Transforming Waste Cheese-Whey into Acetic Acid through a

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Transforming Waste Cheese-Whey into Acetic Acid through a Continuous Membrane-Integrated Hybrid Process Jayato 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 in a multistage membrane-integrated hybrid process. Traditional fermentation allowed cheese-whey, a cheap and waste material, to be used as carbon source where judicious combinations of cross-flow membrane filtration at the micro, ultra, and nano regimes permitted efficient downstream separation and purification of the product. The provision of separation and recycling of microbial cells 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 these provisions 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-neutralizing conditions, and at a dilution rate of 0.102 h−1. Feed dilution was found to have significant impact on product yield and productivity. 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 reduction in product yield. The process involves no phase change and no harsh chemicals and opens up a novel and green route of continuous production of a value-added product (acetic acid) from a low cost by-product of the dairy industry.

1. INTRODUCTION Acetic acid has traditionally been used in a wide range of products like paints, adhesives, foods, textiles, photography, chemicals, and niche application industries. Because of the presence of carboxylic acid (−COOH) group, glacial acetic acid has its own potential to act as an excellent protic solvent. This solvent property is also used for recrystallization of organic compounds to purify them and for the production of terephthalic acid (TPA) as raw material for extensively used polyethylene terephthalate (PET) and also in processes involving carbocations. Acetic acid finds its major application in the production of vinyl acetate monomer which could be further polymerized to polyvinyl acetate, an important component of paints and adhesives. Acetic anhydride is produced by condensation of acetic acid which is mainly exploited for the production of cellulose acetate, a synthetic fiber also used for photographic film. Acetate salts of different metals like sodium, copper(II), aluminum, iron(II), silver are used for various niche applications. The most commonly known use of acetic acid is in the production of vinegar which is an about 4−5% diluted form of acetic acid. Around 90% of the worldwide demand is met through the chemical route of production which is methanol carbonylation or Monsanto process and Cativa process.1,2 These routes, however, involve high energy consumption, high catalyst cost, use of nonrecyclable catalysts, and generation of waste acid.3,4 Production of acetic acid through the fermentative pathway using appropriate bacterial culture on suitable and renewable substrates has always been a preferred one over the chemical routes. The conventional biological pathways adopted for the vinegar production are the Orleans process and the German method5,6 that basically use high purity and good quality grape juice and alcohol for fermentation to produce acetic acid. Use of such expensive raw materials only adds to the production © 2013 American Chemical Society

cost. Moreover, maturation time in such cases is also quite long.7,8 According to the world market statistics, in 2003−2005, global production of acetic acid was estimated at about 6.5 million tons per year9 which doubled within 2010. Amidst evergrowing market demand, fermentative production of acetic acid is gaining importance because of the possibility of use of cheap raw material and evolution of eco-friendly processes. The literature abounds in reports on production of acetic acid from finished carbohydrates like glucose, fructose, lactose, and sucrose.10−12 Production of acetic acid from cheap and renewable resources has been relatively scant. Cheese whey in this perspective has been hardly tried out though it contains a fair amount of carbon source which is renewable and involves very low material cost. This is simply a waste material generated in the dairy industries and needs to be disposed of properly to avoid environmental pollution. Cheese whey is easily available throughout the year and across the countries of the world. Effective separation of protein and fat portions from cheese whey produces clear whey permeates which contain lactose in aqueous phase. Use of additional nutrients during fermentation increases the carbohydrate utilization, but this also increases residual impurities in the fermentation broth necessitating further downstream processing. Dehydration of dilute acetic acid for the purpose of concentration has still remained a problem because of formation of azeotrope during conventional distillation. It was reported that heterogeneous azeotropic distillation was an efficient step for acetic acid dehydration from a number of hydrocarbon mixtures.13 Further investigation Received: Revised: Accepted: Published: 2977

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and an energy-consuming evaporator at the final stage, and the process produced acetate salts. In most of the cases, the carbon source for the fermentative pathway was the synthetic solution of finished carbohydrate glucose or lactose solution or a carbon source supplement like ethanol. Endeavors toward direct production of acetic acid by fermentation of cheese whey permeate are extremely scant. The present experimental study without pH adjustment is an approach toward direct production of acetic acid instead of acetate salts. The main objective of the present study is to develop a green process permitting continuous and direct production of acetic acid in a small, compact, flexible, energysaving, and eco-friendly plant. Such a plant represents a high degree of process intensification. To our knowledge, such a study toward process intensification in acetic acid manufacturing has not been reported.

