Influence of waste water composition on biofilm development in laboratory methanogenic fluidized bed reactors

Summary The influence of the volatile fatty acid composition of waste waters on biofilm development and on the time course of reactor start-up was investigated in laboratory scale fluidized bed reactors. It was found that biofilm development proceeded in a similar way with either acetate, butyrate, propionate or a mixture of these compounds as carbon source in the waste water. Startup was retarded, however, with propionate as sole carbon source. Scanning electron microscopic examination revealed that immobilization of bacteria on the sand used as adhesive support initially occurred in crevices and that thereupon the surface of the sand particles became colonized. The composition of the newly developed biomass was determined when reactors reached steady state. Significant differences in the relative substrate spectra and in the amounts of hydrogenotrophic and acetotrophic methanogenic bacteria were measured. The differences reflected the differences in the composition of the waste waters. The results obtained emphasize the role of the structure of the carrier surface in start-up of methanogenic fluidized bed reactors.Abbreviations used Aw ash weight - COD chemical oxygen demand - EB fluidized bed - hbi vitamin B₁₂-HBI - spt sarcinapterin - UASB upflow anaerobic sludge blanket - VFA volatile fatty acid - VSS volatile suspended solids - Ww wet weight     

Variation of parameters affecting the start-up of methanogenic fluidized bed reactors

Summary The influence of the medium composition, the inoculum and the inoculation procedure on initial biofilm development in methanogenic fluidized bed reactors was studied on laboratory scale. Trace minerals but not vitamins were found to be essential for biofilm development. Inoculation with heterogeneous bacterial cultures of potentially sand-colonizing microorganisms and/or with pure cultures ofMethanothrix soehngenii did not accelerate biofilm development significantly as compared to inoculation with effluent from a fully operative fluidized bed reactor.     

Early stages in biofilm development in methanogenic fluidized-bed reactors

Summary Biofilm development in methanogenic fluidized-bed reactors with sand as the carrier was studied on a laboratory scale. The microorganisms present in consecutive layers of the biofilm of mature sludge granules were preliminarily characterized on the basis of their morphology, element composition and adhesion capacity and were compared to bacteria which take part in the initial colonization of sand. The early phase of biofilm development was monitored with reactors receiving waste-waters containing different mixtures of volatile fatty acids and inoculated with fluidized-bed reactor effluent for different lengths of time. The results obtained indicate that facultative anaerobic bacteria abundantly present in the outermost biofilm layers of mature sludge granules are probably the main primary colonizers of the sand. Methanothrix spp. or other methanogens were rarely observed among the primary colonizers. The course of biofilm formation was comparable under the various start-up conditions employed including variations in waste-water composition, inoculation and anaerobicity. However, omission of waste-water and thus of substrate resulted in rapid wash-out of the attached biomass. Offprint requests to: W. Heinen     

Effects of stratification in a fluidized bed bioreactor during treatment of metalworking wastewater

During wastewater treatment, biofilm-coated sand particles stratified in a fluidized bed bioreactor (FBB); particles coated by thicker biofilm segregated toward the top of the bed. Stratification was so well developed that at least two co-existing regions of significantly different mean biofilm thickness were visually distinct within the operating FBB. The observed stratification is attributed to differences in forces of drag, buoyancy, shear, and collisional impact, as well as differences of collision rate within the different regions. Particles with thick biofilm (thickness >100 μm) near the top of the bed consumed substrate at significantly lower rates per unit biomass than particles with thin biofilm (10-20 μm) near the bottom of the bed, thereby suggesting that substrate mass-transfer resistance through biofilm may limit biodegradation rates in the upper portion of the FBB. Large agglomerates of biomass floc and sand, which formed at the top of the fluidized bed, and sand particles with thick biofilm were susceptible to washout from the FBB, causing operational and treatment instability. Radial injection of supplemental liquid feed near the top of the bed increased shear and mixing, thereby preventing formation and washout of agglomerates and thickly coated sand particles. Supplemental liquid injection caused the mean specific biomass loading on the sand to increase and also increased the total biomass inventory in the FBB. Rates of biodegradation in the FBB appeared to be limited by penetration of substrates into the biofilm and absorption of oxygen from air into the wastewater. Copyright 1999 John Wiley & Sons, Inc.     

