Bioreactor for the study of defined interactions of toxic metals and biofilms

A novel bioreactor system constructed for studies of the interactions of heavy metals and microbial cells at the solid-solution interface is described. The applicability of this experimental system to meet the severe constraints imposed on such an apparatus by the requirements for an unambiguous interpretation of data and for mathematical modeling of these interactions was explored with the trace metal lead and with the marine bacterium Pseudomonas atlantica. A chemically defined medium composed of the major components of seawater, simple salts required for growth, glucose, and the single amino acid glycine was derived. It supported a maximum growth rate several times less than that in a complex medium, but provided growth to high cell densities and the formation of biopolymer and supported the development of a monolayer biofilm. The use of such a medium in conjunction with our bioreactor system minimized trace metal contamination while allowing quantification of the partitioning of lead onto various reactor surfaces. Lead adsorption by reactor walls and model surfaces was linear with equilibrium led concentration up to 6 X 10(-6) mol/liter. Equilibrium lead adsorption due to P. atlantica biofilm surfaces ranged from 20 to 40% at a total lead concentration of 10(-6) mol/liter depending upon solution pH and ionic composition, indicating that biofilms can play an important role in controlling toxic metal concentrations in natural systems.     

Interactions of microbial biofilms with toxic trace metals: 2. Prediction and verification of an integrated computer model of lead (II) distribution in the presence of microbial activity

The interfacial interactions of a toxic trace metal, Pb, with a surface modified by a marine film-forming bacterium, Psedomonas atlantica, were predicted by a structured biofilm model used in conjunction with a chemical speciation model. The validity of the integrated model was tested for batch and continuous operations. Dynamic responses of the biophase due to transient lead concentration increases were also stimulated. The reasonable pre dictions achieved by the model demonstrate its utility in describing trace metal distributions in complex systems where the adsorption properties of inorganic surfaces are modified by adherent bacteria production of extracellular polymers. (c) 1994 John Wiley & Sons, Inc.     

Role of nickel in high rate methanol degradation in anaerobic granular sludge bioreactors

The effect of nickel deprivation from the influent of a mesophilic (30 degrees C) methanol fed upflow anaerobic sludge bed (UASB) reactor was investigated by coupling the reactor performance to the evolution of the Methanosarcina population of the bioreactor sludge. The reactor was operated at pH 7.0 and an organic loading rate (OLR) of 5-15 g COD l(-1) day(-1) for 191 days. A clear limitation of the specific methanogenic activity (SMA) on methanol due to the absence of nickel was observed after 129 days of bioreactor operation: the SMA of the sludge in medium with the complete trace metal solution except nickel amounted to 1.164 (+/-0.167) g CH(4)-COD g VSS(-1) day(-1) compared to 2.027 (+/-0.111) g CH(4)-COD g VSS(-1) day(-1) in a medium with the complete (including nickel) trace metal solution. The methanol removal efficiency during these 129 days was 99%, no volatile fatty acid (VFA) accumulation was observed and the size of the Methanosarcina population increased compared to the seed sludge. Continuation of the UASB reactor operation with the nickel limited sludge lead to incomplete methanol removal, and thus methanol accumulation in the reactor effluent from day 142 onwards. This methanol accumulation subsequently induced an increase of the acetogenic activity in the UASB reactor on day 160. On day 165, 77% of the methanol fed to the system was converted to acetate and the Methanosarcina population size had substantially decreased. Inclusion of 0.5 muM Ni (dosed as NiCl(2)) to the influent from day 165 onwards lead to the recovery of the methanol removal efficiency to 99% without VFA accumulation within 2 days of bioreactor operation.     

Membrane bioreactor with a porous hydrophobic membrane as a gas-liquid contactor for waste gas treatment

A novel type of bioreactor for waste gas treatment has been designed. The reactor contains a microporous hydrophobic membrane to create a large interface between the waste gas and the aqueous phase. To test the new reactor, propene was chosen because of its high air/water partition coefficient, which causes a low water concentration and hampers its removal from air. Propene transfer from air to a suspension of propene-utilizing Xanthobacter Py2 cells in the membrane bioreactor proved to be controlled by mass transfer in the liquid phase. The resistance of the membrane was negligible. Simulated propene transfer rates agreed well with the experimental data. A stable biofilm of Xanthobacter Py2 developed on the membrane during prolonged operation. The propene flux into the biofilm was 1 x 10(-6) mol m(-2) s(-1) at a propene concentration of 9.3 x 10(-2) mol m(-3) in the gas phase. (c) 1995 John Wiley & Sons, Inc.     

