Transforming Waste Cheese-Whey into Acetic Acid through a Continuous Membrane-Integrated Hybrid Process

Transforming Waste Cheese-Whey into Acetic Acid through aContinuous Membrane-Integrated Hybrid ProcessJayato Nayak and Parimal Pal*

Environment & Membrane Technology Laboratory, Department of Chemical Engineering, National Institute of Technology,Durgapur, India-713209

ABSTRACT: Experimental investigations were conducted on fermentative production of acetic acid from waste cheese-whey ina multistage membrane-integrated hybrid process. Traditional fermentation allowed cheese-whey, a cheap and waste material, tobe used as carbon source where judicious combinations of cross-flow membrane filtration at the micro, ultra, and nano regimespermitted efficient downstream separation and purification of the product. The provision of separation and recycling of microbialcells and unconverted carbon source allowed fermentation under high cell density with effective use of a carbon source.Continuous separation and removal of the acid product helped to remove product inhibition to a large extent. All theseprovisions in this novel design resulted in high productivity (4.06 g L−1 h−1), substrate to product yield (0.96 g g−1), purity(94.6%), and final acetic acid concentration of 96.9 g L−1 at 303 K temperature, 150 rpm of agitator speed under non-neutralizingconditions, and at a dilution rate of 0.102 h−1. Feed dilution was found to have significant impact on product yield andproductivity. Productivity could be enhanced to 4.82 g L−1 h−1 at increased dilution of 0.141 h−1 at the cost of a small reductionin product yield. The process involves no phase change and no harsh chemicals and opens up a novel and green route ofcontinuous production of a value-added product (acetic acid) from a low cost by-product of the dairy industry.

1. INTRODUCTIONAcetic acid has traditionally been used in a wide range ofproducts like paints, adhesives, foods, textiles, photography,chemicals, and niche application industries. Because of thepresence of carboxylic acid (−COOH) group, glacial acetic acidhas its own potential to act as an excellent protic solvent. Thissolvent property is also used for recrystallization of organiccompounds to purify them and for the production ofterephthalic acid (TPA) as raw material for extensively usedpolyethylene terephthalate (PET) and also in processesinvolving carbocations. Acetic acid finds its major applicationin the production of vinyl acetate monomer which could befurther polymerized to polyvinyl acetate, an importantcomponent of paints and adhesives. Acetic anhydride isproduced by condensation of acetic acid which is mainlyexploited for the production of cellulose acetate, a syntheticfiber also used for photographic film. Acetate salts of differentmetals like sodium, copper(II), aluminum, iron(II), silver areused for various niche applications. The most commonlyknown use of acetic acid is in the production of vinegar which isan about 4−5% diluted form of acetic acid. Around 90% of theworldwide demand is met through the chemical route ofproduction which is methanol carbonylation or Monsantoprocess and Cativa process.1,2 These routes, however, involvehigh energy consumption, high catalyst cost, use of non-recyclable catalysts, and generation of waste acid.3,4 Productionof acetic acid through the fermentative pathway usingappropriate bacterial culture on suitable and renewablesubstrates has always been a preferred one over the chemicalroutes. The conventional biological pathways adopted for thevinegar production are the Orleans process and the Germanmethod5,6 that basically use high purity and good quality grapejuice and alcohol for fermentation to produce acetic acid. Useof such expensive raw materials only adds to the production

cost. Moreover, maturation time in such cases is also quitelong.7,8 According to the world market statistics, in 2003−2005,global production of acetic acid was estimated at about 6.5million tons per year9 which doubled within 2010. Amidst ever-growing market demand, fermentative production of acetic acidis gaining importance because of the possibility of use of cheapraw material and evolution of eco-friendly processes. Theliterature abounds in reports on production of acetic acid fromfinished carbohydrates like glucose, fructose, lactose, andsucrose.10−12 Production of acetic acid from cheap andrenewable resources has been relatively scant. Cheese whey inthis perspective has been hardly tried out though it contains afair amount of carbon source which is renewable and involvesvery low material cost. This is simply a waste material generatedin the dairy industries and needs to be disposed of properly toavoid environmental pollution. Cheese whey is easily availablethroughout the year and across the countries of the world.Effective separation of protein and fat portions from cheesewhey produces clear whey permeates which contain lactose inaqueous phase. Use of additional nutrients during fermentationincreases the carbohydrate utilization, but this also increasesresidual impurities in the fermentation broth necessitatingfurther downstream processing. Dehydration of dilute aceticacid for the purpose of concentration has still remained aproblem because of formation of azeotrope during conventionaldistillation. It was reported that heterogeneous azeotropicdistillation was an efficient step for acetic acid dehydration froma number of hydrocarbon mixtures.13 Further investigation

Received: December 7, 2012Revised: February 1, 2013Accepted: February 4, 2013Published: February 4, 2013

