The Role of the Liquid Phase in the Degradation of Toluene and m‐Cresol within a Biofilm Trickle‐Bed Reactor

The degradation of toluene and m‐cresol in a biofilm trickle‐bed reactor was experimentally and theoretically investigated. Degradation is the result of the cooperation between suspended and immobilized microorganisms in the trickling film and the biofilm. The role of the trickling film is that of a barrier for mass transfer to the biofilm or that of an additional reaction space. This is the result of physical availability of pollutants to the liquid phase as well as co‐substrate degradation of inherent biomass. An instationary reactor balance model is presented. In addition to this the change in wetting behavior of carrier surface due to biofilm formation is discussed. A partial wetting of biofilm surface by rivulets of the trickling film is proposed. The model was verified by experimental data. The different reactor operation modes denoted as biofilm regime versus trickling film regime for the chosen pollutant system were expressed in terms of dimensionless reactions and transfer numbers. It is shown that the volumetric reaction rates for toluene in a trickling film regime reaches values twice as high as that of a biofilm regime due to the presence of the second substrate m‐cresol. The limiting step in both cases is the mass transfer of oxygen to bacteria in the biofilm or trickling film.     

Biofilm thickness variability investigated with a laser triangulation sensor

Measurement of the surface roughness and thickness of biological films is laborious and usually destructive, thus hampering research in this area. We developed a laser triangulation sensor (LTS) set-up for the fast and nondestructive measurement of these biofilm parameters during growth. Using LTS measurements, the morphological development of a dichloromethane-(DCM) degrading biofilm cultured on a wetted-wall column was studied. The measurements show that the biofilm develops faster at the entrance of the reactor. The biofilm consisted of a base film in which microbial colonies were embedded. The biofilm-surface area gradually increased by 23% compared to the bare surface due to the formation of a large number of these colonies. The number and shape of these colonies were followed in time. Using LTS measurements, biofilms distinctly different in surface roughness could be distinguished in a laboratory trickling filter removing DCM from a waste gas. The consequences of the observed surface characteristics for the reaction-diffusion process in the biofilm and for the falling film hydrodynamics are discussed.     

The influence of NaCl on the degradation rate of dichloromethane byHyphomicrobium sp.

The degradation of dichloromethane by the pure strainHyphomicrobium GJ21 and by an enrichment culture, isolated from a continuously operating biological trickling filter system, as well as the corresponding growth rates of these organisms were investigated in several batch experiments. By fitting the experimental data to generally accepted theoretical expressions for microbial growth, the maximum growth rates were determined. The effect of NaCl was investigated at salt concentrations varying from 0 to 1000 mM. Furthermore the dichloromethane degradation was investigated separately in experiments in which a high initial biomass concentration was applied. The results show that microbial growth is strongly inhibited by increased NaCl concentrations (50% reduction of _max at 200–250 mM NaCl), while a certain degree of adaptation has taken place within an operational system eliminating dichloromethane. A critical NaCl concentration for growth of 600 mM was found for the microbial culture isolated from an operational trickling filter, while a value of 375 mM was found for the pure cultureHyphomicrobium GJ21. The substrate degradation appears to be much less susceptible to inhibition by NaCl. Even at 800 mM NaCl relatively high substrate degradation rates are still observed, although this process is again dependent on the NaCl concentration. Here the substrate elimination is due to the maintenance requirements of the microorganisms. The inhibition of the dichloromethane elimination was also investigated in a laboratory scale trickling filter. The results of these experiments confirmed those obtained in the batch experiments. At NaCl concentrations exceeding 600 mM a considerable elimination of dichloromethane was still observed for during several months of operation. These observations indicate that the inhibition of microbial growth offers a significant control parameter against excessive biomass growth in biological trickling filters for waste gas treatment.     