Degradation of phenol by Pseudomonas putida ATCC 11172 in continuous culture at different ratios of biofilm surface to culture volume

Pseudomonas putida ATCC 11172 was grown in continuous culture with phenol as the only carbon and energy source; a culture practically without biofilm was compared with biofilm cultures of differing surface area/volume ratios. The biofilm did not significantly affect the maximal suspended cell concentration in the effluent, but it increased the maximal phenol reduction rate from 0.23 g/liter per h (without biofilm) to 0.72 g/liter per h at the highest biofilm level (5.5 cm2 of biofilm surface per ml of reactor volume). The increase in phenol reduction rate was linear up to the surface area/volume ratio of 1.4 cm2/ml. The continuous cultures with biofilms could tolerate a higher phenol concentration of the medium (3.0 g/liter) than the nonbiofilm system (2.5 g/liter). At higher dilution rates an intermediate product, 2-hydroxymuconic semialdehyde, accumulated in the culture. When the biomass of the effluent started to decrease, the concentration of 2-hydroxymuconic semialdehyde reached a peak value. We conclude that biofilms in continuous culture have the potential to enhance the aerobic degradation of aromatic compounds.     

Degradation of phenol by Pseudomonas putida ATCC 11172 in continuous culture at different ratios of biofilm surface to culture volume.

Pseudomonas putida ATCC 11172 was grown in continuous culture with phenol as the only carbon and energy source; a culture practically without biofilm was compared with biofilm cultures of differing surface area/volume ratios. The biofilm did not significantly affect the maximal suspended cell concentration in the effluent, but it increased the maximal phenol reduction rate from 0.23 g/liter per h (without biofilm) to 0.72 g/liter per h at the highest biofilm level (5.5 cm2 of biofilm surface per ml of reactor volume). The increase in phenol reduction rate was linear up to the surface area/volume ratio of 1.4 cm2/ml. The continuous cultures with biofilms could tolerate a higher phenol concentration of the medium (3.0 g/liter) than the nonbiofilm system (2.5 g/liter). At higher dilution rates an intermediate product, 2-hydroxymuconic semialdehyde, accumulated in the culture. When the biomass of the effluent started to decrease, the concentration of 2-hydroxymuconic semialdehyde reached a peak value. We conclude that biofilms in continuous culture have the potential to enhance the aerobic degradation of aromatic compounds.     

Treatment of chromium(VI) solutions in a pilot-scale bioreactor through a biofilm of Arthrobacter viscosus supported on GAC

The aim of this work is to evaluate the applicability of a biofilm to the removal of chromium in solution, at a pilot scale. The effect of the initial concentration of metal on the biosorption behavior of an Arthrobacter viscosus biofilm supported on granular activated carbon, in batch and column essays was also analyzed. Six isotherm equations have been tested in the present study. The best fit was obtained with the Freundlich model. It was observed that as the initial chromium concentration increases, the uptake increases too, but the removal percentage decreases, with values between 95.20% (C(0)=5mg/l) and 38.28% (C(0)=1000 mg/l). The batch adsorption studies were used to develop a pilot bioreactor able to remove chromium from aqueous solutions. Data obtained in a pilot-scale reactor showed an average removal percentage of 99.9%, during the first 30 days, for the initial concentration of 10mg/l and an average removal percentage of 72%, for the same period and for the initial concentration of 100mg/l. Uptake values of 11.35 mg/g and 14.55 mg/g were obtained, respectively, for the initial concentration of 10 and 100mg/l. The results obtained are very promising and encourage the utilization of this biofilm in environmental applications.     

Simultaneous Production and Recovery of Fumaric Acid from Immobilized Rhizopus oryzae with a Rotary Biofilm Contactor and an Adsorption Column

An integrated system of simultaneous fermentation-adsorption for the production and recovery of fumaric acid from glucose by Rhizopus oryzae was investigated. The system was constructed such that growing Rhizopus mycelia were self-immobilized on the plastic discs of a rotary biofilm contactor during the nitrogen-rich growth phase. During the nongrowth, production phase, the biofilm was alternately exposed to liquid medium and air upon rotation of the discs in the horizontal fermentation vessel. The product of fermentation, fumaric acid, was removed simultaneously and continuously by a coupled adsorption column, thereby moderating inhibition, enhancing the fermentation rate, and sustaining cell viability. Another beneficial effect of the removal of fumaric acid is release of hydroxyl ions from a polyvinyl pyridine adsorbent into the circulating fermentation broth. This moderates the decrease in pH that would otherwise occur. Polyvinyl pyridine and IRA-900 gave the highest loading for this type of fermentation. This fermentation system is capable of producing fumaric acid with an average yield of 85 g/liter from 100 g of glucose per liter within 20 h under repetitive fed-batch cycles. On a weight yield basis, 91% of the theoretical maximum was obtained with a productivity of 4.25 g/liter/h. This is in contrast to stirred-tank fermentation supplemented with calcium carbonate, whose average weight yield was 65% after 72 h with a productivity of 0.9 g/liter/h. The immobilized reactor was operated repetitively for 2 weeks without loss of biological activity.     