Article

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states that a preconcentrator column becomes essential foracetic acid dehydration to obtain the concentrated form of it.14

The conventional chemical production scheme involvesdownstream processing like filtration, extraction, distillation,crystallization, and evaporation which involve high energyconsumption because of the associated phase change require-ment. Conventional fermentative routes suffer from lowproductivity, high cost of substrate, and cumbersome down-stream purification units. The membrane integrated-hybridreactor system has the potential to overcome all these problemswith a promise to ensure high product yield, high productivity,and high acid concentration in a continuous process.For the continuous fermentative production, a sustained

exponential growth phase along with proper steady stateoperation needs to be maintained. Studies reported onbiological production of acetic acid from finished glucose15

can be referred to as a pioneering ones in this field though theysuffered from drawbacks of high raw material cost, longoperation time, low productivity, and throughput in batchprocesses. Extensive studies had been performed on Clostridiumthermoaceticum16 converting pure glucose solution to acetate.This study shows that by the pH adjustment, product inhibitioncan be controlled, but the resulting product will be acetate saltinstead of direct acetic acid. Another serious attempt wascarried out on the production of acetic acid with Acetogeniumkivui17,18 from pure glucose. In some cases, the whey lactosewas supplemented with additional carbohydrates,19,20 and goodacetic acid yield but low productivity was obtained whileworking in batch mode. High productivity was reported wherea mixture of ethanol and glucose was used as the substrate offermentation in a shallow flow bioreactor.21 Production ofacetic acid using cheap resources like the fermentation ofsucrose present in corn meal hydrolysate in a fibrous bedbioreactor was extensively praised.22 An experimental study onfermentation of whey was performed in a cell recyclemembrane reactor where direct ultrafiltration was applied forproduct purification resulting in severe fouling.23 Extraction ofacetic acid using membrane technology has been a favoredroute after fermentation24 because of the efficiency of recoveryand simplicity of the adopted system. Designs of hollow fibermodules have been well developed and widely used nowadaysin various research works for the extraction of liquid products.25

Extensive study by researchers showed that with a high boresize of the pervaporation membrane in a hollow fiber module,the acetic acid permeability was reduced whereas the separationfactor and water flux were increased.26,27 Despite a highinterfacial mass transfer area28,29 these modules suffer fromsevere fouling problems. Pervaporation was reported to be anefficient technology for the separation of acetic acid-watermixture at 50 °C using an MR-1 membrane made of 25 wt %polyphenylsulfone resulting in high flux of acetic acid.30

Another study on pervaporation was carried out for theseparation of acetic acid water mixture where it was shown thatincreased temperatures resulted in larger fluxes in case of waterpermeation.31 But fundamentally pervaporation is a ratherslower process than use of a cross-flow membrane system. Mostof the reported studies on acetic acid revolve around theseparation of acetic acid from acetic acid-water mixtures.Exhaustive studies on acetate fermentation broth and itspurification using nanofiltration technology stood to be a betteroption in case of downstream processing, and the effect of pHon nanofiltration performance32,33 was also studied byresearchers. But they used fouling-prone dead end modules

and an energy-consuming evaporator at the final stage, and theprocess produced acetate salts. In most of the cases, the carbonsource for the fermentative pathway was the synthetic solutionof finished carbohydrate glucose or lactose solution or a carbonsource supplement like ethanol. Endeavors toward directproduction of acetic acid by fermentation of cheese wheypermeate are extremely scant.The present experimental study without pH adjustment is an

approach toward direct production of acetic acid instead ofacetate salts. The main objective of the present study is todevelop a green process permitting continuous and directproduction of acetic acid in a small, compact, flexible, energy-saving, and eco-friendly plant. Such a plant represents a highdegree of process intensification. To our knowledge, such astudy toward process intensification in acetic acid manufactur-ing has not been reported.

2. MATERIALS AND METHODS2.1. Microorganism. Acetobactor aceti (NCIM-2116), an

acetic acid producing microbe was used throughout this work.It was obtained from National Collection of IndustrialMicroorganisms (NCIM) of the National Chemical Laboratory(Pune, India) in lyophilized condition. The culture wasmaintained in MRS agar slants at 277 K as well as in liquidmedia containing 1 g of CaCO3, 1 g of glucose, 1 g of yeastextract, and 1 g of Tryptone at 303 K in a 250 mL flask with100 mL working volume. The flask was then incubatedovernight at 303 K and used as primary inoculum. To reducethe lag phase of A. aceti (NCIM-2116) in fermentation,inoculated whey permeate with this strain was maintained at303 K overnight and was used as inoculum in a membraneintegrated reactor system.

