novel membrane bioreactor with gas/liquid two-phase flow for high-performance degradation of phenol

Novel Membrane Bioreactor with Gas/Liquid Two-Phase Flow forHigh-Performance Degradation of Phenol

David Leonard, Muriel Mercier-Bonin, Nicholas D. Lindley, andChristine Lafforgue*

Laboratoire de Biotechnologie et Bioprocedes, UMR INSA CNRS 5504, Lab Ass INRA, Centre de BioingenierieGilbert Durand, Institut National des Sciences Appliquees, Complexe Scientifique de Rangueil,31077 Toulouse Cedex 4, France

The use of a membrane bioreactor with cell retention to achieve high biomassconcentrations has been examined for phenol degradation by the bacteria Alcaligeneseutrophus. This process is particularly interesting for toxic substrates as the hydraulicdilution rate and the growth rate are independently controlled. In the case of atransitory excess of phenol, this potentially toxic situation can be overcome bymodifying the substrate concentration or the dilution rate without any loss of cells.The injection of a gas phase at the filter inlet increased both the permeate flow rate(by a factor of 1.75) and the oxygen transfer capacity (by a factor of 1.5). This hasenabled the cell concentration to reach a maximal value of 60 g L-1 with a hydraulicdilution rate of 0.5 h-1 and a phenol feed concentration of 8 g L-1. The volumetricproductivity of this process corresponds to a phenol degradation rate approaching 100kg m-3 day-1. The on-line measurement of the characteristic yellow color of2-hydroxymuconate semialdehyde, a metabolic intermediate of the phenol degradationpathway, in the permeate provides an interesting basis for process control of phenolsupply into the reactor since the color intensity correlates directly to the specific rateof phenol degradation.

IntroductionAmong processes developed to increase both the bio-

mass concentration and the dilution rate (centrifugation,flocculation, and decantation), membrane bioreactorshave so far provided the best results. The membranebioreactor combines a continuous fermentor and a tan-gential filter enabling cell recycle and separation of cellsfrom liquid media. As a consequence, very high biomassconcentrations have been reached and important biocon-version yields obtained. The fermentation broth can beconsidered to be the same throughout the reactor becauseof the important liquid flow inside the tangential filter,and no limitation (i.e., anoxia for aerobic cultures) occursduring the separation of cells and liquid. Moreover, allcells are recycled (no cell loss as for centrifugation) andthe total cell concentration can reach high levels: 300 gL-1 dry weight for yeast (Mota et al., 1986) and more than100 g L-1 dry weight for bacteria (Ferras et al., 1986;Blanc and Goma, 1987). However, the potentiality of thisprocess is limited by the rapid decline in permeate fluxdue to membrane fouling. Various technical solutions,based on generation of turbulence inside the filters, havebeen examined to reduce fouling due to the deposition ofparticles on the membrane surface (Finnigan and Howell,1990; Arroyo and Fonade, 1993; Maranges and Fonade,1997).

Recent work on the use of gas/liquid two-phase flow toreduce membrane fouling during cross-flow filtration hassuccessfully demonstrated the potential of this kind ofunstationarity on the permeate flow rate. With bentonite

and yeast suspensions, a 3-fold increase in permeate flowlevel has been reached with a slug flow (correspondingto a ratio of gas flow to liquid flow of about 100%),compared with classical steady flow (Mercier et al., 1997).Applied to a membrane bioreactor for alcoholic fermenta-tion, this process enabled a 2-fold gain in permeate flowwithout any cell damage (Mercier et al., 1997). Moreover,a comparative evaluation of the economic cost of such aprocess demonstrated a significant energy saving (about50%) for the two-phase flow cross-flow filtration process.This principle could be a useful way to improve theperformance of microbial cultures in membrane bioreac-tors, especially when high air input is essential to avoidanoxic conditions developing at high cell densities.

Cell recycling has been used in membrane reactors tointensify the breakdown of wastewaters with promisingresults though less work has been directed toward thebiodegradation of toxic effluents in such systems. Phe-nolic compounds are frequently detected in industrialeffluents from chemical plants, oil refineries, and agro-chemistry plants (herbicides, pesticides), and althoughmany naturally occurring soil bacteria are capable ofmineralizing aromatic compounds, they persist in theenvironment. Among the naturally occurring phenol-degraders, Alcaligenes eutrophus offers the advantage ofpossessing chromosome-encoded pathways (Johnson andStanier, 1971) and, hence, stable degradative potential.Phenol may be used as sole source of carbon and energyunder aerobic conditions (Hughes and Bayly, 1983),though the presence of alternative substrates (particu-larly organic acids) leads to delayed degradation of thearomatic compound (Ampe et al., 1998).* Corresponding author.

680 Biotechnol. Prog. 1998, 14, 680−688

S8756-7938(98)00069-1 CCC: $15.00 © 1998 American Chemical Society and American Institute of Chemical EngineersPublished on Web 09/02/1998

Physiological investigations have demonstrated thatthe capacity of the degradative pathway to transformphenol to organic acids which are then metabolized viacentral metabolism limits the specific rates of phenolmineralization. Under carbon-limited growth in continu-ous culture this limitation can be visualized by theaccumulation of 2-hydroxymuconate semialdehyde (2-hms) as a function of the rate of phenol degradation(Leonard and Lindley, 1998), suggesting that the meta-bolic bottleneck is situated at the 2-hms dehydrogenasereaction immediately downstream of the two oxygen-consuming reactions (phenol hydroxylase and catecholdioxygenase) leading to the opening of the aromatic ring.Wastewater treatment strategies can be compared tocontinuous culture systems since the concentration ofpollutant in the outflow must be maintained as low as ispossible. Rates of phenol degradation were shown to bedirectly proportional to growth rate, and process perfor-mance can therefore be seen to be dependent upon theamount of active biomass maintained within the reactor.However, the microbial population is sensitive to in-creases in phenol input concentration with inhibition ofsubstrate removal and growth occurring at phenol con-centrations as low as 50 mg L-1 (Hill and Robinson,1975).

