Anammox enrichment from reject water on blank biofilm carriers and carriers containing nitrifying biomass: operation of two moving bed biofilm reactors (MBBR)

The anammox bacteria were enriched from reject water of anaerobic digestion of municipal wastewater sludge onto moving bed biofilm reactor (MBBR) system carriers-the ones initially containing no biomass (MBBR1) as well as the ones containing nitrifying biomass (MBBR2). Duration of start-up periods of the both reactors was similar (about 100?days), but stable total nitrogen (TN) removal efficiency occurred earlier in the system containing nitrifying biomass. Anammox TN removal efficiency of 70% was achieved by 180?days in both 20?l volume reactors at moderate temperature of 26.0°C. During the steady state phase of operation of MBBRs the average TN removal efficiencies and maximum TN removal rates in MBBR1 were 80% (1,000?g-N/m(3)/day, achieved by 308?days) and in MBBR2 85% (1,100?g-N/m(3)/day, achieved by 266?days). In both reactors mixed bacterial cultures were detected. Uncultured Planctomycetales bacterium clone P4, Candidatus Nitrospira defluvii and uncultured Nitrospira sp. clone 53 were identified by PCR-DGGE from the system initially containing blank biofilm carriers as well as from the nitrifying biofilm system; from the latter in addition to these also uncultured ammonium oxidizing bacterium clone W1 and Nitrospira sp. clone S1-62 were detected. FISH analysis revealed that anammox microorganisms were located in clusters in the biofilm. Using previously grown nitrifying biofilm matrix for anammox enrichment has some benefits over starting up the process from zero, such as less time for enrichment and protection against severe inhibitions in case of high substrate loading rates.     

Biofilm reactors: an experimental and modeling study of wastewater denitrification in fluidized-bed reactors of activated carbon particles

A fluidized-bed biofilm reactor using activated carbon particles of 1.69 mm diameter as the support for biomass growth and molasses as the carbon source is used for wastewater denitrification.The start-up of the reactor was successfully achieved in 1 week by using a liquor from garden soil leaching as the inoculum and a superficial velocity u(0) = 5u(mf). Typical biofilm thickness is 800 mum; therefore covered activated carbon particles have 3.3 mm in diameter.Reactor hydrodynamics was studied by tracer (KCl solution) experiments. The analysis based on residence time distribution theory involved a model with axial dispersion flow and tracer diffusion with linear adsorption inside the biofilm. Peclet numbers higher than 100 were found, allowing the plug flow assumption for the reactor model.Experimental profiles of nitrate and nitrite species were explained by a kinetic model of two consecutive zero-order reactions coupled with substrate diffusion inside the biofilm. Under the operating conditons used thick biofilms were obtained working in a diffusion-controlled regime.Comparison is made with results obtained in the same reactor with sand particles as the support for biomass growth. Activated carbon as the support has the following advantages: good adsorptive characteristics, homogeneous biofilm thickness along the reactor, and easy restart-up of the reactor. (c) 1992 John Wiley & Sons, Inc.     

Backwash intensity and frequency impact the microbial community structure and function in a fixed-bed biofilm reactor

Linkages among bioreactor operation and performance and microbial community structure were investigated for a fixed-bed biofilm system designed to remove perchlorate from drinking water. Perchlorate removal was monitored to evaluate reactor performance during and after the frequency and intensity of the backwash procedure were changed, while the microbial community structure was studied using clone libraries and quantitative PCR targeting the 16S rRNA gene. When backwash frequency was increased from once per month to once per day, perchlorate removal initially deteriorated and then recovered, and the relative abundance of perchlorate-reducing bacteria (PRB) initially increased and then decreased. This apparent discrepancy suggested that bacterial populations other than PRB played an indirect role in perchlorate removal, likely by consuming dissolved oxygen, a competing electron acceptor. When backwash intensity was increased, the reactor gradually lost its ability to remove perchlorate, and concurrently the relative abundance of PRB decreased. The results indicated that changes in reactor operation had a profound impact on reactor performance through altering the microbial community structure. Backwashing is an important yet poorly characterized procedure when operating fixed-bed biofilm reactors. Compared to backwash intensity, changes in backwash frequency exerted less disturbance on the microbial community in the current study. If this finding can be confirmed in future work, backwash frequency may serve as the primary parameter when optimizing backwash procedures.     