Microbiology and performance of a methanogenic biofilm reactor during the start-up period

Aims:  To understand the interactions between anaerobic biofilm development and process performances during the start-up period of methanogenic biofilm reactor.
Methods and Results:  Two methanogenic inverse turbulent bed reactors have been started and monitored for 81 days. Biofilm development (adhesion, growth, population dynamic) and characteristics (biodiversity, structure) were investigated using molecular tools (PCR–SSCP, FISH-CSLM). Identification of the dominant populations, in relation to process performances and to the present knowledge of their metabolic activities, was used to propose a global scheme of the degradation routes involved. The inoculum, which determines the microbial species present in the biofilm influences bioreactor performances during the start-up period. FISH observations revealed a homogeneous distribution of the Archaea and bacterial populations inside the biofilm.
Conclusion:  This study points out the link between biodiversity, functional stability and methanogenic process performances during start-up of anaerobic biofilm reactor. It shows that inoculum and substrate composition greatly influence biodiversity, physiology and structure of the biofilm.
Significance and Impact of the Study:  The combination of molecular techniques associated to a biochemical engineering approach is useful to get relevant information on the microbiology of a methanogenic growing biofilm, in relation with the start-up of the process.     

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.     

Simplified Modeling of Simultaneous Reaction Kinetics of Carbon Oxidation and Nitrification in Biofilm Processes

Batch experiments with varying initial substrate concentrations and biomass volumes were performed in a three‐phase fluidized bed biofilm reactor treating simulated domestic wastewater to study the simultaneous carbon oxidation and nitrification in the biofilm process. A simplified mass balance equation for the biofilm was proposed and five different kinetic rate equations were used to match the actual data. The kinetic parameters were obtained by nonlinear regression analysis on a set of two differential equations representing the simultaneous carbon oxidation and nitrification. The competitive inhibition model incorporating the effects of total organic carbon (TOC) concentrations on nitrification rates was the best‐suited model based on the average r². In this model, oxygen concentration and its affinity constants were not included. Instead, it was assumed that the rate of carbon oxidation is independent of the NH₄⁺‐N, while nitrification is affected by TOC. The number of parameters was successfully minimized without reducing its ability to accurately predict the bulk concentration time course, which would reduce computational complexity and possibly enhance the availability for an actual wastewater treatment process.     

Improvement of Process Stability of Microbiological Quinoline Degradation in a Three‐Phase Fluidized Bed Reactor

The biological degradation of quinoline by suspended and immobilized Comamonas acidovorans was studied under continuous and discontinuous operating conditions in a three‐phase fluidized bed reactor. C. acidovorans degrades quinoline into biomass and carbon dioxide. Quinoline and the intermediates of its metabolic pathway are found only by quinoline shockloads. The continuous degradation of quinoline by suspended biomass was only possible, if the dilution rate was less than the growth rate (μ_max =0.42 h^–1) and the concentration of a shockload was less than 1 kg/m³. A concentration greater than 1 kg/m³ led to an irreversible damage of the cells. Hence, two different carrier materials were used for immobilization by attachment, to increase the stability of the process. Using immobilization of biomass on carriers decouples the hydrodynamic retention time and the growth rate of the microorganisms. A comparison of the carrier material showed no differences with respect of activity and stability of the biofilm. The process stability of a three‐phase fluidized bed reactor was increased by immobilized biomass. The degradation of toxic shockloads was only possible with immobilized biomass. A dynamic model has been developed to describe the concentration profile of quinoline, 2‐hydroxyquinoline as metabolite and the suspended biomass. A comparison of the measured and calculated values showed good agreement.     