In the membrane bioreactor apparatus high cell con-centrations can be obtained due to total cell retention.Moreover, the use of cell recycling enables specific growthrates, which are controlled by bleeding directly from thefermentor, to be maintained independently of hydraulicdilution rates corresponding to the permeate outlet.Therefore, higher volumetric throughput can be obtainedin comparison with classical continuous culture setups.Of course, biomass recycling has been traditionallyemployed within the activated sludge system, thoughbiomass retention is only partial in such setups due tothe poor efficiency of settling tanks.

To a great extent the limit of this type of reactor isdependent upon the oxygen requirement, e.g., Bouillotet al. (1990) reported a dry weight bacterial concentrationof 10 g L-1 for Pseudomonas fluorescens grown on organicacids at a hydraulic dilution rate level of 0.9 h-1, whilePierrot et al. (1986) reached 20 g L-1 at a lower hydraulicdilution rate of 0.5 h-1. Considerably higher biomassconcentrations can be reached for anaerobic bacteria, e.g.,Ferras et al. (1986) reached cell concentrations of 125 gL-1 at a dilution rate of 0.33 h-1 in solvent producingcultures of Clostridium acetobutylicum. As cell densityis increased within the bioreactor, two major difficultiesare encountered which are susceptible to diminish theperformance of the system. First, the oxygen transfercapacity will ultimately limit the capacity of the microbialpopulation to support fully aerobic growth. This isparticularly true for substrates such as phenol in whichthe direct incorporation of molecular is involved in thecatabolic pathways in addition to the respiratory chainrequirements. Aeration with pure oxygen is possible buteconomically irrealistic for the treatment of pollutedeffluents for which costs must be maintained low.

Second, the decline in permeate flow as a function ofboth the biomass concentration and the duration of theculture leads to a diminished capacity to maintain anadequate hydraulic dilution rate, thereby reducing thevolumetric productivity and in some cases generatingnutritional limitations.

In this study, the potential of a membrane bioreactorwith enhanced hydraulic performance due to the injectionof air at the filter inlet to provoke slug flow behavior andreduce membrane fouling has been examined. Since such

a system is also reputed to improve oxygen transfercapacity, the performance of cultures of A. eutrophus havebeen assessed for their phenol degrading capacity withinthis high cell density system and compared with datafrom alternative systems from the literature.

Materials and Methods

Bacterial Strain and Growth Conditions. Thestrain used throughout the present study was A. eutro-phus 335 (ATCC 17697) obtained from LMG (Brussels,Belgium).

The culture medium was obtained by on-line dilutionof the concentrated solutions of mineral salts medium,the potassium phosphate solution, and the phenol solu-tion with filtered tap water, each solution being filtersterilized (filter pore size of 0.2 µm). The mineral saltsmedium was that described by Ampe and Lindley (1995),except that the solutions were prepared as 10-foldconcentrated solutions. The stock solution of potassiumphosphate (1 M, pH 7) was added to the mineral saltsmedium to obtain a final concentration of 40 mM. Thephenol solution (50 g L-1) was diluted to obtain therequired substrate concentration with mineral mediumvia a variable-speed peristaltic pump.

The bioreactor was inoculated with a 10% (v/v) late-exponential-phase shake flask culture grown on phenol(5 mM). Growth was under conditions of constanttemperature (30 °C) and pH (automatically maintainedat pH 7.0 by addition of 3 M KOH).

Process Apparatus. The fermentation system (Fig-ure 1) was a continuous stirred tank reactor of 2-Lvolume coupled with an ultrafiltration unit with axisym-metric zircon-coated graphite membranes (6 mm i.d.,1200 mm length) having an average porosity of 50 kDaand an effective filtration surface of 0.0226 m2. To avoidmembrane aging and cleaning introducing possible ex-perimental artifacts, new membranes, calibrated for theirpermeability with distilled water, were used for eachfermentation. Fermentation broth was pumped throughthe filtration membrane by a Mohno type pump (NetzschFrance). The total working volume of the setup (includ-ing the fermentor, the pump, the filtration unit, and thepipes) was 2.5 L. The entire unit was steam sterilizedin situ. The pressure values were measured with Bour-don-type manometers, and the transmembrane pressure(TMP) value taken as the average of the upstream anddownstream pressure values, was 1 bar in all experi-ments. The liquid flow rate value QL, controlled by therotational velocity of the pump, was 0.3 m3 h-1 in allexperiments.

For the culture in steady conditions, air was suppliedinto the reactor at a flow rate of 0.15 m3 h-1, correspond-ing to 1 volume of air, measured at atmospheric pressure,added per unit of liquid volume per minute (VVM). Intwo-phase flow experiments, the air was injected only atthe filter inlet at a gas flow rate, at atmospheric pressure,of 0.63 m3 h-1. No air was added directly into the stirredtank reactor. The gas flow rate was measured at thereactor outflow, at atmospheric pressure, using a flow-meter (Flonic-Schlumberger). The gas flow rate insidethe filter, QG, was obtained by correcting the measuredvalue with the average pressure inside the tube. Theimpeller speed was varied from 300 to 900 rpm (revolu-tions per minute) for steady conditions, to maintain adissolved oxygen concentration in the fermentor at valuesin excess of 50% of saturation. Under two-phase flowconditions, the impeller rotational speed was maintainedat 200 rpm throughout the entire experiment.