Electrochemical biofilm control: mechanism of action

Although it has been previously demonstrated that an electrical current can be used to control biofilm growth on metal surfaces, the literature results are conflicting and there is no accepted mechanism of action. One of the suggested mechanisms is the production of hydrogen peroxide (H(2)O(2)) on metal surfaces. However, there are literature studies in which H(2)O(2) could not be detected in the bulk solution. This is most likely because H(2)O(2) was produced at a low concentration near the surface and could not be detected in the bulk solution. The goals of this research were (1) to develop a well-controlled system to explain the mechanism of action of the bioelectrochemical effect on 316L stainless steel (SS) surfaces and (2) to test whether the produced H(2)O(2) can reduce cell growth on metal surfaces. It was found that H(2)O(2) was produced near 316L SS surfaces when a negative potential was applied. The H(2)O(2) concentration increased towards the surface, while the dissolved oxygen decreased when the SS surface was polarized to?-600 mV(Ag/AgCl). When polarized and non-polarized surfaces with identical Pseudomonas aeruginosa PAO1 biofilms were continuously fed with air-saturated growth medium, the polarized surfaces showed minimal biofilm growth while there was significant biofilm growth on the non-polarized surfaces. Although there was no detectable H(2)O(2) in the bulk solution, it was found that the surface concentration of H(2)O(2) was able to prevent biofilm growth.     

A perfusion system for antibody production by shear-sensitive hybridoma cells in a stirred reactor

Summary A shear-sensitive hybridoma cell line, incapable of growth or antibody production in spinner or shake flasks agitated at 40 rpm, was grown successfully in a perfusion propagation system consisting of a bioreactor (1.5 liter), stirred with a cell-lift impeller at 60 rpm, and a tangential flow filtration unit for removal of spent culture medium from the reactor. The culture was maintained over a 48 day period and cell numbers reached 1.8 × 10⁷ cells/ml. Maximal monoclonal antibody concentration was 800 ug/ml, indicating a productivity of 504 mg/day.     

Tissue plasminogen activator (t-PA) production by human fibroblasts using a bioreactor with t-PA adsorption column

Production of t-PA by human embryonic lung fibroblasts, IMR-90 cells, is regulated by negative feedback control. The increase in the concentration of the extracellular t-PA lead to a reduction of the production. Therefore, we investigated the application of t-PA adsorption column to ceramic bed reactor to promote t-PA production. Amberlite XAD-8 was selected out as an adsorbent, because it is autoclavable and can adsorb 32,000 IU of t-PA per g wet gel. The t-PA adsorption column was located in the medium recirculation line to the vessel. On the other hand, medium was recirculated between the ceramic bed reactor and the vessel using another flow line. The bioreactor system with the adsorption column was about 2.5 times higher with the resulting cumulative t-PA than that without the adsorption column.     

Enhanced Biotransformation of Fluoranthene by Intertidally Derived Cunninghamella elegans under Biofilm-Based and Niche-Mimicking Conditions