Biofilm reactors for industrial bioconversion processes: employing potential of enhanced reaction rates

This article describes the use of biofilm reactors for the production of various chemicals by fermentation and wastewater treatment. Biofilm formation is a natural process where microbial cells attach to the support (adsorbent) or form flocs/aggregates (also called granules) without use of chemicals and form thick layers of cells known as "biofilms." As a result of biofilm formation, cell densities in the reactor increase and cell concentrations as high as 74 gL^-1 can be achieved. The reactor configurations can be as simple as a batch reactor, continuous stirred tank reactor (CSTR), packed bed reactor (PBR), fluidized bed reactor (FBR), airlift reactor (ALR), upflow anaerobic sludge blanket (UASB) reactor, or any other suitable configuration. In UASB granular biofilm particles are used. This article demonstrates that reactor productivities in these reactors have been superior to any other reactor types. This article describes production of ethanol, butanol, lactic acid, acetic acid/vinegar, succinic acid, and fumaric acid in addition to wastewater treatment in the biofilm reactors. As the title suggests, biofilm reactors have high potential to be employed in biotechnology/bioconversion industry for viable economic reasons. In this article, various reactor types have been compared for the above bioconversion processes.     

Modeling biofilm accumulation and mass transport in a porous medium under high substrate loading

A packed bed biofilm reactor inoculated with pure culture Pseudomonas aeruginosa was run under high substrate loading and constant flow rate conditions. The 3.1-cm-diameter cylindrical reactor was 5 cm in length and packed with 1-mm glass beads. Daily observations of biofilm thickness, influent and effluent glucose substrate concentration, and effluent dissolved and total organic carbon were made during the 13-day experiment. Biofilm thickness appeared to rech quasi-steady-state condition after 10 days. A published biofilm process simulation program (AQUASIM) was used to analyze experimental data. Comparison of observed and simulated variables revealed three distinct phases of biofilm accumulation during the experiment: an initial phase, a growth phase, and a mature biofilm phase. Different combinations of biofilm and mass transport process variables were found to be important during each phase. Biofilm detachment was highly correlated with shear at the biofilm surface during all three phases of biofilm development. (c) 1995 John Wiley & Sons, Inc.     

Biofilm formation behaviour of marine filamentous cyanobacterial strains in controlled hydrodynamic conditions

Marine biofouling has severe economic impacts and cyanobacteria play a significant role as early surface colonizers. Despite this fact, cyanobacterial biofilm formation studies in controlled hydrodynamic conditions are scarce. In this work, computational fluid dynamics was used to determine the shear rate field on coupons that were placed inside the wells of agitated 12-well microtiter plates. Biofilm formation by three different cyanobacterial strains was assessed at two different shear rates (4 and 40 s⁻¹) which can be found in natural ecosystems and using different surfaces (glass and perspex). Biofilm formation was higher under low shear conditions, and differences obtained between surfaces were not always statistically significant. The hydrodynamic effect was more noticeable during the biofilm maturation phase rather than during initial cell adhesion and optical coherence tomography showed that different shear rates can affect biofilm architecture. This study is particularly relevant given the cosmopolitan distribution of these cyanobacterial strains and the biofouling potential of these organisms.     

甲烷氧化菌吸附膜反应器中环氧丙烷的连续生物转化

以流化床作为固定化体系 ,在硅藻土颗粒表面构建了混合培养的甲烷氧化细菌的吸附膜。研究发现延迟期后固定化细胞的甲烷单加氧酶活性明显增加。流化床中 90 %以上的甲烷氧化细菌以吸附形式存在。吸附膜浓度为 3.3～3.7 mgdryweightcell gDS。通过批式反应考察了丙烯 甲烷共氧化过程合成环氧丙烷的可能性。研究了甲烷对丙烯环氧化以及丙烯对甲烷氧化细菌生长的影响。通过最佳配比的混合反应气体 (methane :35 % ;propene :20% ;oxygen :45 % )连续循环通入流化床反应器中抽提产物环氧丙烷 ,克服了产物抑制。该生物反应器最初产生环氧丙烷的日产量为 110～ 150μmol d ,连续操作25d ,未观察到环氧丙烷生产能力的明显减小.     