Biotechnol. Prog., 1998, Vol. 14, No. 5 681

Measurements. Biomass concentration was esti-mated by both absorbency measurement at a wavelengthof 600 nm and measured by cell dry weight determinationafter filtration (0.2 µm pore size filter) and drying toconstant weight under partial vacuum (24 h, 200 mmHg,60 °C).

The phenol concentration, in both the feed flow andthe permeate, was determined by HPLC using a Bio-RadAminex HPX-87H column (300 × 7.8 mm) and thefollowing operating conditions: temperature, 65 °C;mobile phase, 5 mM H2SO4 + 7% (v/v) CH3CN; flow rate,0.8 mL/min. Detection was made at 210 nm with avariable-wavelength UV detector, and quantification wasby integration of peak area.

Oxygen uptake and CO2 production were analyzed bychromatographic analysis of inflowing and outflowing gascomposition using a two-column separation technique(Porapack Q followed by a 5-Å molecular sieve column)with catharometer detection.

Color Analysis. The accumulation of 2-hms duringA. eutrophus culture on phenol corresponds with the

appearance of a yellow color (ε375 nm ) 33 000 cm-1). Theon-line determination of 2-hms concentration in thepermeate was achieved according to the method ofSanterre et al. (1994) involving a reflectance measure-ment with a 10° observer under an artificial illuminant(Visual Color Simulator) with a spectrophotometer (Data-color ASC ICS, type CS3). The b-parameter whichdefines the position of a sample on the blue/yellow axisin the CIE Lab Color System (Hunter and Harold, 1987)was used to quantify the 2-hms concentration, C, whichis correlated to b by the following relation:

Results and DiscussionChoice of Experimental Conditions. Previous

studies have indicated that slug flow through the tubularmembrane best increases flux in fermentation experi-ments (Mercier et al., 1997). The ratio QG/QL betweengas and liquid flow rates determines the type of two-

Figure 1. Schematic representation of the experimental setup: 1, fermentor; 2, recirculation pump; 3, ultrafiltration membrane; 4,manometer; 5, gas flowmeter; 6, distilled water with phosphate buffer 40 mM; 7, salt medium 10-fold concentrated; 8, salt + phosphatefeed tank; 9, phenol feed tank (50 g L-1) 10, peristaltic pump; 11, septum for sampling; 12, adjustement valve; 13, reflectancespectrophotometer and dedicated microcomputer; 14, monitoring microcomputer; 15, controller (stirrer speed, pH, temperature, pO2);16, gauge controller.

0.022b - 0.11 ) C (µg L-1)

682 Biotechnol. Prog., 1998, Vol. 14, No. 5

phase flow structure existing in the hollow tubularmembrane. For the membrane diameter used in thisstudy, the slug flow structure is achieved for a ratio QG/QL between 50% and 200% (Mercier et al., 1996). Inprevious studies (Mercier et al., 1997), it has been pointedout that complex hydrodynamic phenomena are observedin a slug flow. A pressure drop occurs when a bubblemoves through the tube that may modify the cakecompacity. Turbulence may be generated in the wakeof the bubbles, improving transverse mixing in the liquidphase. A thin liquid film always remains at the surfaceof the membrane, and these liquid particles usually movein the opposite direction with regard to the main flow,resulting in a modification of the wall shear stress. Allof these phenomena and especially the important varia-tions in wall shear stress and in transmembrane pres-sure, added to the turbulence in the liquid slug betweentwo bubbles, can be expected to improve the filtrationperformances by reducing the fouling due to a particledeposit on the membrane surface.

In previous experiments relating to the alcoholicfermentation (Mercier et al., 1998), it has been observedthat an important part of the fouling was due to theadsorption of medium components onto the filter. Toevaluate the fouling capacity of the fresh medium, duenotably to the chemical affinity of the phenol toward thecarbon filter support, the bioreactor was filled with freshculture medium (phenol concentration of 0.4 g L-1) whichwas pumped through the system with complete recyclingand for 3 h. No noticeable difference in the phenolconcentration within the bioreactor was measured, indi-cating that the phenol does not adsorb selectively ontothe membrane. In a second control experiment, thebioreactor was again filled with fresh culture mediumand maximum permeate flux was 173 L h-1 m-2 prior toinoculation. Following inoculation to achieve an initialbacterial cell concentration of 0.02 g L-1, the reactor wasoperated in batch total recycle mode for a 30-h perioduntil all phenol had been consumed. A bacterial concen-tration of approximately 0.5 g L-1 was achieved andtransitory production of 2-hms was observed, but thepermeate flux value (166 L h-1) remained close to thatof the fresh medium. It may thus be concluded that nosignificant modification of the filter permeability due toadsorption phenomena of phenol, biomass, 2-hms, orother fermentation products occurred.

In the following study two experimental setups havebeen examined. First, a classical membrane bioreactorhas been used to examine the feasibility of improving theperformance of phenol degradation using biomass recy-cling to intensify the process. Second, a gas/liquid two-phase flow has been applied to reduce the fouling of thefilter, thereby increasing the volumetric dilution rate toimprove the performances in terms of productivity.Furthermore, the feasibility of a process control systembased upon the on-line estimation of the permeate colorhas been assessed.