2.2. Collection of Whey Permeate by Ultrafiltration ofCheese Whey. Cheese whey contains 4.5−5% lactose ascarbohydrate, 0.2% casein protein, and 0.6−0.65% wheyprotein.34 42.35 g L−1 of lactose was measured in theinvestigated cheese whey by HPLC. Whey protein wasseparated out by the standard method of ultrafiltration35,36 toavoid inhibition of microbial growth by suspended protein.PES-5 membrane of molecular weight cut off value of 6 KDa atan operating pressure of 6 kg cm−2 was used in a flat sheetcross-flow membrane module. Lactose with smaller molecularweight permeated through the membrane whereas theimpermeable protein portion was recycled back to the feedtank. The separation of this cheese whey protein byultrafiltration was necessary as it is inhibitory for the growthof this specific strain A. Aceti (NCIM 2116) while infermentation, resulting a decline in acetic acid concentrationand substrate to product yield.

2.3. Fermentative Media Preparation. Waste cheesewhey collected from local dairy product manufacturing unitswas first filtered using a flat sheet cross-flow membrane modulefitted with a polyether Sulphone (PES-5) ultrafiltrationmembrane for the removal of suspended particles like proteinsand fats. The obtained whey permeate was found to contain42.35 g L−1 lactose suitable for fermentation. The media wassupplemented with 12 g L−1 yeast extract, 0.2 g L−1

MgSO4·7H2O, 0.05 g L−1 MnSO4·4H2O, 0.5 g L−1 KH2PO4,and 0.5 g L−1 K2HPO4, 0.8 g L−1 NaCl, 0.13 g L−1 CaCl2, and0.011 g L−1 FeSO4·7H2O. All the chemicals used in this study,were from Sigma Aldrich, U.S.A. Solutions prepared on addingsupplements were sterilized at 394 K and 270 kPa pressure for15 min each time before fermentation.

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2.4. Experimental Equipment. Fermentation was carriedout in a 30 L capacity fermentor integrated with flat sheet cross-flow membrane modules each having a 0.012 m2 effectivefiltration surface (Figure 1). The set up was designed andfabricated locally with high grade stainless steel. The temper-ature of the fermentation unit was controlled by circulatingwater from a thermostatically controlled water bath (MetroTech, India). Temperature and agitation speed were main-

tained at 303 K and 150 rpm, respectively, during fermentation.Ultrafiltration of cheese whey was performed by a PES-5membrane having molecular weight cut off value of 6 KDa, andmicrofiltration was accomplished with a Nylon 0.22 membrane(Membrane Solutions, U.S.A.). Composite polyamide NF-2and NF-1 membranes (Sepro Membranes, U.S.A.) were used inthe nanofiltration modules. Detailed characteristics of the usedmembranes have been presented in Table 1. Circulation of

Figure 1. Schematic diagram of membrane integrated reactor system for the continuous production of acetic acid using four stage membranetreatments.

Table 1. Characteristics of the Membranes Used in This Work

parameter PES-5 Nylon 0.22 NF-2 NF-1

membrane type flat-sheet flat-sheet flat-sheet flat-sheetmembrane surface area (m2) 0.012 0.012 0.012 0.012membrane thickness (μm) 165 110−150 165 165nature of filtration ultrafiltration microfiltration nanofiltration nanofiltrationpore size (μm) 0.001 0.22 0.57 0.53maximum process temperature (°C) 50 80 50 50pH resistance 2−11 2−11 2−11 2−11molecular weight cut-off (g mol−1) 6000 5000−100,000 250−300 150−250



fermentation broth through the microfiltration membranemodules was done by peristaltic pump (Entertech, India)whereas a high pressure diaphragm pump (HYDRA-CELL,U.S.A.) was used for the nanofiltration module. The micro-filtered permeate was passed to the nanofiltration modules via aholding vessel in between. An additional nanofiltration modulewas used for further concentration of dilute acetic acid. TheNF-1 membrane with pore size 0.53 nm was employed for thepurpose. Membrane cleaning was performed by back washingand by the treatment with 0.1 N NaOH and 10−2 molar HNO3solutions. Sterilization of the membranes was performed with200 ppm NaOCl solution. After sterilization, membranes wererinsed with Milli-Q water.2.5. Fermentations. The prepared media for fermentation

was inoculated with 10% (v/v) inoculum volume in the 30 Lcapacity fermenter in the membrane integrated pilot plantreactor system. After an initial batch period of around 24 h,fermentation was switched on in continuous mode andconducted at 303 K temperature at 150 rpm of agitatorspeed under non-neutralizing conditions. Effects of feeddilution were investigated by conducting experiments underdifferent dilutions. Dilution rate (h−1) is the rate at which thefresh media is added to the fermentation broth volume presentin the fermenter. It is defined as the ratio of the feed streaminflow rate to the working volume of the reactor. Productivity(g L−1 h−1) is the final product concentration of acetic acid (gL−1) multiplied by the dilution rate (h−1) in the system. This isbasically a measure of output of a plant per unit time.Volumetric flow rate of fluid through membrane module (m3

