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L Raskin

Publications and source records attributed to L Raskin.

At least 19 recordsLinked to original sources

Molecular ecology of anaerobic reactor systems.

Anaerobic reactor systems are essential for the treatment of solid and liquid wastes and constitute a core facility in many waste treatment plants. Although much is known about the basic metabolism in different types of anaerobic reactors, little is known about the microbes responsible for these processes. Only a few percent of Bacteria and Archaea have so far been isolated, and almost nothing is known about the dynamics and interactions between these and other microorganisms. This lack of knowledge is most clearly exemplified by the sometimes unpredictable and unexplainable failures and malfunctions of anaerobic digesters occasionally experienced, leading to sub-optimal methane production and wastewater treatment. Using a variety of molecular techniques, we are able to determine which microorganisms are active, where they are active, and when they are active, but we still need to determine why and what they are doing. As genetic manipulations of anaerobes have been shown in only a few species permitting in-situ gene expression studies, the only way to elucidate the function of different microbes is to correlate the metabolic capabilities of isolated microbes in pure culture to the abundance of each microbe in anaerobic reactor systems by rRNA probing. This chapter focuses on various molecular techniques employed and problems encountered when elucidating the microbial ecology of anaerobic reactor systems. Methods such as quantitative dot blot/fluorescence in-situ probing using various specific nucleic acid probes are discussed and exemplified by studies of anaerobic granular sludge, biofilm and digester systems.

Anaerobiosis↗

Development of a sustainable treatment technology for domestic sewage.

We propose an integrated chemical-physical-biological treatment concept for the low-cost treatment of domestic wastewater. Domestic wastewater was subjected to a chemically enhanced primary sedimentation (CEPS), followed by treatment in an upflow anaerobic sludge blanket (UASB) reactor. In addition, a regenerable zeolite was used to remove NH4+, either after CEPS pretreatment or after biological treatment in the UASB reactor. The CEPS pretreatment consisted of the addition of a coagulant (FeCl3) and an anionic organic flocculant and removed on average 73% of the total chemical oxygen demand (CODt), 85% of the total suspended solids, and 80% of PO4(3-) present in the wastewater. The UASB system, which consequently received a low CODt input of approximately 140 mg/L, was operated using a volumetric loading rate of 0.4 g CODt/L.d (hydraulic retention time [HRT] = 10 h) and 0.7 g CODt/L.d (HRT = 5 h). For these conditions, the system removed about 55% of the CODt in its influent, thus producing an effluent with a low CODt of approximately 50 mg/L. The zeolite, when applied in batch mode before the UASB reactor, removed approximately 45% of the NH4+, whereas its application as a post-treatment cartridge resulted in almost 100% NH4+ removal. The simple design and relatively low operating costs, due to low costs of added chemicals and low energy input (estimated at Euro 0.07-0.1 per m3 wastewater treated), combined with excellent treatment performance, suggest that this system can be used as a novel domestic wastewater treatment system for developing countries. Therefore, the system is called a Low-cost, Integrated Sewage Treatment (LIST) system.

Animals↗

Oligonucleotide probe hybridization and modeling results suggest that populations consuming readily degradable substrate have high cellular RNA levels.

Analyses based on ribosomal RNA (rRNA)-targeted hybridization performed in our laboratory identified two types of bacterial populations: a population with a high RNA level per biomass and a population with a low level of RNA per biomass. To extend these descriptions, the diurnal dynamics of the RNA pool were monitored by rRNA-targeted oligonucleotide probe membrane hybridization. Under the typical diurnal variation in COD loading rate experienced by municipal wastewater treatment plants, the RNA level of the bacterial population with a high level of RNA per biomass varied with changes in the COD loading rate. Under the same conditions, the RNA level of the population with low RNA level per biomass remained constant. A structured biomass model was developed to describe these data. Substrate COD was divided into a readily biodegradable and a slowly biodegradable COD fraction. It was assumed that two specialized populations coexist in municipal activated sludge treatment systems. One population consumes readily degradable COD and the other consumes slowly degradable COD. According to model simulations, the population consuming readily degradable COD has a high level of RNA per biomass under variable substrate concentrations. Comparatively, the population consuming slowly degradable COD has a low level of RNA level per biomass. Furthermore, model simulations reproduced the two diurnal RNA profiles observed in a full-scale municipal activated sludge system. Therefore, we suggest that two populations can be distinguished in municipal activated sludge systems: a population consuming readily degradable substrate and a population consuming slowly degradable substrate.

