- Email: [email protected]
Separation of cells and proteins from fermentation broth using ultrafiltration
Journal of Food Engineering 75 (2006) 574–580 www.elsevier.com/locate/jfoodeng
Separation of cells and proteins from fermentation broth using ultrafiltration Yebo Li a
a,*
, Abolghasem Shahbazi a, Charles T. Kadzere
b
Bioenvironmental Engineering, Department of Natural Resources and Environmental Design, North Carolina A&T State University, Greensboro, NC, USA b Department of Animal Sciences, North Carolina A & T State University, Greensboro, NC, USA Received 27 July 2004; received in revised form 9 April 2005; accepted 26 April 2005 Available online 6 July 2005
Abstract Ultrafiltration of fermented cheese whey broth was studied using a lab scale cross-flow membrane system. The molecular weight cut-offs (MWCO) of the used membranes were 5000 Da and 20,000 Da. The experiments were conducted at five levels of transmembrane pressure (70, 140, 210, 280, and 420 kPa), two temperatures (21 and 37 C) and two cross-flow velocities (1 m/s and 2 m/s). The permeate flow-rate decreased with time due to fouling of the membrane. Higher transmembrane pressure and cross- flow velocity caused higher permeate flux. Transmembrane pressure and cross-flow velocity had no significant effect (P > 0.1) on protein retention ratio. Higher MWCO of the membrane caused higher permeate flux and reduced the crude protein retention ratio. However, the MWCO of membrane had no significant effect on the protein retention ratio. 2005 Elsevier Ltd. All rights reserved. Keywords: Cheese whey; Fermentation; Lactic acid; Membrane; Protein; Ultrafiltration
1. Introduction Manufacturing of cheese produces large volumes of whey as a by-product, which must be disposed of. The United States generates nearly 1.2 billion tons of cheese whey per year (Shahbazi et al., 2005). It is estimated that as much as 40–50% of the whey produced is disposed of as sewage or as fertilizer applied to agricultural lands with the rest being used primarily as animal feed. Cheese whey contains about 4.5–5% lactose, 0.6–0.8% soluble proteins, 0.4–0.5% w/v lipids and varying concentrations of mineral salts (Siso, 1996). Therefore, there is an interest to utilize lactose from cheese whey in the production of value-added products. Lactic acid is one such * Corresponding author. Address: 123 Sockwell Hall, 1601 East Market Street, Greensboro, NC 27411, USA. Tel.: +1 336 334 7787; fax: +1 336 334 7270. E-mail address: [email protected] (Y. Li).
0260-8774/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2005.04.045
value-added product that is produced from processing cheese whey. Lactic acid is a natural organic acid and has many applications in the pharmaceutical, food, and chemical industries. It is used as an acidulant and as a preservative, and also as a substrate in the production of biodegradable plastics and other organic acids (Shahbazi et al., 2005; Tango & Ghaly, 2002). The processes of lactic acid production include two key stages which are (a) fermentation and (b) product recovery. The biggest challenge in lactic acid production lies in the recovery and not in the fermentation step (Atkinson & Mavituna, 1991). Usually, most of the separation of microorganisms from fermentation broth is performed by centrifugation. Recently, cross-flow microfiltration has been used to separate cells in continuous fermentation processes (Persson, Jonsson, & Zacchi, 2001). A successful lactic acid recovery approach has been that of continuous fermentation in a cell-recycled reactor where the cells are separated by a filtration
Y. Li et al. / Journal of Food Engineering 75 (2006) 574–580
unit and returned to the fermentor while the product is removed in the permeate (Jeantet, Maubois, & Boyaval, 1996; Senthuran, Senthuran, Mattiasson, & Kaul, 1997). The long term performance of membrane units at high cell densities is affected by the fouling of filtration membranes, which require extensive cleaning protocols (Hjorleifsdottir, Holst, & Mattiasson, 1991). Ultrafiltration is a pressure-driven membrane separation process that is increasingly being used in water and wastewater treatment processes, and in the pharmaceutical and liquid food industries. Ultrafiltration membranes are characterized by their nominal molecular weight cut-offs (MWCO). All the molecules larger than the MWCO of a particular membrane are retained while those molecules smaller than the MWCO can pass through the membrane (Atkinson & Mavituna, 1991). In cross-flow filtration, the direction of feed flow is tangential to the membrane surface. As a result, accumulation of filtered solids can be minimized by the sharing action of the flow (Goldberg, 1997). Ultrafiltration can remove dissolved macromolecules with MWCO between 1000 and 100,000 Da (Vigneswaran & Kiat, 1988). An important hurdle in the application of membrane technology in whey processing is the decline in permeate flux during the operation. The permeate flux decline during ultrafiltration of cheese whey is attributed to concentration polarization and membrane fouling (Caric, Milanovic, Krstic, & Tekic, 1999). The permeate flux declines from the beginning of filtration, initially falling rapidly and later leveling off to a rate that is dependent on media concentration, membrane MWCO and flow conditions including cross-flow velocity and transmembrane pressure (Marijana, Spasenija, Darko, & Miodrag, 2000). This study was part of a process to develop a method to produce lactic acid from cheese whey, which focused on cell and protein separation and recirculation, the first step in downstream processing of cheese whey. To produce lactic acid from cheese whey, the whey protein must be separated together with cells from the fermentation broth. The objective of these experiments was to study the performance of cross-flow ultrafiltration on protein and cell separation and to evaluate the effects of membrane MWCO, transmembrane pressure, and cross-flow velocity on the permeate flux, and on the retention of protein.