In the first setup, aeration was provided by a perfo-rated air inlet pipe immersed into the liquid phase of thestirred tank reactor. The phenol concentration wasgradually increased so as to maintain the residual phenolconcentration in the permeate equal to zero and aconstant growth rate. In the two-phase flow setupaeration was achieved by injection of air directly into thefilter inlet. No aeration was made into the stirred tank.The values of liquid and gas flow rates were chosenaccording to the slug flow requirements as previouslypublished (Mercier et al., 1997). In the second setup, thephenol concentration in feed flow was modified according

to the measured yellow color (2-hms) in the permeate,indicative of the specific phenol degrading activity of thebacteria (Leonard and Lindley, 1998). The feed profilewas therefore progressively increased so as to avoid anydecrease in yellow color throughout the cultures therebymaintaining high specific biological activity and avoidingthe transient increase in phenol sometimes observed bystepwise changes in feed concentration.

Cell Cultivation and Phenol Degradation. Fer-mentation under Steady Conditions. Under classicalmembrane bioreactor flow conditions with full cell re-cycle, the culture lasted 110 h (Figure 2) and was stoppedbecause oxygen transfer became limiting and furtherincrease of impeller speed and/or aeration flow rate wasnot possible. The final cell concentration was 25 g L-1,and the total mass of phenol degraded during thefermentation was 100 g (Table 1). To overcome thespecific rates decrease (Figure 3) due to the phenollimitation when the biomass concentration increases, thephenol feed concentration was increased. The stepwiseincreases of the phenol concentration provoked transitoryexcesses of phenol at fermentation times of 20, 65, and90 h. The toxic effect of the phenol induced a fall in thespecific rates of both growth and phenol consumption(Figure 3). The dissolved oxygen concentration wasmaintained above 50% saturation by progressively in-creasing the impeller rotational during the fermentationin parallel with the phenol feed concentration but couldnot be maintained at the desired level for unrestrainedgrowth at phenol concentrations in excess of 2.5 g L-1.As can be seen, the sudden increase in phenol inputprovoked transitory inhibition with an immediate in-crease in dissolved oxygen saturation but the washoutof the liquid phase without any loss of cell biomassavoided the collapse of the system. Furthermore, thehigh cell density allows the effects of such transientaccumulations of inhibitory phenol concentrations to beattenuated so long as the system is not operating closeto the maximum capacity of the cells. As a consequence,the perturbations provoked by transient accumulationsof phenol were short-lived and did not have long-termrepercussions on cell activity. The diminished specificrates when phenol became inhibitory lasted for shortperiods and were followed by subsequent increases inboth specific growth and phenol degradation rates once

Figure 2. Variations in cell concentration (b) and phenol feed(4) and permeate (0) concentrations during growth of A.eutrophus on phenol in a classical membrane bioreactor withfull biomass recycling.


noninhibitory phenol concentrations had been restored(Figure 3). It can also be observed that when the increasein phenol concentration was less abrupt an immediateincrease in the specific rates occurred, indicating thatbacterial activity was substrate-limited. This no doubtcontributes to the robustness of such a process butindicates that the full potential of the cells was not beingexploited. In view of these observations, a more efficientprocess could be achieved by coupling the phenol feed rateto the degradative capacity of the cells by a progressiveincrease in the phenol inflow concentration up until theeffective transfer capacity of the reactor is attained.

Fermentation with Two-Phase Flow Conditionsand Process Control by On-Line Color Measure-ments. This setup, with air injection directly into thefilter and a control of the phenol concentration in the feedflow as a function of the yellow color measured on-linein the permeate was run over a shorter time period (90h) than the previous fermentation but enabled a consid-erably higher amount of phenol to be degraded (240 g),with a corresponding increase in the final biomassconcentration (Figure 4) which reached 56 g L-1 (Table1). The use of on-line color intensity measurements topilot the phenol feed concentration enabled a moreefficient coupling of bacterial capacity throughout thisfermentation (Figure 5), explaining to some extent themore efficient degradation of phenol with respect to theduration of the fermentation. Effectively, the transientperiods due to phenol accumulation followed by a de-crease of the cell activity resulting from its toxicity wereavoided.

Compared with the first experiment, the improvedperformance cannot, however, be attributed uniquely tothis more sensitive control of feed concentration input.In both setups, the accumulation of biomass led todecreased specific rates of growth and phenol degradation(Figure 6), and the tight coupling between these twospecific rates was observed in both fermentations.

The high enhancement of the phenol degradation rateΦ, corresponding to an increased volumetric productivity,could have resulted from the increased active biomassconcentration X maintaining a constant specific consump-tion rate qs and/or from the improvement of the hydraulicdilution rate while maintaining the consumed phenolconcentration, (SA - S), constant:

In this experiment, biomass concentrations reached 50g L-1 without problems in maintaining the dissolvedoxygen concentration at an acceptable level, probably dueto a significant enhancement in oxygen transfer capacityin the modified setup (see below). Furthermore, the

Table 1. Balance Sheet at the End of Each Fermentation of A. Eutrophus Grown on Phenol with Full Cell Recycle inClassical Steady Conditions and in Two-Phase Flow Conditions

exptl setupSA maxmM

D(h-1)

duration(h)

XT(g L-1)

amt ofphenol

degraded (g)RSX

(g g-1)

globalproductivity

(kg m-3 day-1)

maximalproductivity

(kg m-3 day-1)

classical 55 0.4 110 25 100 0.62 8.7 50two-phase flow 85 0.5 90 56 240 0.6 26 96

Figure 3. Specific rates of growth (O) and phenol degradation(b) and the dissolved oxygen concentration as % of saturation(]) during growth of A. eutrophus on phenol in a classicalmembrane bioreactor with full biomass recycling.

Figure 4. Variations in cell concentration (b) and phenol feed(4) and permeate (0) concentrations during growth of A.eutrophus on phenol in a two-phase flow membrane bioreactorwith full biomass recycle and feed control by on-line colormeasurement.