Bacteria↗

Who eats what? Classifying microbial populations based on diurnal profiles of rRNA levels.

Identifying the relationships between various bacterial populations and the substrates they consume is central to the understanding of population dynamics and to the development of process control in activated sludge. However, linking a heterotrophic population to its activity in situ is difficult because ribosomal RNA (rRNA) techniques, while allowing the rapid identification of populations, provide little information about their heterotrophic activity. Activated sludge models describe biodegradation kinetics by classifying substrates into two types: readily and slowly degradable substrates. Assuming that bacterial populations specialize in degrading one type of substrate, their growth rate should be affected differently if the COD loading rate varies diurnally as for a municipal activated sludge system. Modeling results suggested that the growth rates of populations consuming readily degradable substrates vary according to variations in COD loading rate. On the other hand, the growth rates of populations consuming slowly degradable substrates do not change despite the variation in COD loading rate. Since the cellular rRNA level is positively correlated with the growth rate, we hypothesized that the rRNA levels of some populations in municipal activated sludge should increase throughout the day, while they should stay constant for other populations. This hypothesis was verified by monitoring the rRNA level of Acinetobacter (a model population consuming readily degradable substrates) and Gordonia (a model population consuming slowly degradable substrates) in the mixed liquor of a full-scale municipal activated sludge reactor for three weeks.

Bacteria↗

Quantifying the impact of wastewater micronutrient composition on in situ growth activity of Acinetobacter spp.

Batch growth studies with pure cultures of Acinetobacter johnsoniiT and Acinetobacter johnsonii strain 210 on various media formulations were used to examine the effects of micronutrient composition on the growth rate of microbial populations in wastewater treatment systems. On nutrient rich Luria-Bertani medium, both strains of A. johnsonii grew with a doubling time of approximately 30 min. On a defined, minimal medium with acetate as the sole carbon source, the doubling time of A. johnsoniiT was 9.62 h and the doubling time of strain 210a was 2.6 h. Using a synthetic wastewater as a growth medium, the type strain had a doubling time of 56 h and strain 210a had a doubling time of 9.62 h. The concentration and redox state of iron appeared to be the principle growth limiting factors with higher doubling times occurring in media containing ferric iron as compared to ferrous iron. Additionally, grab samples from batch growth experiments were analyzed with oligonucleotide hybridization probes targeting the mature 16S ribosomal RNA (rRNA) and precursor 16S rRNA of Acinetobacter spp. Results showed that the precursor 16S rRNA levels responded more rapidly and to a greater extent than total 16S rRNA levels to changes in the micronutrient composition of the growth media. Precursor 16S rRNA levels increased in both strains when overnight cultures were diluted with fresh media and when micronutrient supplements were added to growing cultures. Our results show that the micronutrient composition of the influent wastewater can have a significant impact on the microbial community structure in wastewater treatment systems.

Acinetobacter↗

Water quality factors affecting bromate reduction in biologically active carbon filters.

Biological removal of the ozonation by-product, bromate, was demonstrated in biologically active carbon (BAC) filters. For example, with a 20-min EBCT, pH 7.5, and influent dissolved oxygen (DO) and nitrate concentrations 2.1 and 5.1 mg/l, respectively, 40% bromate removal was obtained with a 20 microg/l influent bromate concentration. In this study, DO, nitrate and sulfate concentrations, pH, and type of source water were evaluated for their effect on bromate removal in a BAC filter. Bromate removal decreased as the influent concentrations of DO and nitrate increased, but bromate removal was observed in the presence of measurable effluent concentrations of DO and nitrate. In contrast, bromate removal was not sensitive to the influent sulfate concentration, with only a slight reduction in bromate removal as the influent sulfate concentration was increased from 11.1 to 102.7 mg/l. Bromate reduction was better at lower pH values (6.8 and 7.2) than at higher pH values (7.5 and 8.2), suggesting that it may be possible to reduce bromate formation during ozonation and increase biological bromate reduction through pH control. Biological bromate removal in Lake Michigan water was very poor as compared to that in tapwater from a groundwater source. Bromate removal improved when sufficient organic electron donor was added to remove the nitrate and DO present in the Lake Michigan water, indicating that the poor biodegradability of the natural organic matter may have been limiting bromate removal in that water. Biological bromate removal was demonstrated to be a sustainable process under a variety of water quality conditions, and bromate removal can be improved by controlling key water quality parameters.