2. Materials and methods 2.1. Cheese whey media Cheese whey media was prepared by dissolving 50 g of deproteinized cheese whey powder (Davisco Foods International, Inc., Eden Prairie, MN) per liter of deionized (DI) water and stirring for 5 min at ambient tem-
575
perature. The composition of the deproteinized cheese whey powder was as follows: crude protein (total nitrogen · 6.38) 6.8%, crude fat 0.8%, lactose 78.6%, ash 9.4%, and moisture 4.4%. The solutions were autoclaved at 103 C for 10 min. 2.2. Microorganism and culture media Bifidobacteria longum was obtained from the National Collection of Food Bacteria (NCFB 2259). Stock culture of this strain was maintained in 50% glycerol and Man Rogosa Sharpe (MRS) broth media at 80 C. Active cultures were propagated in 10 ml MRS broth at a temperature of 37 C for 18–24 h under anaerobic conditions. This was used as a preculture to initiate cell production of higher volume with a 1% inoculation into 100 ml fresh MRS broth, incubated at 37 C for 24 h. 2.3. Fermentation Fermentation was conducted in a stirred 5.0-L bench top fermentor. The pH of the broth was maintained at 6.5 by neutralizing the acid with 5 N ammonium hydroxide during fermentation. The agitation speed of the fermentor was maintained at 150 rpm, while the temperature was maintained at 37 C. Samples were withdrawn every 6 h intervals and analyzed for lactose, lactic acid, and acetic acid. After a fermentation period of 48 h, the broth was pumped into the membrane unit for separation. 2.4. Membrane system The ultrafiltration membrane system consisted of a recirculation pump, cross-flow ultrafiltration module (OPTISEP, North Carolina SRT, Inc., Cary, NC), and an online permeate weighting unit (Fig. 1). The media was fed from the fermentor at constant velocities via the recirculation pump. The concentrate was recycled to the fermentor while permeate was collected in a reservoir placed on an electronic balance. The balance was interfaced via RS232 to a computer that continually recorded time and permeate weight at 30 s intervals. The transmembrane pressure and cross-flow velocity were adjusted by a manual valve and pump controller. The pressure was measured by a standard pressure gauge. The selected cross-flow velocities were 1 and 2 m/s. The time required to pass 1 L of liquid through the membrane unit was 16.6 s and 8.3 s at membrane cross-flow velocities of 1 m/s and 2 m/s, respectively. The time required to circulate 1 L of liquid through the membrane system was set by adjusting the flow-rate of the pump. The pressure levels used in the unit were 70, 140, 210, 280, and 420 kPa. Membranes (PES5 and PES20, Nadir Filtration GmbH, Wiesbaden, Germany) of MWCO of 5000 and
576
Y. Li et al. / Journal of Food Engineering 75 (2006) 574–580 Concentrate back to fermentor
SD
P rofessionalW orkstation 6000
Permeate Tank Recirculation pump
Membrane unit
Balance
Fermentor
Fig. 1. Schematic diagram of the ultrafiltration membrane separation system.