s−1) divided by the cross sectional area of the inlet pipe of thatmodule (m2) gives the value of cross-flow velocity (m s−1)through the membrane.2.6. Assay. Samples for cell growth analysis were collected

at regular intervals and measurement of optical density (OD)was done by UV−vis spectrophotometer (CECIL, 7000 Series,U.K.) at 620 nm. Collected permeates of membrane filtrationunits were first ultracentrifuged (Sigma Instruments) at 10,000rpm for 10 min and then analyzed for acetic acid and residuallactose by HPLC (Agilent, Series 1200, U.S.A.). In the aceticacid measurement, the standard was prepared with 99.99% pureacetic acid (Sigma Aldrich, U.S.A.). Ultron ES-OVM ChiralOrganic Acid Column (Agilent Technologies, U.S.A.) withDiode Array Detector (DAD) was used with mobile phaseacetonitrile (100% pure) and potassium di hydrogen phosphate(20 mili molar aqueous KH2PO4 solution of pH 2.0) at avolume ratio of 1:99 and at a flow rate of 1 mL min−1 withresidence time of 2.46 min and injection volume of 10 μL wasused for acetic acid detection. The assay of the unconvertedcarbohydrates (lactose) was performed with an RID detectorusing Agilent Zorbax carbohydrate analysis column wheretemperature of the column was maintained at 303 K. Themobile phase for carbohydrate analysis comprised 75%acetonitrile (Sigma Aldrich, U.S.A.) and 25% ultrapure waterat a flow rate of 1.4 mL min−1 with injection volume of thesamples of 10 μL. The RT values (residence time) of lactose is7.909 min. Peak purity software tool of HPLC (Agilent, series1200, U.S.A.) was used to assess the purity of the producedacetic acid after nanofiltration. Ions of the minerals like Na+, K+,and Mg2+ were identified and measured with individualelectrodes (Thermo Scientific, U.S.A.). Each of the results ofexperiments and that of analyses were performed in three sets,and the mean values of three sets for individual parameterswere reported. Experimental error was computed to be within

3−4%. The results have been reported in terms ofconcentration, yield, and productivity where the yield is definedas the ratio of gram (g) of product per gram (g) of the substrateconsumed and the productivity is defined as the gram (g) oflactic acid produced per liter of the reactor volume per hour.Productivity may also be computed as

= ×

− −

− −

productivity (g L h )

product concentration (g L ) dilution rate (h )

1 1

1 1

3. RESULTS AND DISCUSSION3.1. Constant Transmembrane Pressure Cross-Flow

Filtration Runs during Fermentation. 3.1.1. Cross FlowMicrofiltration at Constant Transmembrane Pressure. ANylon 0.22 flat sheet membrane used in the cross-flow modulesuccessfully separated cells (as evident in cell analysis of thepermeate) for recycling while generating clear permeate fromthe fermentation broth without significant flux decline for over30 h. It was observed that lower operating pressure facilitatedlong-term filtration without much reduction in flux. Moreover,the steady state flux could be attained quite quickly at lowtransmembrane pressure than at higher operating pressure atthe mentioned operating cross-flow velocities. Thus duringmicrofiltration, operating transmembrane pressure was main-tained at 1 bar, and the experiment was carried out at threecross-flow velocities of 0.53, 0.88, and 1.06 m s−1 withcorresponding volumetric flow rates of 150 L h−1, 250 L h−1,and 300 L h−1. For over 30 h, a steady flux of around 30 L m−2

h−1 (LMH) was obtained at 1.06 m s−1 cross-flow velocity(Figure 2). Greater sweeping action offered by higher operating

cross-flow velocity resulted in higher flux than that attainedduring the operation at lower cross-flow velocity under identicaltransmembrane pressure. The flux behavior of the membranemodules at constant transmembrane pressure and at differentcross-flow velocities was investigated, and it was observed thatthe steady state flux could be attained after 4, 6, and 7 h ofoperation respectively at the above-mentioned three cross-flowvelocities. Hydrodynamic conditions under the investigatedcross-flow velocity regime did not affect viability andphysiological conditions of A. aceti (NCIM-2116) as revealedin the analysis of the cells in the broth.