Bromates↗

Anaerobic codigestion of municipal solid waste and biosolids under various mixing conditions--II: Microbial population dynamics.

Microbial population dynamics were evaluated in anaerobic codigesters treating municipal solid waste and sewage sludge. Ribosomal RNA based oligonucleotide probes were used to characterize changes in population abundance of syntrophic volatile fatty acid degrading bacteria and methanogens. Changes in community structure were linked to traditional performance parameters during the recovery of previously unstable codigesters induced by a reduction in mixing levels. Methanosarcina spp. were the most abundant aceticlastic methanogens in unstable codigesters with high acetate concentrations, while Methanosaeta concilii was dominant in stable systems with low levels of acetate. Growth of Syntrophobacter wolinii was enhanced during stabilization of a codigester with a well-developed population of Methanobacteriaceae, possibly because the presence of adequate numbers of these hydrogenotrophic methanogens encouraged the syntrophic oxidation of propionate. Mesophilic saturated fatty acid beta-oxidizing syntrophs were most abundant in previously unstable codigesters. One minimally mixed reactor became unstable after switching to continuously mixed conditions. After the switch, total archaeal abundance decreased sharply, though Methanobacteriaceae and Methanosarcina spp. levels increased as the fermentation became unbalanced. Based on the results presented here, mixing appears to inhibit the syntrophic oxidation of volatile fatty acids, possibly by disrupting the spatial juxtaposition of syntrophic bacteria and their methanogenic partners.

Biomass↗

Anaerobic codigestion of municipal solid waste and biosolids under various mixing conditions--I. Digester performance.

The feasibility of codigestion of the organic fraction of municipal solid waste, primary sludge, and waste activated sludge was evaluated in mesophilic (37 degrees C), laboratory-scale digesters. In a first experiment, different startup strategies were compared using four digesters, operated under continuously mixed conditions. After two weeks, the experiment was continued under minimally mixed conditions. Results demonstrated that reducing the level of mixing improved digester performance. Therefore, in a second experiment, six digesters were operated to compare performance under continuous mixing and reduced mixing levels at various loading rates and solids levels. The continuously mixed digesters exhibited unstable performance at the higher loading rates, while the minimally mixed digesters performed well for all loading rates evaluated. In a third experiment, it was demonstrated that an unstable, continuously mixed digester was quickly stabilized by reducing the mixing level. These experiments confirmed that continuous mixing was not necessary for good performance and was inhibitory at higher loading rates. In addition, reduction of mixing levels may be used as an operational tool to stabilize unstable digesters.

Oxygen↗

Quantifying filamentous microorganisms in activated sludge before, during, and after an incident of foaming by oligonucleotide probe hybridizations and antibody staining.

Quantitative oligonucleotide probe hybridizations, immunostaining, and a simple foaming potential test were used to follow an incident of seasonal filamentous foaming at the Urbana-Champaign Sanitary District, Northeast Wastewater Treatment Plant. A positive correlation was observed between an increase in foaming potential and the appearance of foam on the surfaces of aeration basins and secondary clarifiers. In addition, during the occurrence of foaming, the mass and activity of Gordonia spp. increased as measured by fluorescence in situ hybridization, antibody staining, and quantitative membrane hybridization of RNA extracts. An increase in Gordonia spp. rRNA levels from 0.25 to 1.4% of total rRNA was observed using quantitative membrane hybridizations, whereas during the same period, the fraction of mixed liquor volatile suspended solids attributed to Gordonia spp. increased from 4% to more than 32% of the total mixed liquor volatile suspended solids. These results indicate that both the activity and biomass level of Gordonia spp. in activated sludge increased relative to the activity aid the biomass level of the complete microbial community during a seasonal occurrence of filamentous foaming. Thus, Gordonia spp. may represent a numerically dominant but metabolically limited fraction of the total biomass, and the role of Gordonia spp. in filamentous foaming may be linked more tightly to the physical presence of filamentous microorganisms than to the metabolic activity of the cells.