The aims of the investigation were to ascertain if surface attachment of Cunninghamella elegans and niche intertidal conditions provided in a bioreactor influenced biotransformation of fluoranthene by C. elegans. A newly designed polymethylmethacrylate (PMMA) conico-cylindrical flask (CCF) holding eight equidistantly spaced rectangular strips mounted radially on a circular disc allowed comparison of fluoranthene biotransformation between CCFs with a hydrophobic surface (PMMA-CCF) and a hydrophilic glass surface (GS-CCF) and a 500-ml Erlenmeyer flask (EF). Fluoranthene biotransformation was higher by 22-fold, biofilm growth was higher by 3-fold, and cytochrome P450 gene expression was higher by 2.1-fold when C. elegans was cultivated with 2% inoculum as biofilm culture in PMMA-CCF compared to planktonic culture in EF. Biotransformation was enhanced by 7-fold with 10% inoculum. The temporal pattern of biofilm progression based on three-channel fluorescence detection by confocal laser scanning microscopy demonstrated well-developed, stable biofilm with greater colocalization of fluoranthene within extracellular polymeric substances and filaments of the biofilm grown on PMMA in contrast to a glass surface. A bioreactor with discs rotating at 2 revolutions per day affording 6-hourly emersion and immersion mimicked the niche intertidal habitat of C. elegans and supported biofilm formation and transformation of fluoranthene. The amount of transformed metabolite was 3.5-fold, biofilm growth was 3-fold, and cytochrome P450 gene expression was 1.9-fold higher in the process mimicking the intertidal conditions than in a submerged process without disc rotation. In the CCF and reactor, where biofilm formation was comparatively greater, higher concentration of exopolysaccharides allowed increased mobilization of fluoranthene within the biofilm with consequential higher gene expression leading to enhanced volumetric productivity.     

Monitoring of a Vibrio natriegens and Desulfovibrio vulgaris marine aerobic biofilm on a stainless steel surface in a laboratory tubular flow system

N.D. BENBOUZID-ROLLET, M. CONTE, J. GUEZENNEC AND D. PRIEUR. 1991. In an aerobic bulk environment sulphate-reducing bacteria (SRB) can find suitable growth conditions on surfaces where other micro-organisms have developed an extensive biofilm. On metal surfaces they may induce or enhance corrosion. A laboratory tubular flow system was designed to study this phenomenon by creating a biofilm on stainless steel under dynamic conditions with Vibrio natriegens and Desulfovibrio vulgaris. The sulphate reducer colonized the surface, constituting approximately 5% of the total population. Its in situ growth rate, calculated by a simplified mathematical model, showed that the attached SRB multiplied at their settling locations. No significant difference with respect to corrosion enhancement was found in the electrochemical reactions of the metal betwen the control and the reactor, where D. vulgaris was present in the biofilm.     

Performance of a membrane bioreactor used for the treatment of wastewater contaminated with heavy metals

In this work the performance of a Membrane bioreactor (MBR) was assessed for the removal of 3-15 mg/l of copper, lead, nickel and zinc from wastewater. The average removal efficiencies accomplished by the MBR system were 80% for Cu(II), 98% for Pb(II), 50% for Ni(II) and 77% for Zn(II). The addition of 5 g/l vermiculite into the biological reactor enhanced metal removal to 88% for copper, 85% for zinc and 60% for nickel due to adsorption of metal ions on the mineral, while it reduced biomass inhibition and increased biomass growth. The metal ions remaining in soluble form penetrated into the permeate, while those attached to sludge flocs were effectively retained by the ultrafiltration membranes. The average heterotrophic biomass inhibition was 50%, while it reduced to 29% when lower metal concentrations were fed into the reactor in the presence of vermiculite. The respective autotrophic biomass inhibition was 70% and 36%. The presence of heavy metals and vermiculite in the mixed liquor adversely impacted on membrane fouling.     

Analysis of a continuous, aerobic, fixed-film bioreactor. I. Steady-state behavior

The analysis of a continuous, aerobic, fixed-film bioreactor is performed by simulating the behavior of penicillin production in a three-phase fluidized bed. Rigorous mathematical models are developed for a fluidized-bed fermentor in which bioparticles are fluidized by the liquid medium and air. The steady-state performance of the fluidized-bed reactor is appraised in terms of penicillin productivity and outlet concentration by considering the two extremes in contacting patterns, complete back-mix and plug flow, in the absence of a growing biofilm. The results show that the complete back-mix contacting pattern is preferred over that of plug flow due to the nature of the penicillin kinetic relationships. It is also shown that for the dual-nutrient (glucose and oxygen) penicillin reaction system the optimum biofilm thickness does not equal the penetration depth of a limiting nutrient, but depends upon the total reactor configuration.     

A new continuous biofilm bioreactor for immobilized oil-degrading filamentous fungi

A new type of horizontal biofilm bioreactor for continuous bioconversion of emulsified oily substrate by immobilized growing biofilm of filamentous fungi was designed, constructed, and feasibility tested. The new reactor design provides "self"-immobilization of homogenized mycelium leading to even biofilm development. This was accomplished by using stainless steel screens of optimal mesh, mounted in parallel and stretching outward from a main rotating axis of a biological rotating contractor. Each screen was equipped with a pair of stainless steel blades mounted on supports allowing for continuous biofilm "shaving" beyond a predetermined thickness, thus retaining freshly growing active biofilm surface. The feasibility of the new bioreactor was demonstrated by decalactone production from emulsified castor oil by immobilized filamentous fungi (Tyromyces sambuceus). The combination of oriented metal screens and moving blades was found to be highly effective for a model system in maintaining stable substrate emulsion in the reactor in either batchwise or continuous processing, as well as maintaining biofilm thickness with continuous removal of excess growing hyphae. (c) 1996 John Wiley & Sons, Inc.     