Toluene degradation kinetics for planktonic and biofilm-grown cells of Pseudomonas putida 54G

Toluene degradation kinetics by biofilm and planktonic cells of Pseudomonas putida 54G were compared in this study. Batch degradation of (14)C toluene was used to evaluate kinetic parameters for planktonic cells. The kinetic parameters determined for toluene degradation were: specific growth rate, mu(max) = 10.08 +/- 1.2/day; half-saturation constant, K(S) = 3.98 +/- 1.28 mg/L; substrate inhibition constant, K(I) = 42.78 +/- 3.87 mg/L. Biofilm cells, grown on ceramic rings in vapor phase bioreactors, were removed and suspended in batch cultures to calculate (14)C toluene degradation rates. Specific activities measured for planktonic and biofilm cells were similar based on toluene degrading cells and total biomass. Long-term toluene exposure reduced specific activities that were based on total biomass for both biofilm and planktonic cells. These results suggest that long-term toluene exposure caused a large portion of the biomass to become inactive, even though the biofilm was not substrate limited. Conversely, specific activities based on numbers of toluene-culturable cells were comparable for both biofilm and planktonically grown cultures. Planktonic cell kinetics are often used in bioreactor models to model substrate degradation and growth of bacteria in biofilms, a procedure we found to be appropriate for this organism. For superior bioreactor design, however, changes in cellular activity that occur during biofilm development should be investigated under conditions relevant to reactor operation before predictive models for bioreactor systems are developed. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 535-546, 1997.     

Nutrient dynamics and successional changes in a lentic freshwater biofilm

SUMMARY 1. Colonisation, species composition, succession of microalgae and nutrient dynamics in biofilms grown under light and dark conditions were examined during the initial phases of biofilm development in a lentic freshwater environment.
2. Biofilms were developed on inert (perspex) panels under natural illuminated and experimental dark conditions and the panels were retrieved for analysis after different incubation periods. Analysed parameters included biofilm thickness, algal density, biomass, chlorophyll a , species composition, total bacterial density and nutrients such as nitrite, nitrate, phosphate and silicate.
3. Biofilm thickness, algal density, biomass, chlorophyll a and species richness were significantly higher in light-grown biofilms, compared with dark-grown biofilms. The light-grown biofilms showed a three-phased succession pattern, with an initial domination of Chlorophyceae followed by diatoms (Bacillariophyceae) and finally by cyanobacteria. Dark-grown biofilms were mostly dominated by diatoms.
4. Nutrients were invariably more concentrated in biofilms than in ambient water. Nutrient concentrations were generally higher in dark-grown biofilms except in the case of phosphate, which was more concentrated in light-grown biofilms. Significant correlations between nutrients and biofilm parameters were observed only in light-grown biofilms.
5. The N : P ratio in the biofilm matrix decreased sharply in the initial 4 days of biofilm growth; ensuing N-limitation status seemed to influence biofilm community structure. The N : P ratios showed significant positive correlations with the chlorophycean fraction in both light and dark-grown biofilms, and low N : P ratio in the older biofilms favoured cyanobacteria. Our data indicate that nutrient chemistry of biofilm matrix shapes community structure in microalgal biofilms.     

Methanotrophic attached-film reactor development and biofilm characteristics

The feasibility of using methanotrophs in an attached-film, fluidized-bed (MAFFB) reactor system has been under investigation since 1987. Mixed culture, methane-utilizing attached biofilms were developed on diatomaceous earth particles and on granular activated carbon. The required feed gases, methane and oxygen, were supplied to the attached biofilm in disolved form using separate gas-liquid aeration columns. Biofilm growth was steady despite low influent dissolved methane concentrations (1 to 3 mg/L). A breeder MAFFB operated consistently for 4.1 years with attached biofilm concentrations as high as 51.7 g VS/L static-bed with minimal biomass wasting and with minimal buffer and nutrient inputs. The maximum biomass concentration observed was 75.6 g VS/L static-bed in a MAFFB reactor treating trichloroethene. Biofilm thickness reached 160 mum with typical values of 70 mum under methane and oxygen growht-rate-limited conditions. Biofilm densities of 120 to 190 g VS/L film were observed. Growth rates varied from <0.01/d to 0.17/d. Greater than 90% of the biomass concentration in the bed was attached, and effluent total suspended solids ranged from 5 to 74 mg/L, with an average of 24 mg/L over 27 runs in four MAFFB systems at upflow velocities of 11.4 to 25 m/h. Heterotrophic attached-film methanotrophs appear to be stable and useful for applications in toxics treatment, and other product manipulations. (c) 1992 John Wiley & Sons, Inc.     

