final SCE yield, whereas at lower BrdU doses the third component seems to be negligible.

On the mechanism of differential Giemsa staining of BrdU-substituted chromatids.

This paper analyses the effect of acid hydrolysis on the differential Giemsa staining of 5-bromo-2'deoxyuridine (BrdU) substituted chromatids in human and plant chromosomes, after treatment with a fluorochrome and light. Human lymphocytes and Allium cepa L. root tips were grown in BrdU for two or three cell cycles. Lymphocytes spreadings and meristem squashes were treated with fluorochrome Hoechst 33258, exposed to sunlight, hydrolysed with 5N HCl and stained with giemsa. This acid hydrolysis improves the differential staining of BrdU substituted and non-substituted chromatin. It also allows the differentiation of sister chromatids with the DNA specific dye azure-A.