Biofilm model calibration and microbial diversity study using Monte Carlo simulations

Mathematical models are useful tools for studying and exploring biological conversion processes as well as microbial competition in biological treatment processes. A single‐species biofilm model was used to describe biofilm reactor operation at three different hydraulic retention times (HRT). The single‐species biofilm model was calibrated with sparse experimental data using the Monte Carlo filtering method. This calibrated single‐species biofilm model was then extended to a multi‐species model considering 10 different heterotrophic bacteria. The aim was to study microbial diversity in bulk phase biomass and biofilm, as well as the competition between suspended and attached biomass. At steady state and independently of the HRT, Monte Carlo simulations resulted only in one unique dominating bacterial species for suspended and attached biomass. The dominating bacterial species was determined by the highest specific substrate affinity (ratio of µ/K_S). At a short HRT of 20 min, the structure of the microbial community in the bulk liquid was determined by biomass detached from the biofilm. At a long HRT of 8 h, both biofilm detachment and microbial growth in the bulk liquid influenced the microbial community distribution. Biotechnol. Bioeng. 2013; 110: 1323–1332. © 2012 Wiley Periodicals, Inc.     

Macrokinetic and quantitative microbial investigation on a bench-scale biofilter treating styrene-polluted gaseous streams

We performed a macrokinetic and quantitative microbial investigation of a continuously operating bench-scale biofilter treating styrene-polluted gases. The device was filled with a mixture of peat and glass beads as packing medium and inoculated with the styrene-oxidizing strain, Rhodococcus rhodochrous AL NCIMB 13259. The experimental data of styrene and microbial concentrations, obtained at different biofilter heights, were used to evaluate the pollutant concentration profiles as well as the influence of styrene loading on biomass distribution along the packing medium. Styrene and biomass concentration profiles permitted detection of a linear relationship between the amount of biomass grown in a given section of the biofilter and that of pollutant removed, regardless of the operating conditions tested. Biomass development in the bed appeared to: depend linearly on pollutant concentration at an inlet styrene concentration of <0.10 g m(-3) in the gaseous stream; achieve a maximum value (7. 10(7) colony forming units per gram of packing material) within a wide styrene concentration range (0.10 to 1.0 g m(-3)); and fall sharply beyond this inhibition threshold. The process followed zeroth-order macrokinetics with respect to styrene concentration, which is consistent with zeroth-order microkinetics with either fully active or not fully active biofilm. The maximal volumetric styrene removal rate was found to be 63 g m(packing material) (-3) h(-1) for an influent pollutant concentration of 0.80 g m(-3) and a superficial gas velocity of 245 m h(-1).     

Coexistence of nitrifiers, denitrifiers and Anammox bacteria in a sequencing batch biofilm reactor as revealed by PCR-DGGE

Aims:  The bacterial diversity in a sequencing batch biofilm reactor (SBBR) treating landfill leachate was studied to explain the mechanism of nitrogen removal.
Methods and Results:  The total microbial DNA was extracted from samples collected from landfill leachate and biofilm of the reactor with the removal efficiencies of NH₄⁺-N higher than 97% and that of chemical oxygen demand (determined by K₂Cr₂O₇, COD_Cr) higher than 86%. Denaturing gradient gel electrophoresis (DGGE) fingerprints based on total community 16S rRNA genes were analyzed with statistical methods, and excised DNA bands were sequenced. The results of phylogenetic analyses revealed high diversity within the SBBR biofilm community, and DGGE banding patterns showed that the community structure in the biofilm remained stable during the running period.
Conclusions:  A coexistence of nitrifiers, including ammonia-oxidizing bacteria and nitrite-oxidizing bacteria, denitrifiers, including aerobic or anaerobic denitrifying bacteria and Anammox bacteria were detected, which might be the real matter of high removal efficiencies of NH₄⁺-N and COD_Cr in the reactor.
Significance and Impact of the Study:  The findings in this study indicated that PCR-DGGE analysis could be used for microbial community detection as prior method, and the SBBR technique could provide preferable growing environment for bacteria with N removal function.     