20,000 Da were used in these experiments. The surface area of the membrane was 0.02 m2. The membrane polymer consisted of permanently hydrophilic polyethersulfone and polysulfone. After each run, the membrane was cleaned by an alkali-acid treatment method in the following steps: (a) fully open the recirculation and permeate valves, (b) flush with tap water for 5 min, (c) circulate 2 L of 4% phosphoric acid for 15 min, (d) rinse with tap water for 5 min, (e) circulate 2 L of 0.1 N NaOH solution for 15 min, and (f) rinse with 5 L of DI water. 2.5. Analyses Lactose, lactic acid, and acetic acid were determined by a high-performance liquid chromatography (Waters, Milford, MA) with a KC-811 ion exclusion column and a Waters 410 differential refractometer detector. The mobile phase was 0.1% H3PO4 solution at a flow-rate of 1 ml/min. The temperatures of the detector and of the column were maintained at 35 C and 60 C respectively. The total nitrogen was analyzed using the macroKjeldahl method. Samples were digested using a block digestion (FOSS Tecator, Sweden) and analyzed for nitrogen on a Tecator Kjeltec auto 2400 analyzer (FOSS Tecator, Sweden) as described in Foss Tecator Application Note, AN300 (Foss Tecator, 1999). When the protein nitrogen was determined, the samples were precipitated using a trichloroacetic (TCA) solution before nitrogen analysis (AOAC, 1995). The digestion and analysis procedure for crude protein was the same as that for total nitrogen analysis. The lactic acid productivity was evaluated by (a) lactic acid yield and (b) conversion ratio. The conversion ratio was expressed as follows: Conversion ratio ð%Þ initial lactose conc: residual lactose conc. ¼ 100% initial lactose conc. ð1Þ
The lactic acid yield was expressed as grams of lactic acid produced per gram of lactose used. Lactic acid yield ðg=gÞ ¼
lactic acid produced lactose used
ð2Þ
The membrane separation of cheese whey was evaluated by using two criteria: (a) permeate flux and (b) protein retention. The permeate flux was calculated by measuring the quantity of permeate collected during a certain time and dividing it by the effective membrane area for filtration. Permeate flux; J permeate volume ¼ ðL m2 h1 Þ membrane area time The protein retention ratio was defined as CP Retention ratio ð%Þ; R ¼ 1 100 CF
ð3Þ
ð4Þ
CF is the concentration of protein in feed stream; CP is the concentration of protein in permeate.
3. Results and discussion 3.1. Fermentation The fermentation time of cheese whey in the production of lactic acid by B. longum was 48 h. The lactose, lactic acid, and acetic acid concentrations shown in Table 1 are average values from three runs. The lactose, lactic acid, and acetic acid concentration in the fermentation broth after 48 h was 2.2, 25.6 and 1.0 g/L, respectively (Table 1). The standard error of the lactose and lactic acid concentration are also shown in Table 1. The results in Table 1 shows that about 94.5% of the lactose was converted and that 0.65 g lactic acid was produced from one gram of lactose used. The production of acetic acid is trivial in comparison to that of lactic
Y. Li et al. / Journal of Food Engineering 75 (2006) 574–580
577
Table 1 The lactose conversion ratio, lactic acid yield and production efficiency using immobilized B. longerm in bioreactor Time (h)
Lactose concentration (g/L)
Lactic acid concentration (g/L)
Acetic acid concentration (g/L)
Conversion ratio (%)
Yield (g lactic acid/g lactose)
0 2 4 6 8 12 24 36 48
39.6±0.2 37.7±0.9 34.6±2.1 32.9±2.1 33.6±1.4 29.7±1.4 22.9±1.7 11.2±1.0 2.2±2.1
1.2±0.1 1.9±0.3 2.8±0.3 3.6±0.4 4.7±0.8 5.8±1.1 10.4±2.1 18.6±1.1 25.6±1.0
0.0 0.4 0.4 0.6 0.6 0.7 0.8 0.9 1.0
4.9 12.7 17.0 15.2 25.1 42.3 71.8 94.5
0.36 0.32 0.35 0.58 0.46 0.54 0.61 0.65
acid production. The lactose conversion ratio is similar to that of other lactic acid producing bacteria such as Lactobacillus helveticus while the lactic acid yield is lower than that of L. helveticus. Tango and Ghaly (2002) obtained a lactose utilization value of 92–95% and a lactic acid yield of 0.86 g lactic acid/g lactose when using immobilized L. helveticus with nutrient supplement at 36 h of fermentation. The current results are better than our previous findings in which we obtained a lactose conversion ratio of 69% and a lactic acid yield of 0.51 g lactic acid/g lactose utilized (Shahbazi et al., 2005). As there was no nutrient supplement used in the current experiments, we postulate that with optimized fermentation conditions which will include the pH and a nutrient supplement, the lactic acid yield could be further improved. 3.2. Permeate flux The performance of two membranes with MWCO of 5000 (PES 5) and 20,000 (PES20) was tested with different cross-flow velocity and transmembrane pressure. Fig. 2 shows the changes in permeate flux over time when a membrane with MWCO of 20,000 Da was used under pressure of 280 kPa at 21 C and 37 C. The fer-
80
Permeate flux (L/m2h)
70
2m/s, 37 oC
60
1m/s, 37 oC
50 40
2m/s, 21 oC 30 20
1m/s, 21 oC
10 0
20
40
60
80
100
Time (min)
Fig. 2. Change of permeate flux over time.