states that a preconcentrator column becomes essential for acetic acid dehydration to obtain the concentrated form of it.14 The conventional chemical production scheme involves downstream processing like filtration, extraction, distillation, crystallization, and evaporation which involve high energy consumption because of the associated phase change requirement. Conventional fermentative routes suffer from low productivity, high cost of substrate, and cumbersome downstream purification units. The membrane integrated-hybrid reactor system has the potential to overcome all these problems with 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 state operation needs to be maintained. Studies reported on biological production of acetic acid from finished glucose15 can be referred to as a pioneering ones in this field though they suffered from drawbacks of high raw material cost, long operation time, low productivity, and throughput in batch processes. Extensive studies had been performed on Clostridium thermoaceticum16 converting pure glucose solution to acetate. This study shows that by the pH adjustment, product inhibition can be controlled, but the resulting product will be acetate salt instead of direct acetic acid. Another serious attempt was carried out on the production of acetic acid with Acetogenium kivui17,18 from pure glucose. In some cases, the whey lactose was supplemented with additional carbohydrates,19,20 and good acetic acid yield but low productivity was obtained while working in batch mode. High productivity was reported where a mixture of ethanol and glucose was used as the substrate of fermentation in a shallow flow bioreactor.21 Production of acetic acid using cheap resources like the fermentation of sucrose present in corn meal hydrolysate in a fibrous bed bioreactor was extensively praised.22 An experimental study on fermentation of whey was performed in a cell recycle membrane reactor where direct ultrafiltration was applied for product purification resulting in severe fouling.23 Extraction of acetic acid using membrane technology has been a favored route after fermentation24 because of the efficiency of recovery and simplicity of the adopted system. Designs of hollow fiber modules have been well developed and widely used nowadays in various research works for the extraction of liquid products.25 Extensive study by researchers showed that with a high bore size of the pervaporation membrane in a hollow fiber module, the acetic acid permeability was reduced whereas the separation factor and water flux were increased.26,27 Despite a high interfacial mass transfer area28,29 these modules suffer from severe fouling problems. Pervaporation was reported to be an efficient technology for the separation of acetic acid-water mixture 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 the separation of acetic acid water mixture where it was shown that increased temperatures resulted in larger fluxes in case of water permeation.31 But fundamentally pervaporation is a rather slower process than use of a cross-flow membrane system. Most of the reported studies on acetic acid revolve around the separation of acetic acid from acetic acid-water mixtures. Exhaustive studies on acetate fermentation broth and its purification using nanofiltration technology stood to be a better option in case of downstream processing, and the effect of pH on nanofiltration performance32,33 was also studied by researchers. But they used fouling-prone dead end modules

2. MATERIALS AND METHODS 2.1. Microorganism. Acetobactor aceti (NCIM-2116), an acetic acid producing microbe was used throughout this work. It was obtained from National Collection of Industrial Microorganisms (NCIM) of the National Chemical Laboratory (Pune, India) in lyophilized condition. The culture was maintained in MRS agar slants at 277 K as well as in liquid media containing 1 g of CaCO3, 1 g of glucose, 1 g of yeast extract, and 1 g of Tryptone at 303 K in a 250 mL flask with 100 mL working volume. The flask was then incubated overnight at 303 K and used as primary inoculum. To reduce the lag phase of A. aceti (NCIM-2116) in fermentation, inoculated whey permeate with this strain was maintained at 303 K overnight and was used as inoculum in a membrane integrated reactor system. 2.2. Collection of Whey Permeate by Ultrafiltration of Cheese Whey. Cheese whey contains 4.5−5% lactose as carbohydrate, 0.2% casein protein, and 0.6−0.65% whey protein.34 42.35 g L−1 of lactose was measured in the investigated cheese whey by HPLC. Whey protein was separated out by the standard method of ultrafiltration35,36 to avoid inhibition of microbial growth by suspended protein. PES-5 membrane of molecular weight cut off value of 6 KDa at an operating pressure of 6 kg cm−2 was used in a flat sheet cross-flow membrane module. Lactose with smaller molecular weight permeated through the membrane whereas the impermeable protein portion was recycled back to the feed tank. The separation of this cheese whey protein by ultrafiltration was necessary as it is inhibitory for the growth of this specific strain A. Aceti (NCIM 2116) while in fermentation, resulting a decline in acetic acid concentration and substrate to product yield. 2.3. Fermentative Media Preparation. Waste cheese whey collected from local dairy product manufacturing units was first filtered using a flat sheet cross-flow membrane module fitted with a polyether Sulphone (PES-5) ultrafiltration membrane for the removal of suspended particles like proteins and fats. The obtained whey permeate was found to contain 42.35 g L−1 lactose suitable for fermentation. The media was supplemented 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, and 0.011 g L−1 FeSO4·7H2O. All the chemicals used in this study, were from Sigma Aldrich, U.S.A. Solutions prepared on adding supplements were sterilized at 394 K and 270 kPa pressure for 15 min each time before fermentation. 2978