Actinomycetales↗

Deafness heterogeneity in a Druze isolate from the Middle East: novel OTOF and PDS mutations, low prevalence of GJB2 35delG mutation and indication for a new DFNB locus.

About 60% of congenital hearing impairment cases in developed countries are due to genetic defects. Data on the molecular basis of hereditary hearing reflects vast genetic heterogeneity. There are >400 disorders in which hearing impairment is one of the characteristic traits of a syndrome. Linkage studies have identified more than 40 human chromosomal loci associated with non-syndromic hearing loss. So far, 16 of these 40 non-syndromic hearing impairment genes have been identified. We have studied the molecular basis of hearing impairment in four Druze families from the same village in Northern Galilee. The Druze are a small, isolated population in the Middle East practising endogamous marriage. Thus it was expected that a single mutation would account for hearing impairments in all these families. Our results show that at least four different genes are involved. Hearing impairment was caused in one family by a novel mutation in the recently identified OTOF (the DFNB9 gene), by a novel Pendred syndrome mutation (Thr193Ile) in another family, and by a GJB2 mutation (35delG also known as 30delG) in the third family. In the fourth family linkage was excluded from all known hearing impairments loci (recessive and dominant) as well as from markers covering chromosomes 11-22, pointing therefore to the existence of another non-syndromic recessive hearing loss (NSRD) locus on chromosomes 1-10.

Carrier Proteins↗

Desulfotomaculum genus- and subgenus-specific 16S rRNA hybridization probes for environmental studies.

Based on comparative analysis of 16S rRNA sequences and the recently established phylogeny of the genus Desulfotomaculum, a set of phylogenetically nested hybridization probes was developed and characterized. A genus-specific probe targets all known Desulfotomaculum species (with the exception of Desulfotomaculum acetoxidans), and five specific probes target subclusters within the Desulfotomaculum genus. The dissociation temperature of each probe was determined experimentally. Probe specificities were verified through hybridizations with pure culture rRNA isolated from a wide variety of target and non-target organisms and through an evaluation of probe 'nesting' using samples obtained from four different environments. Fixation and hybridization conditions for fluorescence in situ hybridizations were also optimized. The probes were used in quantitative membrane hybridizations to determine the abundance of Desulfotomaculum species in thermophilic anaerobic digesters, in soil, in human faeces and in pig colon samples. Desulfotomaculum rRNA accounted for 0.3-2.1% of the total rRNA in the digesters, 2.6-6.6% in soil, 1.5-3.3% in human faeces and 2.5-6.2% in pig colon samples.

Animals↗

The presence of humic substances and DNA in RNA extracts affects hybridization results.

RNA extracts obtained from environmental samples are frequently contaminated with coextracted humic substances and DNA. It was demonstrated that the response in rRNA-targeted oligonucleotide probe hybridizations decreased as the concentrations of humic substances and DNA in RNA extracts increased. The decrease in hybridization signal in the presence of humic substances appeared to be due to saturation of the hybridization membrane with humic substances, resulting in a lower amount of target rRNA bound to the membrane. The decrease in hybridization response in the presence of low amounts of DNA may be the result of reduced rRNA target accessibility. The presence of high amounts of DNA in RNA extracts resulted in membrane saturation. Consistent with the observations for DNA contamination, the addition of poly(A) to RNA extracts, a common practice used to prepare RNA dilutions for membrane blotting, also reduced hybridization signals, likely because of reduced target accessibility and membrane saturation effects.