Gas-phase methyl ethyl ketone biodegradation in a tubular biofilm reactor: microbiological and bioprocess aspects

A novel type of bioreactor was designed to clean VOCs-containing air.The operation of this reactor consists in mixing the polluted gas and a mistof nutrient solution in the presence of microorganisms in order to maximizecontact and transfer between gas, liquid and microorganisms and to promotethe degradation kinetics and the relative removal efficiency of thepollutant. A bacterial consortium acclimatized to MEK and containing apreponderance of Alcaligenes denitrificans was established under non-axenicconditions. On the tubular reactor's glass walls, a continuous biofilm wasdeveloped. This biofilm was rapidly contaminated by two fungi able todegrade MEK: Geotrichum candidum and Fusarium oxysporum. Their abundance inthe reactor is probably linked to the acidic conditions inside the biofilmand to their broader tolerance for low pH values concomitant with MEKdegradation. In the reactor, a maximum volumetric degradation rate of 3.5 kgMEK/m³ _reactor·d was obtained for arelative removal efficiency of 35%, whereas the latter was maintainedat 70% for more modest applied loadings of 1.5 kgMEK/m³ _reactor ·d. In liquid batchcultures, a biomass originating from the biofilm was able to degrade 0.40g_MEK/g_DCW·h at the optimal pH of 7. Aregular cycle of detachment-recolonization was observed during the operationof the bioreactor. The maximal degradation activity was obtained with a thinbiofilm and was not increased as the biofilm grew in thickness. The overalldegradation rate of the process did not appear to be limited by thediffusion of oxygen inside the biofilm. Over short periods of time, the MEKtransfer from the gaseous phase to the biofilm was neither affected by thepresence of the mist nor by the wetting of the biofilm. A better control ofthe biofilm pH led to improved performance in terms of removal rate but notin terms of relative elimination efficiency.     

Effect of vapor-phase bioreactor operation on biomass accumulation, distribution, and activity: linking biofilm properties to bioreactor performance

Excess biomass accumulation and activity loss in vapor-phase bioreactors (VPBs) can lead to unreliable long-term operation. In this study, temporal and spatial variations in biomass accumulation, distribution and activity in VPBs treating toluene-contaminated air were monitored over a 96-day period. Two laboratory-scale bioreactors were subjected to a toluene loading rate of 45.8 g/m(3)-h with one VPB operating in a unidirectional (UD) mode and a second identical VPB operating in a directionally switching (DS) mode. In the UD bioreactor, the contaminated air stream was continuously fed to the bottom of the reactor, while, in the DS bioreactor, the direction of the contaminated gas flow was reversed every three days. Overall, the DS system performed better with respect to biomass distribution and microbial activity across the bioreactor, resulting in more stable bioreactor performance. In contrast, most of the biomass accumulation and activity was confined to the front half of the UD bioreactor column which caused high pressure drops, rapid activity loss and eventually toluene breakthrough. A carbon balance reveals that excess biomass accumulated continuously in both bioreactors, and biomass yield coefficients were very similar (0.59 g dry biomass/g toluene for the UD and 0.63 g dry biomass/g toluene for the DS). The viable biomass population remained relatively constant in both bioreactors over the operational period, while the inactive biomass fraction steadily increased over the same time frame. Biodegradation activity determined by the dehydrogenase enzyme activity assay was found to be a function of biomass accumulation and reflected pollutant removal profiles along the columns. In addition, biomass activity correlated well with the toluene-degrading fraction of the total bacterial population.     

Biological denitrification in a fluidized bed

A fluidized bed biofilm reactor using sand as the carrier particle was employed to study the effects of superficial velocity on the removal of nitrates as well as on the growth of the biofilm. Velocity was found to affect significantly both nitrate removal and biofilm growth. An analysis based on heterogenous catalysis was used to describe the denitrification process. There is good agreement between analysis and experimental measurements for startup and steady-state operating conditions.     