Degradation of diesel oil by immobilized <Emphasis Type="Italic">Candida tropicalis</Emphasis> and biofilm formed on gravels

The performance of diesel oil degradation by Candida tropicalis immobilized on various conventional matrices (sodium alginate, carboxyl methyl cellulose, chitosan) and biowaste materials (wheat bran, sawdust, peanut hull powder) was investigated using the method of entrapment and physical adsorption. The yeast species immobilized in wheat bran showed enhanced efficiency in degrading diesel oil (98%) compared to free cells culture (80%) over a period of 7 days. Copious amount of exopolysaccharides were also produced in the presence of diesel oil. The biofilm forming ability of C. tropicalis on PVC strips was evaluated using XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) reduction assay and monitored by scanning electron microscopy and atomic force microscopy. Yeast biofilm formed on gravels showed 97% degradation of diesel oil over a period of 10 days. The potential use of the biofilms for preparing trickling filters (gravel particles), for attenuating hydrocarbons in oily liquid wastes before their disposal in the open environment is suggested and discussed. This is the first successful attempt for ‘artificially’ establishing hydrocarbon degrading yeast biofilm on solid substrates.     

Microbial biogeography across a full-scale wastewater treatment plant transect: evidence for immigration between coupled processes

Wastewater treatment plants use a variety of bioreactor types and configurations to remove organic matter and nutrients. Little is known regarding the effects of different configurations and within-plant immigration on microbial community dynamics. Previously, we found that the structure of ammonia-oxidizing bacterial (AOB) communities in a full-scale dispersed growth activated sludge bioreactor correlated strongly with levels of NO₂ ^? entering the reactor from an upstream trickling filter. Here, to further examine this puzzling association, we profile within-plant microbial biogeography (spatial variation) and test the hypothesis that substantial microbial immigration occurs along a transect (raw influent, trickling filter biofilm, trickling filter effluent, and activated sludge) at the same full-scale wastewater treatment plant. AOB amoA gene abundance increased >30-fold between influent and trickling filter effluent concomitant with NO₂ ^? production, indicating unexpected growth and activity of AOB within the trickling filter. Nitrosomonas europaea was the dominant AOB phylotype in trickling filter biofilm and effluent, while a distinct “Nitrosomonas-like” lineage dominated in activated sludge. Prior time series indicated that this “Nitrosomonas-like” lineage was dominant when NO₂ ^? levels in the trickling filter effluent (i.e., activated sludge influent) were low, while N. europaea became dominant in the activated sludge when NO₂ ^? levels were high. This is consistent with the hypothesis that NO₂ ^? production may cooccur with biofilm sloughing, releasing N. europaea from the trickling filter into the activated sludge bioreactor. Phylogenetic microarray (PhyloChip) analyses revealed significant spatial variation in taxonomic diversity, including a large excess of methanogens in the trickling filter relative to activated sludge and attenuation of Enterobacteriaceae across the transect, and demonstrated transport of a highly diverse microbial community via the trickling filter effluent to the activated sludge bioreactor. Our results provide compelling evidence that substantial immigration between coupled process units occurs and may exert significant influence over microbial community dynamics within staged bioreactors.     

Activity of Toluene-degrading Pseudomonas putida in the early growth phase of a biofilm for waste gas treatment

A biological trickling filter for treatment of toluene-containing waste gas was studied. The overall kinetics of the biofilm growth was followed in the early growth phase. A rapid initial colonization took place during the first three days. The biofilm thickness increased exponentially, whereas the incease of active biomass and polymers was linear. In order to investigate the toluene degradation, various toluene degraders from the multispecies biofilm were isolated, and a Pseudomonas putida was chosen as a representative of the toluene-degrading population. A specific rRNA oligonucleotide probe was used to follow the toluene-degrading P. putida in the multispecies biofilm in the filter by means of number and cellular rRNA content. P. putida appeared to detach from the biofilm during the first three days of growth, after which P. putida was found at a constant level of 10% of the active biomass in the biofilm. Based on the rRNA content, the in situ activity was estimated to be reduced to 20% of cells grown at maximum conditions in batch culture. The toluene degraded by P. putida was estimated to be a minor part (11%) of the overall toluene degradation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 131-141, 1997.     