DNA, Bacterial↗

Monitoring precursor 16S rRNAs of Acinetobacter spp. in activated sludge wastewater treatment systems.

Recently, Cangelosi and Brabant used oligonucleotide probes targeting the precursor 16S rRNA of Escherichia coli to demonstrate that the levels of precursor rRNA were more sensitive to changes in growth phase than the levels of total rRNA (G. A. Cangelosi and W. H. Brabant, J. Bacteriol. 179:4457-4463, 1997). In order to measure changes in the levels of precursor rRNA in activated sludge systems, we designed oligonucleotide probes targeting the 3' region of the precursor 16S rRNA of Acinetobacter spp. We used these probes to monitor changes in the level of precursor 16S rRNA during batch growth of Acinetobacter spp. in Luria-Bertani (LB) medium, filtered wastewater, and in lab- and full-scale wastewater treatment systems. Consistent with the previous reports for E. coli, results obtained with membrane hybridizations and fluorescence in situ hybridizations with Acinetobacter calcoaceticus grown in LB medium showed a more substantial and faster increase in precursor 16S rRNA levels compared to the increase in total 16S rRNA levels during exponential growth. Diluting an overnight culture of A. calcoaceticus grown in LB medium with filtered wastewater resulted in a pattern of precursor 16S rRNA levels that appeared to follow diauxic growth. In addition, fluorescence in situ hybridizations with oligonucleotide probes targeting total 16S rRNA and precursor 16S rRNA showed that individual cells of A. calcoaceticus expressed highly variable levels of precursor 16S rRNA when adapting from LB medium to filtered sewage. Precursor 16S rRNA levels of Acinetobacter spp. transiently increased when activated sludge was mixed with influent wastewater in lab- and full-scale wastewater treatment systems. These results suggest that Acinetobacter spp. experience a change in growth activity within wastewater treatment systems.

Acinetobacter↗

Flexible community structure correlates with stable community function in methanogenic bioreactor communities perturbed by glucose.

Methanogenic bioreactor communities were used as model ecosystems to evaluate the relationship between functional stability and community structure. Replicated methanogenic bioreactor communities with two different community structures were established. The effect of a substrate loading shock on population dynamics in each microbial community was examined by using morphological analysis, small-subunit (SSU) rRNA oligonucleotide probes, amplified ribosomal DNA (rDNA) restriction analysis (ARDRA), and partial sequencing of SSU rDNA clones. One set of replicated communities, designated the high-spirochete (HS) set, was characterized by good replicability, a high proportion of spiral and short thin rod morphotypes, a dominance of spirochete-related SSU rDNA genes, and a high percentage of Methanosarcina-related SSU rRNA. The second set of communities, designated the low-spirochete (LS) set, was characterized by incomplete replicability, higher morphotype diversity dominated by cocci, a predominance of Streptococcus-related and deeply branching Spirochaetales-related SSU rDNA genes, and a high percentage of Methanosaeta-related SSU rRNA. In the HS communities, glucose perturbation caused a dramatic shift in the relative abundance of fermentative bacteria, with temporary displacement of spirochete-related ribotypes by Eubacterium-related ribotypes, followed by a return to the preperturbation community structure. The LS communities were less perturbed, with Streptococcus-related organisms remaining prevalent after the glucose shock, although changes in the relative abundance of minor members were detected by morphotype analysis. A companion paper demonstrates that the more stable LS communities were less functionally stable than the HS communities (S. A. Hashsham, A. S. Fernandez, S. L. Dollhopf, F. B. Dazzo, R. F. Hickey, J. M. Tiedje, and C. S. Criddle, Appl. Environ. Microbiol. 66:4050-4057, 2000).

Anaerobiosis↗

Quantification of syntrophic fatty acid-beta-oxidizing bacteria in a mesophilic biogas reactor by oligonucleotide probe hybridization.