Simultaneous nitrification and denitrification by controlling vertical and horizontal microenvironment in a membrane-aerated biofilm reactor

Nitrogen and carbon components in domestic modified wastewater were completely removed by simultaneous nitrification and denitrification using a membrane-aerated biofilm reactor where biofilm was fixed on a hollow-fiber membrane. To measure the spatial distribution of pH, ammonium and nitrate ions and to observe microbes inside the biofilm fixed on the membrane, microelectrodes and the fluorescence in situ hybridization (FISH) method were applied. Due to plug flow in the vertical direction (from the bottom to the top of the reactor), ammonium nitrogen was gradually removed and negligible nitrate nitrogen was detected throughout the reactor. FISH revealed that ammonia-oxidizing bacteria were mainly distributed inside the biofilm and other bacteria, which included denitrifying bacteria, were mainly distributed outside the biofilm and over the suspended sludge. In order to characterize bacterial activity in the vertical direction of the reactor, nitrification rates at lower, central and upper points were calculated using microelectrode data. The nitrification rate at the lower point was 7 and 125 times higher than those at the central and upper points, respectively. These results show that the removal of carbon and nitrogen compounds was accomplished efficiently by using various kinds of bacteria distributed vertically and horizontally in a single reactor.     

Influence of hydrodynamic conditions on biofilm behavior in a methanogenic inverse turbulent bed reactor

This paper presents a study about the influence of gas velocity on a methanogenic biofilm in an inverse turbulent bed reactor. Experimental results indicate a dynamic response of the growing attached biomass to the changes of hydrodynamic conditions, mainly attrition constraints. Short but intensive increases of gas velocity (U(g)) are shown to induce more detachment than a high but constant gas flow rate. Hydrodynamic conditions control the composition of the growing biofilm in terms of cells and exocellular polymeric substances (EPS). The cell fraction within the biofilm (R(cell)) was found to be inversely proportional to the gas velocity. The specific activity expressed in methane production rate or COD removal rate is higher in biofilms formed under high hydrodynamic constraints. The control of the hydrodynamic conditions in a biofilm reactor should make it possible to obtain a resistant and active biofilm.     

Mathematical model for a three-phase fluidized bed biofilm reactor in wastewater treatment

A mathematical model for a three phase fluidized bed bioreactor (TFBBR) was proposed to describe oxygen utilization rate, biomass concentration and the removal efficiency of Chemical Oxygen Demand (COD) in wastewater treatment. The model consisted of the biofilm model to describe the oxygen uptake rate and the hydraulic model to describe flow characteristics to cause the oxygen distribution in the reactor. The biofilm model represented the oxygen uptake rate by individual bioparticle and the hydrodynamics of fluids presented an axial dispersion flow with back mixing in the liquid phase and a plug flow in the gas phase. The difference of settling velocity along the column height due to the distributions of size and number of bioparticle was considered. The proposed model was able to predict the biomass concentration and the dissolved oxygen concentration along the column height. The removal efficiency of COD was calculated based on the oxygen consumption amounts that were obtained from the dissolved oxygen concentration. The predicted oxygen concentration by the proposed model agreed reasonably well with experimental measurement in a TFBBR. The effects of various operating parameters on the oxygen concentration were simulated based on the proposed model. The media size and media density affected the performance of a TFBBR. The dissolved oxygen concentration was significantly affected by the superficial liquid velocity but the removal efficiency of COD was significantly affected by the superficial gas velocity. An erratum to this article can be found online at .     

Disturbance Frequency Determines Morphology and Community Development in Multi-Species Biofilm at the Landscape Scale

Many natural and engineered biofilm systems periodically face disturbances. Here we present how the recovery time of a biofilm between disturbances (expressed as disturbance frequency) shapes the development of morphology and community structure in a multi-species biofilm at the landscape scale. It was hypothesized that a high disturbance frequency favors the development of a stable adapted biofilm system while a low disturbance frequency promotes a dynamic biofilm response. Biofilms were grown in laboratory-scale reactors over a period of 55-70 days and exposed to the biocide monochloramine at two frequencies: daily or weekly pulse injections. One untreated reactor served as control. Biofilm morphology and community structure were followed on comparably large biofilm areas at the landscape scale using automated image analysis (spatial gray level dependence matrices) and community fingerprinting (single-strand conformation polymorphisms). We demonstrated that a weekly disturbed biofilm developed a resilient morphology and community structure. Immediately after the disturbance, the biofilm simplified but recovered its initial complex morphology and community structure between two biocide pulses. In the daily treated reactor, one organism largely dominated a morphologically simple and stable biofilm. Disturbances primarily affected the abundance distribution of already present bacterial taxa but did not promote growth of previously undetected organisms. Our work indicates that disturbances can be used as lever to engineer biofilms by maintaining a biofilm between two developmental states.     