Occurrence of Cytophagas in Sewage Plants

With the application of plate count methods and of the KOH-flexirubin test, bacteria belonging to the Cytophaga group were proved to occur regularly in samples from biological sewage treatment facilities. Generally, the percentage of Cytophaga colonies of the total heterotrophic colonies was lowest in the inflow sewage water as compared with the values found in activated sludge, trickling filter, and effluent samples. During an observation period of 16 months, the highest percentages of cytophagas were found in winter samples from activated sludge and trickling filters. Furthermore, cytophagas were shown to have high percentages of the bacteria lytic to polymeric substrates such as cellulose, chitin, dextran, pectin, xylan, and gelatin. Thus, it is suggested that cytophagas may contribute to sewage purification, especially at cold temperatures and by polymer breakdown. Cytophaga strains isolated were shown to have gliding motility, flexirubin pigmentation, and a low guanine plus cytosine base ratio in common. The strains were roughly subdivided into a spreading, a nonspreading, and a cellulolytic group.     

Optimization of <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> (KU923381) for diesel oil degradation using response surface methodology (RSM)

Efficiency of Enterobacter cloacae KU923381 isolated from petroleum hydrocarbon contaminated soil was evaluated in batch culture and bioreactor mode. The isolate were screened for biofilm formation using qualitative and quantitative assays. Response surface methodology (RSM) was used to study the effect of pH, temperature, glucose concentration, and sodium chloride on diesel degradation. The predicted values for diesel oil degradation efficiency by the statistical designs are in a close agreement with experimental data (R ² = 99.66%). Degradation efficiency is increased by 36.78% at pH = 7, temperature = 35°C, glucose = 5%, and sodium chloride concentration = 5%. Under the optimized conditions, the experiments were performed for diesel oil degradation by gas chromatographic mass spectrometric analysis (GC-MS). GC-MS analysis confirmed that E. cloacae had highly degrade hexadecane, heptadecane, tridecane, and docosane by 99.71%, 99.23%, 99.66%, and 98.34% respectively. This study shows that rapid bioremoval of hydrocarbons in diesel oil is acheived by E. cloacae with abet of biofilm formation. The potential use of the biofilms for preparing trickling filters (gravel particles) for the degradation of hydrocarbons from petroleum wastes before their disposal in the open environment is highly suggested. This is the first successful attempt for artificially establishing petroleum hydrocarbon degrading bacterial biofilm on solid substrates in bioreactor.     

Microbial film development in a trickling filter

The transmission and scanning electron microscopes were employed to visualize the sequence of the biofilm development in the trickling wastewater filter. After the deposit of a small amount of debris upon a hard surface, the bacterial cells attach and develop the matrix on which the biofilm is formed. Protozoa invade the basic layer where they feed on the bacteria. The algae are seeded upon the bacterial matrix and grow so profusely that the bacteria must develop aerial colonies in the competition for food and oxygen. Destruction of the bacteria in the matrix and the weight and hydraulic pressure cause detachment of the biofilm and a new matrix must be developed.     

Dynamics of specific ammonia-oxidizing bacterial populations and nitrification in response to controlled shifts of ammonium concentrations in wastewater

Ammonia-oxidizing bacteria (AOB) are essential for the nitrification process in wastewater treatment. To retain these slow-growing bacteria in wastewater treatment plants (WWTPs), they are often grown as biofilms, e.g., on nitrifying trickling filters (NTFs) or on carriers in moving bed biofilm reactors (MBBRs). On NTFs, a decreasing ammonium gradient is formed because of the AOB activity, resulting in low ammonium concentrations at the bottom and reduced biomass with depth. To optimize the NTF process, different ammonium feed strategies may be designed. This, however, requires knowledge about AOB population dynamics. Using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy, we followed biomass changes during 6 months, of three AOB populations on biofilm carriers. These were immersed in aerated MBBR tanks in a pilot plant receiving full-scale wastewater. Tanks were arranged in series, forming a wastewater ammonium gradient mimicking an NTF ammonium gradient. The biomass of one of the dominating Nitrosomonas oligotropha-like populations increased after an ammonium upshift, reaching levels comparable to the high ammonium control in 28 days, whereas a Nitrosomonas europaea-like population increased relatively slowly. The MBBR results, together with competition studies in NTF systems fed with wastewater under controlled ammonium regimes, suggest a differentiation between the two N. oligotropha populations, which may be important for WWTP nitrification.     