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

Table 1. Characteristics of the Membranes Used in This Work parameter

PES-5

Nylon 0.22

NF-2

NF-1

membrane type membrane surface area (m2) membrane thickness (μm) nature of filtration pore size (μm) maximum process temperature (°C) 50 pH resistance molecular weight cut-off (g mol−1)

flat-sheet 0.012 165 ultrafiltration 0.001 80 2−11 6000

flat-sheet 0.012 110−150 microfiltration 0.22 50 2−11 5000−100,000

flat-sheet 0.012 165 nanofiltration 0.57 50 2−11 250−300

flat-sheet 0.012 165 nanofiltration 0.53

2.4. Experimental Equipment. Fermentation was carried out in a 30 L capacity fermentor integrated with flat sheet crossflow membrane modules each having a 0.012 m2 effective filtration surface (Figure 1). The set up was designed and fabricated locally with high grade stainless steel. The temperature of the fermentation unit was controlled by circulating water from a thermostatically controlled water bath (Metro Tech, India). Temperature and agitation speed were main-

2−11 150−250

tained at 303 K and 150 rpm, respectively, during fermentation. Ultrafiltration of cheese whey was performed by a PES-5 membrane having molecular weight cut off value of 6 KDa, and microfiltration was accomplished with a Nylon 0.22 membrane (Membrane Solutions, U.S.A.). Composite polyamide NF-2 and NF-1 membranes (Sepro Membranes, U.S.A.) were used in the nanofiltration modules. Detailed characteristics of the used membranes have been presented in Table 1. Circulation of 2979

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

fermentation broth through the microfiltration membrane modules 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 microfiltered permeate was passed to the nanofiltration modules via a holding vessel in between. An additional nanofiltration module was used for further concentration of dilute acetic acid. The NF-1 membrane with pore size 0.53 nm was employed for the purpose. Membrane cleaning was performed by back washing and by the treatment with 0.1 N NaOH and 10−2 molar HNO3 solutions. Sterilization of the membranes was performed with 200 ppm NaOCl solution. After sterilization, membranes were rinsed with Milli-Q water. 2.5. Fermentations. The prepared media for fermentation was inoculated with 10% (v/v) inoculum volume in the 30 L capacity fermenter in the membrane integrated pilot plant reactor system. After an initial batch period of around 24 h, fermentation was switched on in continuous mode and conducted at 303 K temperature at 150 rpm of agitator speed under non-neutralizing conditions. Effects of feed dilution were investigated by conducting experiments under different dilutions. Dilution rate (h−1) is the rate at which the fresh media is added to the fermentation broth volume present in the fermenter. It is defined as the ratio of the feed stream inflow rate to the working volume of the reactor. Productivity (g L−1 h−1) is the final product concentration of acetic acid (g L−1) multiplied by the dilution rate (h−1) in the system. This is basically 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 that module (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 filtration units were first ultracentrifuged (Sigma Instruments) at 10,000 rpm for 10 min and then analyzed for acetic acid and residual lactose by HPLC (Agilent, Series 1200, U.S.A.). In the acetic acid measurement, the standard was prepared with 99.99% pure acetic acid (Sigma Aldrich, U.S.A.). Ultron ES-OVM Chiral Organic Acid Column (Agilent Technologies, U.S.A.) with Diode Array Detector (DAD) was used with mobile phase acetonitrile (100% pure) and potassium di hydrogen phosphate (20 mili molar aqueous KH2PO4 solution of pH 2.0) at a volume ratio of 1:99 and at a flow rate of 1 mL min−1 with residence time of 2.46 min and injection volume of 10 μL was used for acetic acid detection. The assay of the unconverted carbohydrates (lactose) was performed with an RID detector using Agilent Zorbax carbohydrate analysis column where temperature of the column was maintained at 303 K. The mobile phase for carbohydrate analysis comprised 75% acetonitrile (Sigma Aldrich, U.S.A.) and 25% ultrapure water at a flow rate of 1.4 mL min−1 with injection volume of the samples of 10 μL. The RT values (residence time) of lactose is 7.909 min. Peak purity software tool of HPLC (Agilent, series 1200, U.S.A.) was used to assess the purity of the produced acetic acid after nanofiltration. Ions of the minerals like Na+, K+, and Mg2+ were identified and measured with individual electrodes (Thermo Scientific, U.S.A.). Each of the results of experiments and that of analyses were performed in three sets, and the mean values of three sets for individual parameters were reported. Experimental error was computed to be within