Figure 5. Specific rates of growth (0) and phenol degradation(9), dissolved oxygen concentration as % of saturation (]), andcolor intensity (4) during growth of A. eutrophus on phenol ina two-phase flow membrane bioreactor with full biomass recycleand feed control by on-line color measurement.

Φ ) Xqs ) D(SA - S)


hydraulic dilution rate could be maintained at a highlevel despite the increased biomass concentration.

Improved Filtration Characteristics. As in theconventional single-phase cross-flow filtration system, theinitial decline in flux can still be observed for the two-phase flow filtration system (Figure 7), but it should benoticed that once this initial period has ended the fluxwas maintained at a significantly higher level (70 L h-1

m-2 against 45 L h-1 m-2) despite the considerably higheramount of biomass within the two-phase flow setup. Atbiomass levels exceeding 50 g L-1, other phenomena nodoubt contributed to the decrease in permeate flowprobably linked to the bulk consistency and the rheo-logical characteristics of the liquid.

To quantify the influence of the unsteady two-phaseflow, the results have been expressed in terms of the ratioKj between the flux obtained at the same biomassconcentration, in unsteady and steady conditions (Figure8). The unsteady setup systematically enhanced thepermeate flux: this effect becoming more pronounced asthe total cell concentration increased. As suspended

particulate matter increased in the broth, the fouling dueto particle deposit would become preponderant. Thegenerated unsteady conditions was used to reduce theparticle deposit on the membrane surface, and it istherefore not surprising that the reduction of this foulinglayer strongly improved permeate flux. A 1.75 improve-ment of permeate flow was reached which remainedconstant up until the maximum biomass concentration(25 g L-1) achieved in steady conditions.

By incorporating a biomass bleed to limit the cellconcentration to a value of 45 g L-1, a hydraulic dilutionrate could be maintained at a value of 0.65 h-1 andtransform the system to a continuous process. Further-more, the efficient control of the phenol load, madepossible by the on-line measurement of color intensity,would enable some further optimization of the substrateconcentration to be attained, further improving thealready high volumetric productivity of this system.

Evaluation of Oxygen Transfer Capacity of theBioreactor. Since the liquid film resistance dominates,oxygen transfer has been assumed to take place in theliquid phase. Moreover oxygen consumption was relatedto the biomass activity located in the liquid phase. Inthe following discussion all of the oxygen mass transferrates are referred to the liquid volume.

Oxygen Transfer under Steady Conditions: De-termination of the Volumetric Transfer Rate. Aresidence time distribution trial realized previously withthe same apparatus (Mota et al., 1986) has shown thatthis bioreactor can be considered to be perfectly mixed.Under the experimental conditions chosen in this studyfor the cell culture, corresponding to the impeller rota-tional speed of 900 rpm and an aeration rate of 1 VVM,the gas hold-up localized uniquely in the fermentor, wasaround 5%. The average oxygen transfer rate value withpure water, determined according to the “gassing-out”method (Rainer, 1990), and taking into account theoxygen probe response characteristics, was equal to 190h-1.

Additional experiments were run using fresh culturemedium under the same operating conditions to take intoaccount the effect of the medium components on thetransfer (notably salts since outflowing phenol wasalways extremely low). The obtained transfer rate valuewas 5% higher than that obtained with pure water. Thisdifference, which is in the order of magnitude of theexperimental error, could be attributed to the salt effect(K2HPO4) corresponding both to an increase in theinterfacial area and to a decrease in oxygen solubility(Schumpe, 1985).

Under culture conditions, the gas hold-up variationwas not noticeable, as it remained in the order of

Figure 6. Specific rates of growth (open symbols) and phenoldegradation (closed symbols) as a function of the accumulatedbiomass concentrations during growth of A. eutrophus on phenolwith full cell recycle using a classical membrane bioreactor (O,b) or a two-phase flow membrane bioreactor (0, 9).

Figure 7. Permeate fluxes during growth of A. eutrophus onphenol in a classical membrane bioreactor (0) or a two-phaseunsteady slug flow bioreactor (9) as a function of the total cellconcentration.

Figure 8. Influence of the biomass concentrations on the ratio(Kj) of permeate flux values in slug flow and classical operatingconditions.


magnitude of the volume measurement. As the bioreac-tor is assumed to be perfectly mixed and as the oxygentransfer takes place mainly in the liquid phase, thefollowing equation at equilibrium translates that the totaltransferred oxygen is equal to the cell uptake for cellgrowth:

The value of the overall oxygen volumetric transfer ratecan be calculated from experimental data at a fermenta-tion time of 50 h:

The calculated volumetric transfer rate of oxygen is then

This value is almost 2-fold higher than that estimatedwith pure water, but this enhancement, frequentlyobserved when using fermentation media (Merchuk,1977), has been attributed to an alternative route ofoxygen uptake by cells situated within the relativestagnant liquid region adjacent to gas bubbles (Sobotkaet al., 1981). At identical gas hold-up, this oxygen uptakeroute is mainly significant for small gas bubbles havinga corresponding high specific exchange area but remainsnegligible for large bubbles (Votruba et al., 1981).

Oxygen transfer under two-phase flow condi-tions: evaluation of the volumetric transfer rate.Under two-phase flow conditions, the bioreactor has beendivided into two parts according to the oxygen transfercharacteristics: (1) the slug flow in the membranefiltration unit plus the return pipe leading to the stirredtank and (2) the perfectly mixed stirred tank itself. Thetransfer equations in each part of the bioreactor arededuced from the balance of the oxygen transfer rate andmicrobial uptake. In the reactor, oxygen transfer wasdue to the jet created by the two-phase flow return insidethe perfectly mixed reactor. In the filtration unit, as thebubbles were very large (Mercier et al., 1997), the oxygentransfer in the gas/liquid film interface was assumed totake place mainly in the aerated slugs as postulated byVoturba et al. (1981), which were considered to beperfectly mixed. In both parts, the volumetric transferrates are related to the liquid phase; the hold-up wasarround 10% of the total volume in the fermentor partand approximatively 50% of the total volume in thefiltration unit as the gas volume was compressed (Mercieret al., 1997).