Gallionella spp. in trickling filtration of subsurface aerated and natural groundwater

The growth of iron-oxidizing bacteria, generally regarded as obligate microaerophilic at neutral pH conditions, has been reported in a wide range of environments, including engineered systems for drinking water production. This research focused on intensively aerated trickling filters treating deep anaerobic and subsurface aerated groundwater. The two systems, each comprising groundwater abstraction and trickling filtration, were monitored over a period of 9 months. Gallionella spp. were quantified by qPCR with specifically designed 16S rRNA primers and identified directly in the environmental samples using clone libraries with the same primers. In addition, enrichments in gradient tubes were evaluated after DGGE separation with general bacterial primers. No other iron-oxidizing bacteria than Gallionella spp. were found in the gradient tubes. qPCR provided an effective method to evaluate the growth of Gallionella spp. in these filter systems. The growth of Gallionella spp. was stimulated by subsurface aeration, but these bacteria hardly grew in the trickling filter. In the uninfluenced, natural anaerobic groundwater, Gallionella spp. were only present in low numbers, but they grew extensively in the trickling filter. Identification revealed that Gallionella spp., growing in the trickling filter were phylogenetically distinct from the species found growing during subsurface aeration, indicating that the different conditions in both systems selected for niche organisms, while inhibiting other groups. The results suggest a minor direct significance for inoculation of Gallionella spp. during filtration of subsurface aerated groundwater.     

Influence of physiologically relevant parameters on biomass formation in a trickle-bed bioreactor used for waste gas cleaning

 Limitation of biomass formation in a mixed culture immobilised in a trickle-bed bioreactor without substantially affecting the biological degradation of organic compounds in waste gas streams was investigated. As carbon source, the industrially relevant volatile organic compounds ethyl acetate and toluene were used. The temporal biofilm composition was investigated by means of transmission electron microscopy of ultrathin sections cut along the film height. Physiologically relevant parameters were varied. In this context the effect of (a) the type of nitrogen source, (b) the concentration of inert salt and (c) limiting the availability of essential nutrients by intermittent trickling was studied. The effect of these parameters on both biomass formation and degradation was expressed in terms of the ratio R which was defined as the fractional inhibition of biomass formation related to the fractional decrease of degradation. Using nitrate as nitrogen source instead of ammonium, R was 0.71, which means that the fractional inhibition of biomass formation was less than the fractional inhibition of degradation. When the concentration of NaCl as inert salt was adjusted to 0.4 M, the R became 1.32, showing that the fractional inhibition of biomass formation was stronger than the fractional inhibition of degradation. Limiting the availability of nutrients by intermittent trickling, the pressure drop fell by 50% whereas the degradation efficiency decreased by 30%. In summary, intermittent trickling and addition of an inert salt were observed to be advantageous unlike the impact of the type of nitrogen source. Received: 20 March 1995/Received last revision: 27 September 1995/Accepted: 4 October 1995     

Enumeration of Aeromonas hydrophila from domestic wastewater treatment plants and surface waters

Influents, effluents and sludges from sewage purification plants and surface water samples were examined quantitatively for Aeromonas hydrophila on the mA medium of Rippey and Cabelli. Between 10(4) and 10(6)/ml A. hydrophila were found in domestic wastewaters. On the average 99.975% were removed by activated sludge and 98.25% by trickling filters. Only 20.9% of A. hydrophila end up in the primary sludge, which contained up to 10(7)/g dry sludge. After 3 months, anaerobically (methane) fermented and partially dried sludge from trickling filters contained more than 10(6) A. hydrophila/g dry sludge. Surface water receiving raw sewage contained several hundreds of A. hydrophila/ml, comparable with the numbers found in effluent waters, while surface receiving no municipal wastewater and destined for the preparation of drinking water contained only small and negligible numbers. It was concluded that A. hydrophila was omnipresent in surface water.     