3.1.2. Continuous Fermentation with Microfiltration andNanofiltration. Prior to continuous production, fermentation

Figure 2. Permeate flux decline in Nylon 0.22 microfiltrationmembrane operated at fixed transmembrane pressure of 1 bar anddifferent cross-flow velocities.



was initiated in batch mode at 303 K in a membrane-integratedfermentor of 30 L volume and allowed to continue in the modefor 24 h. During this stage of operation, microorganisms werein an active state of exponential growth phase. The changes inthe concentrations of cell, carbohydrates, acetic acid, and pH ofthe medium with time as observed have been presented inFigure 3. No effective period of lag phase of microorganismswas observed at the beginning of fermentation because of thedirect use of preinoculated and nutrient-supplemented whey

permeates as inoculum. During this preliminary batchfermentation stage, the pH of the fermentation broth droppedfrom 4.2 to 2.73 when the concentration of produced aceticacid rose to 32.2 g L−1, cell mass concentration, 2.62 g L−1,acetic acid productivity, 1.34 g L−1 h−1, and product yieldreached 76%. Figure 2 shows that the steady state conditionwas reached within 4 h after starting of microfiltration cellrecycle at a cross-flow velocity 0.53 m s−1, where the dilutionrate was maintained at 0.102 h−1 and the time requirement for

Figure 3. Continuous acetic acid production at different cross-flow and feed dilution rates. (a) Cross flow velocity = 0.53 m s−1, dilution = 0.102 h−1;(b) cross flow velocity = 0.88 m s−1, dilution = 0.128 h−1; (c) cross flow velocity = 1.06 m s−1, dilution = 0.141 h−1.

Table 2. Comparison of the Results Obtained during Microfiltration of the Fermentation Broth at Three Different WorkingConditions, Prior to First Stage Nanofiltration during Continuous Runa

run conditions average acetic acid concentration (g L−1) product yield (%) (product/substrate) × 100 productivity (g/L/h)

1. u = 0.53 m s−1, D = 0.102 h−1 40.65 96.0 4.142. u = 0.88 m s−1, D = 0.128 h−1 37.65 88.9 4.193. u = 1.06 m s−1, D = 0.141 h−1 34.2 80.75 4.82

au = cross flow velocity; D = feed dilution rate.



reaching steady state increased with increase in cross-flowvelocity and dilution rate. Because of the introduction of freshmedium with higher dilution rates of 0.128 h−1 and 0.141 h−1,overall cell mass concentration decreased in the first few hoursof microfiltration cell recycle which was gradually recoveredwith microbial adaption with the conditions of fermentationbroth as exhibited in the above-mentioned figures. Acetic acidconcentration (40.65 g L−1) with maximum product yield of96% was achieved under steady state conditions at a cross-flowvelocity of 0.53 m s−1. Product concentration, yield, andproductivity achieved while microfiltration at three differentcross-flow velocities and dilution, prior to nanofiltration arepresented in Table 2. The highest productivity achieved was4.82 g L−1 h−1 during continuous operation with a cross-flowvelocity of u = 1.06 m s−1 and dilution rate of D = 0.141 h−1.The concentration of acetic acid of the fully membrane-integrated system in those three runs after nanofiltrationreached final concentrations of 35.37 g L−1, 32.76 g L−1, and31.8 g L−1 at cross-flow velocities of 0.53 m s−1, 0.88 m s−1, and1.06 m s−1, respectively. Overall system productivitiescomputed after nanofiltration stage were observed to beslightly less than the fermentative productivities and reached3.61 g L−1 h−1, 4.2 g L−1 h−1, and 4.48 g L−1 h−1 at thecorresponding dilution rates of 0.102 h−1, 0.128 h−1, and 0.141h−1, respectively, with 96% product purity. For the finalconcentration of the produced acid, another downstreamnanofiltration membrane module was pressed into service. Anexperimentally selected NF-1 nanofiltration membrane of poresize 0.53 nm concentrated acetic acid to the level of 96.9 g L−1.The produced sample, after carrying out the long hours ofcontinuous fermentation and two stage membrane treatment(MF and NF), contains only acetic acid and the remainingamount of unconverted lactose in an aqueous solution. TheNF-1 membrane operated at high transmembrane pressureallowed water to permeate through it while retaining acetic acid(85%) and the residual unconverted carbohydrate. After 14 h ofnanofiltration in recycling mode, acetic acid with initialconcentration of 35.37 g L−1 was concentrated to the level of96.9 g L−1 . This ultimate product was found to be 94.6% pureas measured by the peak purity software tool of HPLC.3.1.3. Constant Transmembrane Pressure Cross-Flow