(i) For the stirred tank reactor at atmospheric pressure(corresponding liquid volume of 0.5 L):

(ii) For the slug flow tubular filtration module andreturn pipe, at a 2 bar mean pressure:

where VR is the reactor volume corresponding to theliquid volume where the cells are growing, Ce is thedissolved oxygen concentration at the outlet of the stirredtank, Cs is the dissolved oxygen concentration at the inletof the stirred tank, q is the liquid flow rate crossing thefiltration unit (0.3 m3 h-1), CR

* and CTF* are the dissolved

oxygen concentration at saturation of the liquid phasein the reactor part and in the filter part, respectively (0.22and 0.44 mM, respectively), and (KLa)R and (KLa)TF arethe overall volumetric transfer rates for oxygen (h-1) inthe reactor part and in the filter part, respectively.

For the stirred tank reactor part the value of thevolumetric rate of oxygen transfer ((KLa)R) has beendeduced from the study of Rainer (1992), who assumedthat with a jet, in similar working conditions (liquid flowand gas flow values and reactor geometry), a KLa meanvalue around 100 h-1 was attained with pure water.Taking into account the 2-fold enhancement factor dueto the biological medium, established in the study ofoxygen transfer in steady conditions (see above), avolumetric rate of oxygen transfer in cell culture condi-tions of 200 h-1 may be assumed. The value of thedissolved oxygen concentration at the return pipe, Cs, canthen be evaluated using experimental data. At a cellculture age of 50 h, the measured experimental dataswere

Resolving eq 2 gives the value of Cs ) 0.27 mM.For the filtration unit and return pipe, it has been

assumed that, at initial time (t ) 0), the dissolved oxygenconcentration in the liquid phase is equal to zero. Theintegration of eq 3 gives the time value of the dissolvedoxygen concentration C(t):

This equation was solved between t0 and (t0 + ∆t), where∆t is the time necessary for a new slug to pass throughthe entire length of the membrane filtration module andreturn pipe. According to the experimental conditionsand data, we have

The deduced value of the overall volumetric rate ofoxygen transfer referred to the liquid volume in this partof the bioreactor is therefore

This high level could be attributed to a high oxygentransfer potential, probably as a result of the complexhydrodynamic conditions generated in the slug flow.However, these values of transfer rates were deducedfrom the estimated values of oxygen concentration in thedifferent parts of the setup. These results should beverified by appropriate measurements of dissolved oxy-

KLa(CR* - C) ) qO2

X (1)

CR* ) 0.22 mM at a total pressure of 1 bar

X ) 12.5 g L-1

qO2) 4.5 mmol g-1 h-1

C ) 0.07 mM

KLa ) 375 h-1

(KLa)R(CR* - Ce)VR + q(CS - Ce) ) qO2

XVR (2)

ddt

C(t) ) (KLa)TF[CTF* - C(t)] - qO2

X (3)

X ) 20 g L-1

Ce ) 0.18 mM

qO2) 3 mmol g-1 h-1

C(t) ) (CTF* -

qO2X

(KLa)TF)(1 - e-(KLa)TFt) (4)

∆t ) 2.4 s

C(t0) ) Ce

C(t0 + ∆t) ) Cs

(KLa)TF ) 950 h-1


gen concentration in further experiments exclusivelydevoted to the study of oxygen transfer in such abioreactor.

Evaluation of the Maximal Attainable BiomassConcentration without Oxygen Limitation. Understeady flow conditions, the maximal biomass concentra-tion reached without oxygen limitation can be evaluatedassuming that all the transferred oxygen in the fermentoris immediately consumed for cell growth:

Considering a specific oxygen uptake close to 3 mmolg-1 h-1 according to the experimental data, with anoverall transfer rate of KLa ) 375 h-1 and a dissolvedoxygen concentration at saturation of the liquid phaseof CR

* ) 0.22 mM, the maximal biomass concentrationwould be approximate 27 g L-1. This value is thatobtained at the end of the cell culture after 110 h, whena decrease in dissolved oxygen concentration (Figure 3)was observed.

Under two-phase flow conditions, the value of Cs andthe maximal cell concentration X when Ce was equal tozero (maximal oxygen uptake) were calculated with theeqs 2 and 4.

The volume of the fermentor part was 0.5 L, the transferrate in this part was 200 h-1, and the dissolved oxygenconcentration at saturation of the liquid phase was CR

*

) 0.22 mM. In the filter unit, the transfer rate, the liquidflow rate, and the dissolved oxygen concentration wererespectively 950 h-1 and 300 L h-1 and CTF

* ) 0.44 mM(since the pressure in the filtration unit was 2 bar).

Assuming a similar specific oxygen uptake qO2 of 3mmol g-1 h-1, the maximal cell concentration value beforeoxygen limitation would limit cell metabolism would beapproximatively 45 g L-1. This value, close to theexperimental results, seems to confirm that the gain inphenol biodegradation performance could be attributedto the gain in both permeate flux and oxygen transfercapacity, thus enabling higher biomass concentrations tobe obtained without oxygen limitations deleteriouslyaffecting specific metabolic rates.