productivity (g L−1 h−1) = product concentration (g L−1) × dilution rate (h−1)

3. RESULTS AND DISCUSSION 3.1. Constant Transmembrane Pressure Cross-Flow Filtration Runs during Fermentation. 3.1.1. Cross Flow Microfiltration at Constant Transmembrane Pressure. A Nylon 0.22 flat sheet membrane used in the cross-flow module successfully separated cells (as evident in cell analysis of the permeate) for recycling while generating clear permeate from the fermentation broth without significant flux decline for over 30 h. It was observed that lower operating pressure facilitated long-term filtration without much reduction in flux. Moreover, the steady state flux could be attained quite quickly at low transmembrane pressure than at higher operating pressure at the mentioned operating cross-flow velocities. Thus during microfiltration, operating transmembrane pressure was maintained at 1 bar, and the experiment was carried out at three cross-flow velocities of 0.53, 0.88, and 1.06 m s−1 with corresponding 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

Figure 2. Permeate flux decline in Nylon 0.22 microfiltration membrane operated at fixed transmembrane pressure of 1 bar and different cross-flow velocities.

cross-flow velocity resulted in higher flux than that attained during the operation at lower cross-flow velocity under identical transmembrane pressure. The flux behavior of the membrane modules at constant transmembrane pressure and at different cross-flow velocities was investigated, and it was observed that the steady state flux could be attained after 4, 6, and 7 h of operation respectively at the above-mentioned three cross-flow velocities. Hydrodynamic conditions under the investigated cross-flow velocity regime did not affect viability and physiological conditions of A. aceti (NCIM-2116) as revealed in the analysis of the cells in the broth. 3.1.2. Continuous Fermentation with Microfiltration and Nanofiltration. Prior to continuous production, fermentation 2980

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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 Working Conditions, Prior to First Stage Nanofiltration during Continuous Runa run 1. 2. 3. a

conditions −1

average acetic acid concentration (g L−1)

product yield (%) (product/substrate) × 100

productivity (g/L/h)

40.65 37.65 34.2

96.0 88.9 80.75

4.14 4.19 4.82

−1

u = 0.53 m s , D = 0.102 h u = 0.88 m s−1, D = 0.128 h−1 u = 1.06 m s−1, D = 0.141 h−1

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

permeates as inoculum. During this preliminary batch fermentation stage, the pH of the fermentation broth dropped from 4.2 to 2.73 when the concentration of produced acetic acid 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 yield reached 76%. Figure 2 shows that the steady state condition was reached within 4 h after starting of microfiltration cell recycle at a cross-flow velocity 0.53 m s−1, where the dilution rate was maintained at 0.102 h−1 and the time requirement for

was initiated in batch mode at 303 K in a membrane-integrated fermentor of 30 L volume and allowed to continue in the mode for 24 h. During this stage of operation, microorganisms were in an active state of exponential growth phase. The changes in the concentrations of cell, carbohydrates, acetic acid, and pH of the medium with time as observed have been presented in Figure 3. No effective period of lag phase of microorganisms was observed at the beginning of fermentation because of the direct use of preinoculated and nutrient-supplemented whey 2981