Small-subunit rRNA sequences were obtained for two saturated fatty acid-beta-oxidizing syntrophic bacteria, Syntrophomonas sapovorans and Syntrophomonas wolfei LYB, and sequence analysis confirmed their classification as members of the family Syntrophomonadaceae. S. wolfei LYB was closely related to S. wolfei subsp. wolfei, but S. sapovorans did not cluster with the other members of the genus Syntrophomonas. Five oligonucleotide probes targeting the small-subunit rRNA of different groups within the family Syntrophomonadaceae, which contains all currently known saturated fatty acid-beta-oxidizing syntrophic bacteria, were developed and characterized. The probes were designed to be specific at the family, genus, and species levels and were characterized by temperature-of-dissociation and specificity studies. To demonstrate the usefulness of the probes for the detection and quantification of saturated fatty acid-beta-oxidizing syntrophic bacteria in methanogenic environments, the microbial community structure of a sample from a full-scale biogas plant was determined. Hybridization results with probes for syntrophic bacteria and methanogens were compared to specific methanogenic activities and microbial numbers determined with most-probable-number estimates. Most of the methanogenic rRNA was comprised of Methanomicrobiales rRNA, suggesting that members of this order served as the main hydrogen-utilizing microorganisms. Between 0.2 and 1% of the rRNA was attributed to the Syntrophomonadaceae, of which the majority was accounted for by the genus Syntrophomonas.

Bacteria, Anaerobic↗

Methanogenic population dynamics during start-up of anaerobic digesters treating municipal solid waste and biosolids.

An aggressive start-up strategy was used to initiate codigestion in two anaerobic, continuously mixed bench-top reactors at mesophilic (37 degrees C) and thermophilic (55 degrees C) conditions. The digesters were inoculated with mesophilic anaerobic sewage sludge and cattle manure and were fed a mixture of simulated municipal solid waste and biosolids in proportions that reflect U.S. production rates. The design organic loading rate was 3.1 kg volatile solids/m3/day and the retention time was 20 days. Ribosomal RNA-targeted oligonucleotide probes were used to determine the methanogenic community structure in the inocula and the digesters. Chemical analyses were performed to evaluate digester performance. The aggressive start-up strategy was successful for the thermophilic reactor, despite the use of a mesophilic inoculum. After a short start-up period (20 days), stable performance was observed with high gas production rates (1.52 m3/m3/day), high levels of methane in the biogas (59%), and substantial volatile solids (54%) and cellulose (58%) removals. In contrast, the mesophilic digester did not respond favorably to the start-up method. The concentrations of volatile fatty acids increased dramatically and pH control was difficult. After several weeks of operation, the mesophilic digester became more stable, but propionate levels remained very high. Methanogenic population dynamics correlated well with performance measures. Large fluctuations were observed in methanogenic population levels during the start-up period as volatile fatty acids accumulated and were subsequently consumed. Methanosaeta species were the most abundant methanogens in the inoculum, but their levels decreased rapidly as acetate built up. The increase in acetate levels was paralleled by an increase in Methanosarcina species abundance (up to 11.6 and 4.8% of total ribosomal RNA consisted of Methanosarcina species ribosomal RNA in mesophilic and thermophilic digesters, respectively). Methanobacteriaceae were the most abundant hydrogenotrophic methanogens in both digesters, but their levels were higher in the thermophilic digester.

Anaerobiosis↗

A Comparison of the Use of In Vitro-Transcribed and Native rRNA for the Quantification of Microorganisms in the Environment.

Abstract Nearly full-length, small subunit (SSU) rRNA was transcribed in vitro from clones of SSU rDNA genes. Comparing the use of in vitro-transcribed and native rRNA indicated that, when in vitro-transcribed rRNA was used as a standard for quantitative hybridizations with oligonucleotide probes, the population was consistently underestimated. The population abundance was expressed as a percentage of specific target SSU rRNA (determined with a specific oligonucleotide probe), relative to the total SSU rRNA (measured with a universal probe). Differences in hybridization signals could be related to specific probe target locations and rRNA denaturation conditions, suggesting that higher order structure is important in quantitative membrane hybridizations. Therefore, in vitro-transcribed rRNA cannot always be used for the absolute quantification of microbial populations, but can be employed as a standard to quantify shifts in population abundance over time, and to compare community structure in various environments.

Journal Article↗