Vertical distribution of nitrifying populations in bacterial biofilms from a full-scale nitrifying trickling filter

Cryosectioned biofilm from three depths (0.5, 3.0 and 6.0 m) in a full-scale nitrifying trickling filter (NTF) were studied using fluorescence in situ hybridization (FISH). A large number of sections were used to determine how the biofilm thickness, structure and community composition varied with depth along the ammonium concentration gradient in the NTF, and how the ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were distributed vertically within the biofilm. Both the biofilm thickness and relative biomass content of the biofilm decreased with depth, along with structural differences such as void size and surface roughness. Four AOB populations were found, with two Nitrosomonas oligotropha populations dominating at all depths. A smaller population of Nitrosomonas europaea was present only at 0.5 m, while a population of Nitrosomonas communis increased with depth. The two N. oligotropha populations showed different vertical distribution patterns within the biofilm, indicating different ecophysiologies even though they belong to the same AOB lineage. All NOB were identified as Nitrospira sp., and were generally more associated with the biofilm base than the surface-associated dominating AOB population. Additionally, a small population of anaerobic ammonia-oxidizers was found at 6.0 m, even though the biofilm was well aerated.     

The Removal of Salmonellas in Conventional Sewage Treatment Processes

The numbers of salmonellas in raw sewage entering a treatment plant varied hourly and diurnally; their peak concentration preceded the peak influent waste water flow into the plant by about 2 hours. Salmonellas were detected in all raw influent samples collected from 2 sewage works and the mean population level at the daily peak period was 3000 organisms/1. On average. 70–80% of the salmonellas were removed during primary sedimentation when upwards of 74–84% of solids were removed. Biological treatment and secondary sedimentation removed a further 70–100% of the pathogen. Compared with the activated sludge process the trickling filters were less efficient in removing salmonellas and they were adversely affected by increased loading following rainfall. Considering the whole treatment process, the Guildford works with its activated sludge treatment removed an average of 99–83% salmonellas while the Woking works with its trickling filter plant removed an average of 93–04% of the organisms. The large variance in the numbers of salmonellas in the final effluent from the trickling filters suggest that greater emphasis should be placed on the actual quality of the effluent rather than on percentage removal efficiencies.     

Microbial Communities Involved in Biological Ammonium Removal from Coal Combustion Wastewaters

The efficiency of a novel integrated treatment system for biological removal of ammonium, nitrite, nitrate, and heavy metals from fossil power plant effluent was evaluated. Microbial communities were analyzed using bacterial and archaeal 16S rRNA gene clone libraries (Sanger sequences) and 454 pyrosequencing technology. While seasonal changes in microbial community composition were observed, the significant (P?=?0.001) changes in bacterial and archaeal communities were consistent with variations in ammonium concentration. Phylogenetic analysis of 16S rRNA gene sequences revealed an increase of potential ammonium-oxidizing bacteria (AOB), Nitrosomonas, Nitrosococcus, Planctomycetes, and OD1, in samples with elevated ammonium concentration. Other bacteria, such as Nitrospira, Nitrococcus, Nitrobacter, Thiobacillus, ε-Proteobacteria, Firmicutes, and Acidobacteria, which play roles in nitrification and denitrification, were also detected. The AOB oxidized 56 % of the ammonium with the concomitant increase in nitrite and ultimately nitrate in the trickling filters at the beginning of the treatment system. Thermoprotei within the phylum Crenarchaeota thrived in the splitter box and especially in zero-valent iron extraction trenches, where an additional 25 % of the ammonium was removed. The potential ammonium-oxidizing Archaea (AOA) (Candidatus Nitrosocaldus) were detected towards the downstream end of the treatment system. The design of an integrated treatment system consisting of trickling filters, zero-valent iron reaction cells, settling pond, and anaerobic wetlands was efficient for the biological removal of ammonium and several other contaminants from wastewater generated at a coal burning power plant equipped with selective catalytic reducers for nitrogen oxide removal.     

Assay for determination of α-glucosidase and peptidase activity and location in a nitrifying trickling filter

Enzymatic alpha-glucosidase and peptidase activity in a nitrifying trickling filter (NTF) at the Rya wastewater treatment plant, G?teborg, Sweden, was investigated to evaluate whether these activities can be used as indicators of heterotrophic activity and polymer degradation. Samples of the biofilm were taken from the NTF and incubated in sterile filtered effluent water from the NTF with the addition of soluble starch, peptone, and ammonium chloride. In order to determine the distribution of enzyme activities, the alpha-glucosidase and peptidase activities were measured in the biofilm samples, in the filtered effluent water from the NTF and in the water phase in which the biofilm was incubated. Activities of both enzymes were found both in the effluent water from the NTF and in the biofilm. The enzyme activities were elevated in the samples when starch and peptone were present. In addition, there was a significant inhibition of ammonium oxidation in samples incubated with starch and peptone. Thus, the presence of starch, peptone and ammonium resulted in increased activity of heterotrophs, which lead to an inhibition of the nitrifiers, probably via competition for available oxygen.     