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reaching steady state increased with increase in cross-flow velocity and dilution rate. Because of the introduction of fresh medium with higher dilution rates of 0.128 h−1 and 0.141 h−1, overall cell mass concentration decreased in the first few hours of microfiltration cell recycle which was gradually recovered with microbial adaption with the conditions of fermentation broth as exhibited in the above-mentioned figures. Acetic acid concentration (40.65 g L−1) with maximum product yield of 96% was achieved under steady state conditions at a cross-flow velocity of 0.53 m s−1. Product concentration, yield, and productivity achieved while microfiltration at three different cross-flow velocities and dilution, prior to nanofiltration are presented in Table 2. The highest productivity achieved was 4.82 g L−1 h−1 during continuous operation with a cross-flow velocity of u = 1.06 m s−1 and dilution rate of D = 0.141 h−1. The concentration of acetic acid of the fully membraneintegrated system in those three runs after nanofiltration reached final concentrations of 35.37 g L−1, 32.76 g L−1, and 31.8 g L−1 at cross-flow velocities of 0.53 m s−1, 0.88 m s−1, and 1.06 m s−1, respectively. Overall system productivities computed after nanofiltration stage were observed to be slightly less than the fermentative productivities and reached 3.61 g L−1 h−1, 4.2 g L−1 h−1, and 4.48 g L−1 h−1 at the corresponding dilution rates of 0.102 h−1, 0.128 h−1, and 0.141 h−1, respectively, with 96% product purity. For the final concentration of the produced acid, another downstream nanofiltration membrane module was pressed into service. An experimentally selected NF-1 nanofiltration membrane of pore size 0.53 nm concentrated acetic acid to the level of 96.9 g L−1. The produced sample, after carrying out the long hours of continuous fermentation and two stage membrane treatment (MF and NF), contains only acetic acid and the remaining amount of unconverted lactose in an aqueous solution. The NF-1 membrane operated at high transmembrane pressure allowed water to permeate through it while retaining acetic acid (85%) and the residual unconverted carbohydrate. After 14 h of nanofiltration in recycling mode, acetic acid with initial concentration of 35.37 g L−1 was concentrated to the level of 96.9 g L−1 . This ultimate product was found to be 94.6% pure as 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 size of 0.57 nm) in the nanofiltration module resulted in high permeation of acetic acid with retention of about 97% of the residual lactose. The flux profile of the nanofiltration membrane as shown in Figure 4 exhibits a positive correlation of cross-flow velocity with permeate flux at a constant transmembrane pressure. Very little fouling problem was encountered during nanofiltration because of filtration of a low viscosity fluid at this stage and the very flat sheet cross-flow modular design of the system. In Figures 2 and 4, the permeate flux decline characteristics of Nylon 0.22(microfiltration) and NF-2 (nanofiltration) with respect to time are shown. The flux during both microfiltration and nanofiltration, first decreases, then reaches a steady state and sustains over a longer period and then finally begins declining. At the starting of the process, all the pores remain open and the permeation starts with a high magnitude permeate flux. During the present investigations, after around 30 and 26 h respectively, concentration polarization in nanofiltration membranes and pore blocking in microfiltration membranes started with the onset of flux decline. Thus these 26 or 30 h are not necessarily indicators of the maximum operating time. Rather these time periods are merely indicators of the

Figure 4. Permeate flux decline in the NF-2 nanofiltration membrane operated at fixed transmembrane pressure of 12 bar and different cross-flow velocities.