Energy Consumption Considerations. Energy con-sumption with air injection was estimated and comparedwith classical operation for the same filtration perfor-mances. The total consumed power is equal to the sumof the hydraulic power, the power for air compression andfor agitation by the impeller:

The hydraulic power consumed for fluid motion in thefiltration system, WH, is equal to the product of the

upstream pressure and the liquid flow rate (withouttaking into account pump efficiency). The energy for aircompression, WC, has to be taken into account, particu-larly for experiments under unsteady conditions. Ifcompression operation is considered as adiabatic, therelationship given by Perry and Chilton (1973) can beapplied to calculate the consumed power (without takinginto account compressor performance), taking into ac-count the pressure at the bottom of the filter. The powerfor impeller agitation was calculated according to Van’tRiet and Tramper (1991) and took into account thegeometry of both the reactor and the impeller.

For each operating condition, the total power input hasbeen estimated (Table 2). The specific energy consump-tion, defined as the consumed energy per volume ofpermeate has then been calculated for a cell concentra-tion of 25 g L-1 (Table 2). The energy requirement forfiltration is the same in both systems. Since, the oxygentransfer capacity is higher in the two-phase unsteadysetup, the oxygen-consuming rate of degradation can beincreased without any increase in energy cost. Thus, theunsteady two-phase flow system can support both ahigher rate of degradation (by using a higher biomassconcentration and/or by maintaining a higher specificactivity) but can also ensure a more rapid volumetricthroughput due to diminished filter fouling.

Conclusion

This work has demonstrated the potential of a mem-brane bioreactor for continuous phenol degradation usingA. eutrophus. The adaptability of this continuous cellculture process, in which the hydraulic dilution rate caneasily be modified independently of the cell growth rate,facilitates the modulate of the phenol load with respectto the bacterial degradative potential. This can beachieved by modifying the dilution rate or the phenol feedconcentration. With such a system the possibility toeconomize the amount of water used to dilute concen-trated wastes exists by incorporating a simple feedbackloop to reuse the treated waste for on-line dilution.

The impact of a gas/liquid two-phase flow as a meansto increase both the permeate flux and the oxygentransfer rate in membrane bioreactors has been demon-strated. The use of air injection into the membranemodule is a good compromise between simplicity andefficiency for improving both filtration and oxygen trans-fer. The flux enhancement lasts for an extended period,even at increasing biomass concentrations, because of thepermanent effect of the unsteadiness generated by thetwo-phase flow. Moreover, the active cell concentrationcan be increased by a factor of 2 without encounteringoxygen depletion. In this study, a cell concentration of60 g L-1 was reached with a hydraulic dilution rate of0.5 h-1.

The on-line control of the phenol feed concentration,using the yellow color of 2-hms in the permeate, therebyavoiding rate-diminishing transitory inhibition periodsdue to excessive buildup of phenol, enabled the produc-tivity to be greatly improved. The process as it standsis capable of complete degradation of a phenol load of 96

Table 2. Power Required for Filtration and Oxygen Transfer during Fermentation of A. Eutrophus Grown on Phenolwith Full Cell Recycle in Classical Steady Conditions and in Two-Phase Flow Conditions

exptl setupQL

(m3 h-1)

QG(m3 h-1)at TMP

TMP(bar)

WH(W)

WC(W)

WA(W)

WT(W)

permeate flux J(L h-1) for XT )

25 g L-1WT/J

(kWh m-3)

classical 0.3 0.15 1 11.7 3.7 1.5 17 42 18two-phase flow 0.3 0.31 1 11.7 17 0.02 29 70 18

KLaCR* ) qO2

X

(KLa)RCR* VR + qCs ) qO2

XVR (2)

Cs ) (CTF* -

qO2X

(KLa)TF)(1 - e-(KLa)TF∆t) (4)

WT ) WH + WC + WA


kg m-3 day-1 at a phenol feed concentration of 8 g L-1.The limitation of the biomass concentration under 45 gL-1 should permit to maintain the dilution rate at 0.65h-1, thus a phenol degradation rate of 120 kg m-3 day-1

could be easily reached with a phenol feed concentrationof 8 g L-1.

These performance data, significantly higher thanthose seen in the scientific literature (Hughes andCooper, 1996; Leonard and Lindley, 1998) could no doubtbe further improved with a better optimization of theoperating conditions (liquid and gas flow rates and phenolfeed concentration).

NotationX cell concentration (g L-1)SA phenol concentration in the feed (g L-1)S phenol concentration in the permeate (g L-1)D dilution rate (h-1)V total volume of the bioreactor (L)VR volume of the fermentor (L)VM volume of the filtration unit (L)YXS biomass conversion yield from substrate (g g-1)µ specific growth rate (g g-1 h-1)qs specific phenol uptake rate (g g-1 h-1)Φ phenol degradation rate (g L-1 h-1)γ specific heat ratio (CP/CV)J permeate flux (L h-1 m-2)Kj ratio between unsteady and steady fluxes at the

same biomass concentrationPatm atmospheric pressure (bar)PU upstream pressure (bar)QG gas flow rate (m3 h-1)QL liquid flow rate (m3 h-1)TMP transmembrane pressure (bar)WA consumed power for impeller (W)WC consumed power for air compression (W)WH consumed hydraulic power under steady condi-

tions (W)WT total consumed power (W)KLa overall volumetric transfer rate for oxygen (h-1)C* dissolved oxygen concentration at saturation of

the liquid phase (g L-1)C dissolved oxygen concentration in the bulk (g

L-1)qO2 specific oxygen uptake rate (g g-1 h-1)

References and NotesAmpe, F.; Lindley, N. D. Acetate utilisation is inhibited by

benzoate in Alcaligenes eutrophus: evidence for transcrip-tional control of the expression of acoE coding for acetyl-CoAsynthetase. J. Bacteriol. 1995, 177, 5826-5833.