Nanofiltration Runs. Use of the NF-2 membrane (pore sizeof 0.57 nm) in the nanofiltration module resulted in highpermeation of acetic acid with retention of about 97% of theresidual lactose. The flux profile of the nanofiltration membraneas shown in Figure 4 exhibits a positive correlation of cross-flowvelocity with permeate flux at a constant transmembranepressure. Very little fouling problem was encountered duringnanofiltration because of filtration of a low viscosity fluid at thisstage and the very flat sheet cross-flow modular design of thesystem. In Figures 2 and 4, the permeate flux declinecharacteristics of Nylon 0.22(microfiltration) and NF-2 (nano-filtration) with respect to time are shown. The flux during bothmicrofiltration and nanofiltration, first decreases, then reaches asteady state and sustains over a longer period and then finallybegins declining. At the starting of the process, all the poresremain open and the permeation starts with a high magnitudepermeate flux. During the present investigations, after around30 and 26 h respectively, concentration polarization innanofiltration membranes and pore blocking in microfiltrationmembranes started with the onset of flux decline. Thus these 26or 30 h are not necessarily indicators of the maximum operatingtime. Rather these time periods are merely indicators of the

time when the corresponding membranes should be cleanedand reused to allow operation with reasonable flux over a muchlonger time. Used membranes may be cleaned (as mentioned insection 2.4, Experimental Equipment) and reused. By the verychoice of flat sheet cross-flow membrane modules, foulingproblems could be largely overcome because of the highsweeping action of the fluid on the membrane surface but,because of operation over a prolonged time with such a densefermentation broth containing microbes and nutrients, cloggingof the porous membranes may take place. Thus after 26 h in thecase of the Nylon 0.22 microfiltration membrane and after 30 hinthe case of the NF-2 membrane, the permeate fluxconsiderably decreases. Rejection trends for lactose and aceticacid at increasing transmembrane pressure have been shown inFigure 5. While using the NF-2 membrane, acetic acid rejection

increased from 10% to 15% with almost 98% rejection oflactose when transmembrane pressure was increased from 12bar to 15 bar. Lower cross-flow velocity of 0.53 m s−1

maintained during microfiltration turned out to be morehelpful in this system to generate more concentrated acetic acidthan a higher cross-flow velocity of 1.06 m s−1.The maximum average acetic acid concentration achieved

was 40.65 g L−1 with maximum product yield of 96% andproductivity 4.14 g L−1 h−1 at a dilution rate of 0.102 h−1.However, maximum productivity (4.82 g L−1 h−1) wasassociated with a highest dilution rate of 0.141 h−1. Duringcontinuous operation, if the cross-flow velocity is kept at a lowvalue (0.53 m s−1) the permeate flux decreases leading to

Figure 4. Permeate flux decline in the NF-2 nanofiltration membraneoperated at fixed transmembrane pressure of 12 bar and differentcross-flow velocities.

Figure 5. Rejection characteristics shown by the nanofiltrationmembranes used in this work.



considerable decline in the rate of dilution (0.102 h−1).Subsequently, the rate of addition of fresh feed into thefermenter is low allowing long enough residence time tomicrobes to intake nutrients and substrates, resulting in higherproduct concentration (40.65 g L−1) and product yield (96%)after microfiltration treatment than those obtained whileoperating at higher cross-flow velocities. By setting highercross-flow velocities during microfiltration, higher permeate fluxis obtained because of the high sweeping action on themembrane surface. Thus increase of cross-flow velocity (0.106m s−1) increases feed dilution (0.141 h−1) or the rate at whichfresh feed is added to the fermenter. This condition allows lowresidence time to the microbes to produce the desired aceticacid resulting in a low acetic acid concentration (34.2 g L−1)and low product yield (80.75%).Though the productivity or the output from the fermenter

per unit time is high (4.82 g L−1 h−1) while operating at highercross-flow velocity than that obtained (4.14 g L−1 h−1) in caseof lower cross-flow velocity, it is better to operate the system atsome lower cross-flow velocity during microfiltration to achievethe maximum conversion of carbohydrate to acetic acid in acontinuous fermentation process. Higher productivity has beenreported in some literatures, but those were achieved using anexpensive carbon source. In many such cases,17−19 theproduction process is a batch fermentation one resulting inlow productivity (0.66−0.7 g L−1 h−1) though a yield of 80−98% has been reported. With the use of additional lactose asfeed supplement, the reported maximum yield of acetic acid is0.55 g g−1 with a productivity of 0.15 g L−1 h−1 where a singlestage membrane system was utilized.23 Though, most of thecase studies were carried out using pure carbohydrate solutionsas substrate for fermentation but ended up with lowproductivities. From that perspective, this work of continuousproduction of acetic acid, exploiting cheese whey in amembrane integrated hybrid reactor system, may be claimedto be a novel one because of it promises high product yield(96%), productivity (4.14 g L−1 h−1), and product purity(94.6%) at the lowest operating cross-flow velocity (0.53 m s−1)and dilution rate (0.102 h−1). The simplicity in design, use ofwaste raw material as carbon source, non-neutralizing operationconditions ensuring direct production of acetic acid, theflexibility of the associated modular design and capability ofcontinuous production of acetic acid with high concentration,product yield, productivity, and purity have all contributed todevelopment of a novel process of acetic acid production fromcheese whey.