time when the corresponding membranes should be cleaned and reused to allow operation with reasonable flux over a much longer time. Used membranes may be cleaned (as mentioned in section 2.4, Experimental Equipment) and reused. By the very choice of flat sheet cross-flow membrane modules, fouling problems could be largely overcome because of the high sweeping action of the fluid on the membrane surface but, because of operation over a prolonged time with such a dense fermentation broth containing microbes and nutrients, clogging of the porous membranes may take place. Thus after 26 h in the case of the Nylon 0.22 microfiltration membrane and after 30 h inthe case of the NF-2 membrane, the permeate flux considerably decreases. Rejection trends for lactose and acetic acid at increasing transmembrane pressure have been shown in Figure 5. While using the NF-2 membrane, acetic acid rejection

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

increased from 10% to 15% with almost 98% rejection of lactose when transmembrane pressure was increased from 12 bar to 15 bar. Lower cross-flow velocity of 0.53 m s−1 maintained during microfiltration turned out to be more helpful in this system to generate more concentrated acetic acid than 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% and productivity 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) was associated with a highest dilution rate of 0.141 h−1. During continuous operation, if the cross-flow velocity is kept at a low value (0.53 m s−1) the permeate flux decreases leading to 2982

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considerable decline in the rate of dilution (0.102 h−1). Subsequently, the rate of addition of fresh feed into the fermenter is low allowing long enough residence time to microbes to intake nutrients and substrates, resulting in higher product concentration (40.65 g L−1) and product yield (96%) after microfiltration treatment than those obtained while operating at higher cross-flow velocities. By setting higher cross-flow velocities during microfiltration, higher permeate flux is obtained because of the high sweeping action on the membrane surface. Thus increase of cross-flow velocity (0.106 m s−1) increases feed dilution (0.141 h−1) or the rate at which fresh feed is added to the fermenter. This condition allows low residence time to the microbes to produce the desired acetic acid 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 higher cross-flow velocity than that obtained (4.14 g L−1 h−1) in case of lower cross-flow velocity, it is better to operate the system at some lower cross-flow velocity during microfiltration to achieve the maximum conversion of carbohydrate to acetic acid in a continuous fermentation process. Higher productivity has been reported in some literatures, but those were achieved using an expensive carbon source. In many such cases,17−19 the production process is a batch fermentation one resulting in low 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 as feed supplement, the reported maximum yield of acetic acid is 0.55 g g−1 with a productivity of 0.15 g L−1 h−1 where a single stage membrane system was utilized.23 Though, most of the case studies were carried out using pure carbohydrate solutions as substrate for fermentation but ended up with low productivities. From that perspective, this work of continuous production of acetic acid, exploiting cheese whey in a membrane integrated hybrid reactor system, may be claimed to 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 of waste raw material as carbon source, non-neutralizing operation conditions ensuring direct production of acetic acid, the flexibility of the associated modular design and capability of continuous production of acetic acid with high concentration, product yield, productivity, and purity have all contributed to development of a novel process of acetic acid production from cheese whey.

and fast production of acetic acid overcoming the difficulties of substrate-product inhibitions, and involvement of multiple steps of separation and purification. Membrane separation has helped to achieve a high degree of separation and purification also. Membrane separation at the microfiltration regime has helped to separate and recycle microbial cells from the fermentation medium facilitating fermentation with high cell density. Pretreatment of cheese whey by ultrafiltration eliminates the chance of lag phase in microbes during fermentation. The post treatment unit of nanofiltration concentrates the produced acetic acid further without involving any energy-intensive evaporation step. The whole process involves no phase change and no harsh chemicals thus representing a high degree of process intensification which is very significant and being sought urgently by chemical process industries across the world.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +91343-2754078. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge financial support under DST-FIST Program of the Government of India. Sincere thanks are also extended to National Chemical Laboratory, Pune, India, for providing the necessary microbial strains.

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REFERENCES

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5. CONCLUSIONS High purity acetic acid was produced in a continuous process without any requirement of pH adjustment in a fully membrane-integrated fermentation process using waste raw material. The modular design of those flat sheet cross-flow modules used throughout the study permitted use of any number of active modules in it for the ultrafiltration, microfiltration, or nanofiltration step during steady state operation. Steady operation could be attained for a constant transmembrane operating pressure as well as for constant fermentor capacity. The study culminates in the development of a novel process of acetic acid production where conventional fermentative production of acetic acid from a waste material has been integrated with downstream membrane-based separation and purification. Membrane-integration has permitted continuous 2983

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