Ampe, F.; Leonard, D.; Lindley, N. D. Repression of phenolcatabolism by organic acids in Ralstonia eutrophus. Appl.Environ. Microbiol. 1998, in press.

Arroyo, G.; Fonade, C. Use of intermittent jets to enhance fluxin crossflow filtration. J. Membr. Sci. 1993, 80, 117-129.

Blanc, P.; Goma, G. Propionic acid fermentation: improvementof performances by coupling continuous fermentation andultrafiltration. Bioprocess Eng. 1987, 2, 137-139.

Bouillot, P.; Canales, A.; Pareilleux, A.; Huyard, A.; Goma, G.Membrane bioreactors for the evaluation of maintenancephenomena in wastewater treatment. J. Ferment. Bioeng.1990, 69, 178-183.

Ferras, E.; Minier, M.; Goma, G. Acetonobutylic fermentation:improvement of performances by coupling continuous fer-mentation and ultrafiltration. Biotechnol. Bioeng. 1986, 28,523-533.

Finnigan, S. M.; Howell, J. A. The effect of pulsatile flow onUF fluxes in a baffled tubular membrane system. Desalina-tion 1990, 79, 181-202.

Hill, G. A.; Robinson, C. W. Substrate inhibition kinetics: phenoldegradation by Pseudomonas putida. Biotechnol. Bioeng.1975, 17, 1599-1615.

Hughes, E. J. L.; Bayly R. C. Control of catechol meta-cleavagein Alcaligenes eutrophus. J. Bacteriol. 1983, 154, 1363-1370.

Hughes, S. M.; Cooper D. G. Biodegradation of phenol using theself-cycling fermentation (SCF) process. Biotechnol. Bioeng.1996, 51, 112-119.

Hunter, R. S.; Harold, R. W. In The measurement of appearance,2nd ed.; Hunter, R. S., Harold, R. W., Eds.; Wiley Inter-science: New York, 1987; pp 177-180.

Johnson, B. F.; Stainer, R. Y. Dissimilation of aromatic com-pounds by Alcaligenes eutrophus. J. Bacteriol. 1971, 107,468-475.

Leonard, D.; Lindley, N. D. Carbon and energy flux constraintsin continuous cultures of Alcaligenes eutrophus grown onphenol. Microbiology 1998, 241-248.

Maranges, C.; Fonade, C. Flux enhancement in crossflowfiltration using an unsteady jet. J. Membr. Sci. 1997, 123,1-8.

Merchuk, J. C. Further considerations on the enhancementfactor for oxygen absorption into fermentation broth. Bio-technol. Bioeng. 1977, 19, 1885-1889.

Mercier, M.; Fonade, C.; Lafforgue-Delorme, C. Influence of theflow regime on the efficiency of a gas-liquid two-phase flowmedium. Biotechnol. Tech. 1995, 9, 853-858.

Mercier, M.; Fonade, C.; Lafforgue-Delorme, C. How slug flowcan enhance the ultrafiltration flux in mineral tubularmembranes. J. Membr. Sci. 1997, 128, 103-113.

Mercier, M.; Maranges, C.; Fonade, C.; Lafforgue-Delorme, C.Yeast suspension filtration: flux enhancement using anupward gas/liquid slug flowsApplication to continuous alco-holic fermentation with cell recycle. Biotechnol. Bioeng. 1998,58, 47-57.

Mota, M.; Lafforgue, C.; Strehaiano, P.; Goma, G. Fermentationcoupled with microfiltration: kinetics of ethanol fermentationwith cell recycle. Bioprocess. Eng. 1986, 2, 65-68.

Perry, R. H.; Chilton, C. H. In Chemical Engineer’s Handbook;McGraw Hill: New York, 1973; pp 6-16.

Pierrot, P.; Fick, M.; Engasser, J. M. Continuous acetone-butanol fermentation with high productivity by cell ultrafil-tration and recycling. Biotechnol. Lett. 1986, 8, 253-256.

Rainer, B. W. Determination methods of the volumetric oxygentransfer coefficient kLa in bioreactors. Chem. Biochem. Eng.Q. 1990, 4, 185-196.

Rainer, B. W. Etude des performances d’aeration d’un nouveautype d’hydroejecteur (Study of the aeration performances ofa new hydroejector system). Ph.D. Thesis, INSA Toulouse,France, 1992.

Santerre, A. L.; Queinnec, I.; Destruhaut , C.; Pourciel, J. B.;Blanc, P. J. On-line evaluation of fermentation broth colour.Biotechnol. Tech. 1994, 8, 319-324.

Schumpe, A. Gas solubilities in biomedia. In Biotechnology;Rehm, H. J., Reed, G., Eds.; VCH Publishers: Weinheim,Germany, 1985; Vol. 2, pp 160-170.

Sobotka, M.; Votruba, J.; Prokop, A. A two-phase oxygen uptakemodel of aerobic fermentations. Biotechnol. Bioeng. 1981, 23,1193-1202.

Van’t Riet, K.; Tramper, H. Power consumption. In Basicbiorector design; M. Dekker Inc.: New York, 1991; pp 303-316.

Voturba, J.; Sobotka, M.; Prokop, A. A two-phase oxygen uptakemodel of aerobic fermentation. Biotechnol. Bioeng. 1981, 23,1193-1202.

Accepted July 20, 1998.

BP980069Z


novel membrane bioreactor with gas/liquid two-phase flow for high-performance degradation of phenol

Documents