Biofilm microbial community of a thermophilic trickling biofilter used for continuous biohydrogen production

Molecular methods were employed to investigate the microbial community of a biofilm obtained from a thermophilic trickling biofilter reactor (TBR) that was operated long-term to produce H(2). Biomass concentration in the TBR gradually decreased as reactor bed height increased. Despite this difference in biomass concentration, samples from the bottom and middle of the TBR bed revealed similar microbial populations as determined by PCR-DGGE analysis of 16S rRNA genes. Nucleotide sequences of most DGGE bands were affiliated with the classes Clostridia and Bacilli in the phylum Firmicutes, and the most dominant bands showed a high sequence similarity to Thermoanaerobacterium thermosaccharolyticum.     

Long-term performance and microbial dynamics of an up-flow fixed bed reactor established for the biodegradation of fluorobenzene

An up-flow fixed bed reactor (UFBR) was established to investigate the biodegradation of fluorobenzene (FB) under a number of operating conditions, which included variation in the concentration of FB in the feed stream (up to 180 mg l⁻¹) and temporary suspension of feeding. Degradation of FB was followed for a period of 8 months under a continuous flow regime. During the operation of the UFBR, FB was never detected in the reactor effluent, being biodegraded by the microbial biofilm or adsorbed to the granular activated carbon (GAC). Biodegradation of FB was observed from the beginning of the reactor operation, and overall, it accounted for 50% of the total amount fed to the bioreactor. High organic loads of FB (210–260 mg d⁻¹ dm⁻³) were found to affect the biological removal efficiency, possibly due to an inhibitory effect caused by the higher FB concentrations fed to the bioreactor (149–179 mg l⁻¹). When FB feeding was suspended for 1 month, biodegradation continued, indicating that the adsorbed FB became bioavailable. Biofilm bacterial dynamics were followed throughout the UFBR operation by denaturing gradient gel electrophoresis and plate-counting techniques, showing that a quite stable community was found in the bioreactor, and this was mainly attributed to the high selective pressure exerted by the presence of FB.     

Air-lift internal loop biofilm reactor for realized simultaneous nitrification and denitrification

Simultaneous nitrification and denitrification (SND) was realized by means of a novel air-lift internal loop biofilm reactor, in which aeration was set in middle of the reactor. During operation, the aeration was adjusted to get appropriate dissolve oxygen (DO) in bulk solution and let aerobic and anoxic zone coexist in one reactor. When aeration was at 0.6 and 0.2 L/min, corresponding to DO of 5.8 and 2.5 mg/L in bulk solution, ammonia nitrogen removal percentage reached about 80 and 90 %, but total nitrogen removal percentage was lower than 25 %. While the aeration was reduced to 0.1 L/min, aerobic and anoxic zones existed simultaneously in one reactor to get 75 % of ammonia nitrogen and 50 % of total nitrogen removal percentage. Biofilms were, respectively, taken from aerobic and anoxic zone to verify their function of nitrification and denitrification in two flasks, in which ammonia nitrogen was transferred into nitrate completely by aerobic biofilm, and nitrate was removed more than 80 % by anoxic biofilm. Microelectrode was used to measure the DO distribution inside biofilms in anoxic zone corresponding to different aerations. When aeration was at 0.6 and 0.2 L/min, DO inside biofilm was more than 1.5 mg/L, but the DO inside biofilm decreased to anoxic status with depth of biofilm increasing corresponding to aeration of 0.1 L/min. The experimental results indicated that SND could be realized because of simultaneous existence of aerobic and anoxic biofilms in one reactor.     