5. CONCLUSIONSHigh purity acetic acid was produced in a continuous processwithout any requirement of pH adjustment in a fullymembrane-integrated fermentation process using waste rawmaterial. The modular design of those flat sheet cross-flowmodules used throughout the study permitted use of anynumber of active modules in it for the ultrafiltration,microfiltration, or nanofiltration step during steady stateoperation. Steady operation could be attained for a constanttransmembrane operating pressure as well as for constantfermentor capacity.The study culminates in the development of a novel process

of acetic acid production where conventional fermentativeproduction of acetic acid from a waste material has beenintegrated with downstream membrane-based separation andpurification. Membrane-integration has permitted continuous

and fast production of acetic acid overcoming the difficulties ofsubstrate-product inhibitions, and involvement of multiplesteps of separation and purification. Membrane separation hashelped to achieve a high degree of separation and purificationalso. Membrane separation at the microfiltration regime hashelped to separate and recycle microbial cells from thefermentation medium facilitating fermentation with high celldensity. Pretreatment of cheese whey by ultrafiltrationeliminates the chance of lag phase in microbes duringfermentation. The post treatment unit of nanofiltrationconcentrates the produced acetic acid further without involvingany energy-intensive evaporation step. The whole processinvolves no phase change and no harsh chemicals thusrepresenting a high degree of process intensification which isvery significant and being sought urgently by chemical processindustries across the world.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected]. Fax: +91343-2754078.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors acknowledge financial support under DST-FISTProgram of the Government of India. Sincere thanks are alsoextended to National Chemical Laboratory, Pune, India, forproviding the necessary microbial strains.

■ REFERENCES(1) Cheung, H.; Tanke, R. S.; Torrence, G. P. Acetic Acid. InUllmann’s Encyclopedia of Industrial Chemistry, 6th ed; Wiley-VCH:Weinheim, Germany, 2005; Vol. 1.(2) Monsanto, U.S. patent 3769329, 1973.(3) Yoneda, N.; Kusano, S.; Yasui, M.; Pujado, P.; Wilcher, S. Recentadvances in process and catalysts for the production of acetic acid.Appl. Catal. 2001, 22, 253−265.(4) Howard, M. J.; Jones, M. D.; Roberts, M. S.; Taylor, S. A. Catal.Today 1993, 18, 325.(5) Ndoye, B.; Lebeccque, S.; Destain, J.; Guiro, A. T.; Thonart, P. Anew pilot plant scale acetifier designed for vinegar production in Sub-Saharan Africa. Process Biochem. 2007, 42, 1561−1565.(6) Sokollek, J. S.; Hertel, C.; Hammes, W. P. Cultivation andpreservation of vinegar bacteria. J. Biotechnol. 1998, 60, 195−206.(7) Hidalgo, C.; Vegas, C.; Mateo, E.; Tesfaye, W.; Cerezo, A. B.;Callejon, R. M.; Poblet, M.; Guillamon, J. M.; Mas, A.; Torija, M. J.Effect of barrel design and the inoculation of Acetobacter pasteurianusin wine vinegar production. Int. J. Food Microbiol. 2010, 141, 56−62.(8) Morales, M. L.; Benitez, B.; Troncoso, A. M. Accelerated aging ofwine vinegars with oak chips: evaluation of wood flavour compounds.Food Chem. 2004, 88, 305−315.(9) Production report, Chem. Eng. News, 11 July 2005, 67−76.(10) Wang, G.; Wang, D. I. C. Elucidation of growth inhibition andacetic acid production by Clostridium thermoaceticum. Appl. Environ.Microbiol. 1984, 47, 294−298.(11) Wang, D. I. C.; Fleishchaker, R. J.; Wang, G. Y. A novel route tothe production of acetic acid by fermentation. AIChE Symp. Ser. 1811978, 74, 105−110.(12) Schwartz, R. D.; Keller, F. A., Jr. Isolation of a strain ofClostridium thermoaceticum capable of growth and acetic acidproduction at pH 4.5. Appl. Environ. Microbiol. 1982, 43, 117−123.(13) Chien, I. L.; Zeng, K. L.; Chao, H. Y.; Liu, J. H. Design andcontrol of acetic acid dehydration system via heterogeneous azeotropicdistillation. Chem. Eng. Sci. 2004, 59, 4547−4567.(14) Chien, I. L.; Kuo, C. L. Investigating the need of a pre-concentrator column for acetic acid dehydration system via