Kinetic model for dynamic response of three-phase fluidized bed biofilm reactor for wastewater treatment

Step changes in inlet concentration has been introduced into the completely mixed three-phase fluidized bed biofilm reactor treating simulated domestic wastewater to study the dynamic behavior of the system and to establish the suitable kinetic model from the response curve. Three identical reactors having different biomass volumes were operated in parallel. It was found that the response curves showed second-order characteristics, and thus at least two first-order differential equations are necessary to simulate the substrate and biomass response curves. Nonlinear regression analysis was performed using different types of rate equations and their corresponding kinetic parameters were used to simulate the theoretical response curve using the Runge–Kutta numerical integration method. As a result, although various types of conventional biokinetic models such as Monod, Haldane and Andrew types were examined, all the theoretical substrate response curves underestimated time constants compared to the actual substrate response plots. On the other hand, the theoretical curve of the kinetic model that incorporates adsorption term has best fit to the actual response in most of the cases. Thus, it was concluded that adsorption of substrate onto biofilm and carrier particles has significant effect on the dynamic response in biofilm processes.     

The use of cellulase in inhibiting biofilm formation from organisms commonly found on medical implants

A study was made of the use of cellulase to inhibit biofilm formation by a pathogenic bacterium commonly found in medical implants. A Pseudomonas aeruginosa biofilm was grown on glass slides in a parallel flow chamber for 4 d with glucose as the nutrient source. Biofilm development was assessed by measuring the colony forming units (CFU) and biomass areal density. Biofilm was grown at pH 5 and 7 in the presence of three different cellulase concentrations, 9.4, 37.6 and 75.2 units ml-1. In addition, a control study using deactivated cellulase was performed. The results show that cellulase is effective in partially inhibiting biomass and CFU formation by P. aeruginosa on glass surfaces. The effect of cellulase depended on concentration and was more effective at pH 5 than pH 7. The experiment was further extended by investigating the effect of cellulase on the apparent molecular weight of purified P. aeruginosa exopolysaccharides (EPS). The observation of EPS using size exclusion chromatography showed a decrease in apparent molecular weight when incubated with enzyme. An increase in the amount of reducing sugar with time when the purified EPS were incubated with enzyme also supports the hypothesis that cellulase degrades the EPS of P. aeruginosa. While cellulase does not provide total inhibition of biofilm formation, it is possible that the enzyme could be used in combination with other treatments or in combinations with other enzymes to increase effectiveness.     

Degradation of aromatic compounds by Pseudomonas putida

The influence of different process kinetics on the course of phenol degradation has been studied as well as the influence of axial dispersion in the liquid phase on the reactor height with relatively large biofilm thickness in a conventional fluidized bed and air-lift bioreactor. The object of this was to achieve a high conversion of substrate in a device of real size in real process time. For calculating the mathematical model, the method of orthogonal collocation with the STIFF integration routine has been used.     

Biofilm development during the start-up period of anaerobic biofilm reactors: the biofilm Archaea community is highly dependent on the support material

To evaluate the impact of the nature of the support material on its colonization by a methanogenic consortium, four substrata made of different materials: polyvinyl chloride, 2 polyethylene and polypropylene were tested during the start-up of lab-scale fixed-film reactors. The reactor performances were evaluated and compared together with the analysis of the biofilms. Biofilm growth was quantified and the structure of bacterial and archaeal communities were characterized by molecular fingerprinting profiles (capillary electrophoresis-single strand conformation polymorphism). The composition of the inoculum was shown to have a major impact on the bacterial composition of the biofilm, whatever the nature of the support material or the organic loading rate applied to the reactors during the start-up period. In contrast, the biofilm archaeal populations were independent of the inoculum used but highly dependent on the support material. Supports favouring Archaea colonization, the limiting factor in the overall process, should be preferred.