Bioreactor with a semi-fixed packing: anaerobic lactose to lactic acid fermentation

The bioreactor with a semi-fixed packing consists of frames with stretched “Raschell”-type sacks on them. This construction enables the attainment of a larger interfacial area of the biofilm carrier per unit reactor volume as well as the easy removal of the exausted biomass at certain biofilm thickness. In the present paper the batch anaerobic conversion of lactose to lactic acid in this reactor with immobilized bacteria Lactobacillus casei is studied. The dilution rate of the feeding substrate solution was 1?mm/s. Comparison of our experimental results with data, obtained for free cells and other construction of bioreactors with immobilized cells is made. It is shown that the used immobilised biocatalyst is superior to free bacterial cells, or attached to the inner pores of polyurethane foam.     

Biofilm development in laboratory methanogenic fluidized bed reactors

Biofilm development on sand with different heterogeneous inocula was studied in laboratory-scale methanogenic fluidized bed reactors. Both the course of biofilm formation during reactor start-up and the bacterial composition of newly developed biofilms at steady-state were found to be similar irrespective of the type of inoculum applied. Biofilm formation proceeded according to a fixed pattern that could be subdivided in three consecutive phases, designated as the lag phase, biofilm production phase, and steady-state phase. Methanogenic activity and biomass content of the fluidized bed granules were found to be accurate parameters of the course of biofilm formation. More indirect parameters monitored did not give unambiguous results in all instances. The composition of the newly developed biomass as assessed on the basis of potential methanogenic activities on different substrates and of the concentration of specific methanogenic cofactors was consistent with electron microscopic observations.     

Possible complication regarding phosphorus removal with a continuous flow biofilm system: diffusion limitation

Diffusion limitation of phosphate possibly constitutes a serious problem regarding the use of a biofilm reactor for enhanced biological phosphorus removal. A lab-scale reactor for simultaneous removal of phosphorus and nitrate was operated in a continuous alternating mode of operation. For a steady-state operation with excess amounts of carbon source (acetate) during the anaerobic phase, the same amount of phosphate was released during the anaerobic phase as was taken up during the anoxic phase. The measured phosphorus content of the biomass that detached during backwash after an anoxic phase was low, 2.4 +/- 0.4% (equal to 24 +/- 4 mg P/g TS). A simplified computer model indicated the reason to be phosphate diffusion limitation and the model revealed a delicate balance between the obtainable phosphorus contents of the biomass and operating parameters, such as backwash interval, biofilm thickness after backwash, and phase lengths. The aspect of diffusion is considered of crucial importance when evaluating the performance of a biofilter for phosphate removal.     

Sequencing batch membrane biofilm reactor for simultaneous nitrogen and phosphorus removal: novel application of membrane-aerated biofilm

A sequencing batch membrane biofilm reactor (SBMBfR) was developed for simultaneous carbon, nitrogen, and phosphorus removal from wastewater. This reactor was composed of two functional parts: (1) a gas-permeable membrane on which a nitrifying biofilm formed and (2) a bulk solution in which bacteria, mainly denitrifying polyphosphate-accumulating organisms (DNPAOs), were suspended. The reactor was operated sequentially under anaerobic condition and then under membrane aeration condition in one cycle. During the anaerobic period, organic carbon was consumed by DNPAOs; this was accompanied by phosphate release. During the subsequent membrane aeration period, nitrifying bacteria utilized oxygen supplied directly to them from the inside of the membrane. Consequently, the nitrite and nitrate products diffused into the bulk solution, where they were used by DNPAOs as electron acceptors for phosphate uptake. In a long-term sequencing batch operation, the mean removal efficiencies of total organic carbon (TOC), total nitrogen (T-N), and total phosphorus (T-P) under steady-state condition were 99%, 96%, and 90%, respectively. In addition, fluorescence in situ hybridization (FISH) clearly demonstrated the difference in bacterial community structure between the membrane biofilm and the suspended sludge: ammonia-oxidizing bacteria belonging to the Nitrosomonas group were dominant in the region adjacent to the membrane throughout the operation, and the occupation ratio of the well-known polyphosphate-accumulating organism (PAO) Candidatus "Accumulibacter phosphates" in the suspended sludge gradually increased to a maximum of 37%.