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heterogeneous azeotropic distillation. Chem. Eng. Sci. 2006, 61, 569−585.(15) Schwartz, R. D.; Keller, F. A., Jr. Acetic Acid Production byClostridium thermoaceticum in pH Controlled Batch Fermentations atAcidic pH. Appl. Environ. Biotechnol. 1982, 43, 1385−1392.(16) Witjitra, K.; Shah, M.; Cheryan, M. Effect of nutrient sources ongrowth and acetate production by Clostridium thermoaceticum. EnzymeMicrob. Technol. 1996, 19, 322−327.(17) Klemps, R.; Schoberth, S.; Sahm, H. Production of acetic acid byAcetogenium kivui. Appl. Microbiol. Biotechnol. 1987, 27, 229−234.(18) Esymondt, J.; Vasic-Racki, D. Wandrey, Ch. Acetic acidproduction by Acetogenium kivui in continuous culture-kinetic studiesand computer simulations. Appl. Microbiol. Biotechnol. 1990, 34, 334−349.(19) Tang, I.; Yang, S.; Okos, M. Acetic acid production from wheylactose by the co culture of Streptococcus lactis and Clostridiumformicoaceticum. Appl. Microbiol. Biotechnol. 1988, 28, 138−143.(20) Parrondo, J.; Herrero, M.; Garcia, L.; Dιaz, M. A Note −Production of Vinegar from Whey. J. Inst. Brew. 2003, 109 (4), 356−358.(21) Toda, K.; Park, Y.; Asakura, T.; Cheng, C.; Ohtake, H. High-rateacetic acid production in a shallow flow bioreactor. Appl. Microbiol.Biotechnol. 1989, 30, 559−563.(22) Huang, Y.; Wu, Z.; Zhang, L.; Cheung, C.; Yang, S. Productionof carboxylic acids from hydrolyzed corn meal by immobilized cellfermentation in a fibrous-bed bioreactor. Bioresour. Technol. 2002, 82,51−59.(23) Mostafa, N. A. Production of acetic acid and glycerol from saltedand dried whey in a membrane cell recycle bioreactor. Energy Convers.Manage. 2001, 42, 1133−1142.(24) Grzenia, D. L.; Schell, D. J.; Wickramasinghe, S. R. Membraneextraction for removal of acetic acid from biomass hydrolysates. J.Membr. Sci. 2008, 322, 189−195.(25) Wickramasinghe, S. R.; Semmens, M. J.; Cussler, E. L. Masstransfer in various hollow fiber geometries. J. Membr. Sci. 1992, 69,235−250.(26) Zhou1, F.; Koros, W. J. Characterization of bore pressurechange effects on Matrimid® fiber performance in pervaporation ofacetic acid and water mixtures. Chem. Eng. Sci. 2006, 61, 3736−3745.(27) Zhou, F.; Koros, W. J. Pervaporation Using Hollow-FiberMembranes for Dehydrating and Acetic Acid Water Mixtures. Ind. Eng.Chem. Res. 2006, 45 (5), 1787−1796.(28) Frank, G. T.; Sirkar, K. K. Alcohol production and yeastfermentation by membrane extraction. Biotechnol. Bioeng. Symp. Ser.1985, 15, 621−631.(29) D’Elia, N. A.; Dahuron, L.; Cussler, E. L. Liquid − Liquidextractions with microporous hollow fibers. J. Membr. Sci. 1986, 29,309−319.(30) Jullok, N.; Deforche, T.; Luis, P.; Bruggen, B. V. Sorption anddiffusivity study of acetic acid and water in polymeric membranes.Chem. Eng. Sci. 2012, 78, 14−20.(31) Gorri, D.; Urtiga, A.; Ortiz, I. Pervaporative Recovery of AceticAcid from an Acetylation Industrial Effluent Using CommercialMembranes. Ind. Eng. Chem. Res. 2005, 44 (4), 977−985.(32) Han, I. S.; Cheryan, M. Nanofiltration of model acetatesolutions. J. Membr. Sci. 1995, 107, 107−113.(33) Han, I. S.; Cheryan, M. Downstream processing of AcetateFermentation Broths by Nanofiltration. Appl. Microbiol. Biotechnol.1995, 57/58, 19−28.(34) Yang, S., Ed.; Bioprocessing for Value-Added Products fromRenewable Resources: New Technologies and Applications, 1st ed.;Elsevier Science Publishers: Amsterdam, The Netherlands, 2007.(35) Bhattacharjee, S.; Ghosh, S.; Datta, S.; Bhattacharjee, C. Studieson ultrafiltration of casein whey using a rotating disk module: effects ofpH and membrane disk rotation. Desalination 2006, 195, 95−108.(36) Sarkar, P.; Ghosh, S.; Dutta, S.; Sen, D.; Bhattacharjee, C. Effectof different operating parameters on the recovery of proteins fromcasein whey using a rotating disc membrane ultrafiltration cell.Desalination 2009, 249, 5−11.



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Transforming Waste Cheese-Whey into Acetic Acid through a Continuous Membrane-Integrated Hybrid Process