Propagation of Rhizopus javanicus Biosorbent

After propagation of Rhizopus javanicus in defined media containing glucose, urea, and mineral salts in deionized distilled water, the ability of the nonliving biomass to sequester cupric ion was assayed. Growth, uptake capacity (saturation uptake at >1 mM Cu²⁺ concentration in solution), and biosorptive yield (biomass concentration × uptake capacity) were increased by augmentation of the growth medium with mineral salts once growth was under way. In the stationary phase, the uptake capacity of mycelia, which were normally a poor biosorbent, was improved within 4 h of trace metal addition to the growth medium. Growth of the culture was inhibited by excessive concentrations (0.04 to 40 μM) of metals in the medium in the following order: Cu > Co ≥ Ni > Mn > Mo; zinc was not inhibitory at 40 μM, and chromium was stimulatory at 0.53 μM but slightly inhibitory at higher levels. Iron and potassium phosphate stimulated growth at levels of 0.53 and 40 mM, respectively. When R. javanicus was propagated in a medium with a high salt concentration, exponential growth (0.23 h⁻¹) to a biomass concentration of >3 g/liter and a biosorptive yield of >500 μmol/liter was achieved. It is evident that the powerful biosorbent characteristics of Rhizopus biomass led to depletion of available trace minerals in suspension culture, which in turn limited growth.     

Hydrophobic interactions within biofilms of nitrifying and denitrifying bacteria in biofilters

Hydrophobicity of the solid surface and microbial cell surface is important factor for the development of biofilms applied in bioengineering systems. An adsorption of phenanthrene was used for analysis of the hydrophobicity of support fibers and bacterial cell surfaces within the biofilter of wastewater. The adsorption of phenanthrene was measured by synchronous fluorescence spectrometry. Cell surface hydrophobicity does not depend on the fixation procedure, pH of microbial suspension, and has no clear correlation with an adherence of the cells to hexadecane droplets. Notwithstanding high hydrophobicity of bacterial cells, the hydrophobicity of intact biofilm is determined by the hydrophobicity of the support fibers. New indexes were proposed to evaluate the reactor performance related with hydrophobic interactions within the biofilm. These indexes showed that significant share of hydrophobic sites within the nitrifying biofilm is protected from the hydrophobic interactions between the cells and environment.     

Zinc deprivation of methanol fed anaerobic granular sludge bioreactors

The effect of omitting zinc from the influent of mesophilic (30 degrees C) methanol fed upflow anaerobic sludge bed (UASB) reactors, and latter zinc supplementation to the influent to counteract the deprivation, was investigated by coupling the UASB reactor performance to the microbial ecology of the bioreactor sludge. Limitation of the specific methanogenic activity (SMA) on methanol due to the absence of zinc from the influent developed after 137 days of operation. At that day, the SMA in medium with a complete trace metal solution except Zn was 3.4 g CH4-COD g VSS(-1) day(-1), compared to 4.2 g CH4-COD g VSS(-1) day(-1) in a medium with a complete (including zinc) trace metal solution. The methanol removal capacity during these 137 days was 99% and no volatile fatty acids accumulated. Two UASB reactors, inoculated with the zinc-deprived sludge, were operated to study restoration of the zinc limitation by zinc supplementation to the bioreactor influent. In a first reactor, no changes to the operational conditions were made. This resulted in methanol accumulation in the reactor effluent after 12 days of operation, which subsequently induced acetogenic activity 5 days after the methanol accumulation started. Methanogenesis could not be recovered by the continuous addition of 0.5 microM ZnCl2 to the reactor for 13 days. In the second reactor, 0.5 microM ZnCl2 was added from its start-up. Although the reactor stayed 10 days longer methanogenically than the reactor operated without zinc, methanol accumulation was observed in this reactor (up to 1.1 g COD-MeOH L(-1)) as well. This study shows that zinc limitation can induce failure of methanol fed UASB reactors due to acidification, which cannot be restored by resuming the continuous supply of the deprived metal.