An alpha-proteobacterium converts linear alkylbenzenesulfonate surfactants into sulfophenylcarboxylates and linear alkyldiphenyletherdisulfonate surfactants into sulfodiphenylethercarboxylates

The surfactant linear alkylbenzenesulfonate (LAS; 0.5 mM) or linear monoalkyldiphenyletherdisulfonate (LADPEDS; 0.5 mM) in salts medium was easily degraded in laboratory trickling filters, whereas carbon-limited, aerobic enrichment cultures in suspended culture with the same inocula did not grow. We took portions of the trickling filters which degraded LADPEDS, shook the organisms from the solid support (polyester), and found that growth in suspended culture in LADPEDS-salts medium occurred only in the presence of some solid support (polyester fleece or glass wool), though little biomass was immobilized on the support. The end products in suspended culture were identical with those from the trickling filters. There was low plating efficiency of LADPEDS-grown cultures on complex medium, and no picked colony or mixture of colonies grew in LADPEDS-salts-glass wool medium. However, selective plates containing LADPEDS-salts medium solidified with agarose yielded LADPEDS-dependent, pinpoint colonies which could be picked singly and subcultured in selective liquid medium. Isolate DS-1 was a bacterium which showed 93% sequence homology (16S ribosomal DNA) to its nearest phylogenetic neighbor, an alpha-proteobacterium. Strain DS-1 grew heterotrophically in LADPEDS-salts-glass wool medium and converted the set of aryl-substituted alkanes to the corresponding aryl-substituted carboxylic acids of shorter chain length. Similarly, strain DS-1 grew heterotrophically with commercial LAS, converting it to a set of sulfophenylcarboxylates. Growth with a single isomer of LAS [3-(4-sulfophenyl)dodecane] was concomitant with excretion of 4-(4-sulfophenyl)hexanoate, which was identified by matrix-assisted laser desorption ionization mass spectrometry. The growth yield (6.4 g of protein/mol of C) indicated mass balance, which, with the specific growth rate (0.05 h(-1)), indicated a specific utilization rate of LAS of 2.2 mkat/kg of protein.     

Enumeration of Aeromonas hydrophila from domestic wastewater treatment plants and surface waters

R. POFFÉ AND E. OP DE BEECK. 1991. Influents, effluents and sludges from sewage purification plants and surface water samples were examined quantitatively for Aeromonas hydrophila on the mA medium of Rippey and Cabelli. Between 10⁴ and 10⁶/ml A. hydrophila were found in domestic wastewaters. On the average 99.975% were removed by activated sludge and 98.25% by trickling filters. Only 20.9% of A. hydrophila end up in the primary sludge, which contained up to 10⁷/g dry sludge. After 3 months, anaerobically (methane) fermented and partially dried sludge from trickling filters contained more than 10⁶ A. hydrophila /g dry sludge. Surface water receiving raw sewage contained several hundreds of A. hydrophila /ml, comparable with the numbers found in effluent waters, while surface water receiving no municipal wastewater and destined for the preparation of drinking water contained only small and negligible numbers. It was concluded that A. hydrophila was omnipresent in surface water.     

Microelectrode studies of oxygen transfer in trickling filter slimes*

Slime-covered rocks and samples of process waters from two trickling filters for treatment of municipal wastes were brought to the laboratory for probing with microelectrodes to determine dissolved oxygen (DO). Slime thickness was 0.4–1.5 mm. Flow rate of medium over the slime had a minor effect on slime respiration, but pH 5 or below was strongly inhibitory. Increasing temperature showed lower oxygen concentration throughout a slime, although 27°C had results little different from those at 22°C. Medium concentration had a profound effect on oxygen concentration profiles, and either oxygen-limited or substrate limited respiration could be demonstrated. Illumination of slimes from the top of the trickling filter developed oxygen supersaturation because oxygen from photosynthesis could not diffuse away rapidly.