120
mentation broth was maintained at pH 6.5 during separation. The permeate flux values shown in Fig. 2 are average values of duplicate tests. The standard errors of the permeate flux for the four tests at 2 m/s, 37 C; 1 m/s, 37 C; 2 m/s, 21 C; and 1 m/s, 21 C were 4.56, 3.43, 1.81, and 0.50, respectively. It is evident from Fig. 2, that an increase of cross-flow velocity caused a higher permeate flux. The decrease in permeate flux over time is a result of fouling of the membranes. These results show that the permeate flux of cheese whey broth was higher at a higher temperature (Fig. 2). Increasing the cross-flow velocity also resulted in an increase of the permeate flow-rate. These results are in agreement with the findings of Suki, Fane, and Fell (1984) who reported a permeate flux decrease in protein ultrafiltration and also with Barhate, Subramanian, Nandini, and Hebbar (2003) who demonstrated that the permeate flux decreased during the processing of honey using polymeric ultrafiltration membranes. The results are also consistent with the findings of Vigneswaran and Kiat (1988) who showed that increased temperature and larger MWCO caused higher permeate flux during ultrafiltration of polyvinyl alcohol. Fig. 3 shows the effects of transmembrane pressure, cross-flow velocity and MWCO on the permeate flux at 21 C. The fermentation broth was obtained by fermentation for 48 h at pH 6.5. Each separation test lasted 2 h and the permeate flux was calculated based on the permeate volume collected in the 2-h test. The permeate flux values in Fig. 3 are the average of two duplicate tests. It can be discerned that increased transmembrane pressure caused an increase of the permeate flux. Beyond a certain pressure, the increase in permeate flux with pressure was negligible which indicates that there is an optimum pressure to obtain the maximum permeate flux. Similar results were also reported by Vigneswaran and Kiat (1988) who obtained the optimum pressure for maximum permeate flux during the ultrafiltration of polyvinyl alcohol solution at different concentrations. Results in Fig. 3 indicate that higher cross-flow velocity caused higher permeate flux for the membrane with
578
Y. Li et al. / Journal of Food Engineering 75 (2006) 574–580 35
30
Permeate Flux (L/m2h)
Permeate Flux (L/m2h)
35
Cross flow velocity 2 m/s
25 20 15 10 5
Cross flow velocity 1 m/s
25 20 15 10 Cross flow velocity 1 m/s
5
0
0 0
100
a
200
300
400
500
0
100
b
Pressure (kPa)
200
300
400
500
Pressure (kPa) 35
30
Permeate Flux (L/m2h)
35
Permeate Flux (L/m2h)
Cross flow velocity 2 m/s
30
20,000 Da
25 20 15 10 5
30
20,000 Da
25 20 15 10
5,000 Da
5
5,000 Da
0
0 0
100
c
200
300
400
0
500
d
Pressure (kPa)
100
200
300
400
500
Pressure (kPa)
Fig. 3. Effect of transmembrane pressure, cross-flow velocity, and membrane cutoff on permeate flux: (a) MWCO: 5000 Da, (b) MWCO: 20,000 Da, (c) cross-flow velocity: 1 m/s and (d) cross-flow velocity: 2 m/s.
Table 2 Analysis of variance of the permeate flux Source
DF
SS
F-value
P>F
MWCO Velocity MWCO * velocity Pressure MWCO * pressure Velocity * pressure MWCO * velocity * pressure
1 1 1 4 4 4 4
526.1 88.8 54.2 1560.5 29.5 17.2 10.4
149.2 25.2 15.4 110.6 2.1 1.2 0.7
<0.0001 <0.0001 0.0009 <0.0001 0.1223 0.3360 0.5780
DF: Degree of freedom, SS: sum of squares.
MWCO of both 5000 and 20,000 Da. At the same crossflow velocity, the membrane with MWCO of 20,000 Da had a higher permeate flux than that with MWCO of 5000 Da. Table 2 shows the results of the analysis of variance performed on the permeate flux data using a statistical package from the SAS System (SAS Institute, Cary, NC). The MWCO, pressure and cross-flow velocity showed significant (P < 0.0001) effects on the permeate flux. Most of the interactions between the parameters were not significant. 3.3. Protein retention Fig. 4 shows the effect of transmembrane pressure and cross-flow velocity on the crude protein retention ratio. Table 3 shows the results of the analysis of variance performed on the data of crude protein retention ratio. Only the MWCO had a significant (P < 0.0001) ef-
fect on the crude protein retention ratio flux. The higher MWCO caused a significantly lower protein retention ratio. The effect of pressure and cross-flow velocity had no significant (P > 0.5) effect on the crude protein retention ratio. The interactions between these parameters had no significant effects (P > 0.004) on the crude protein retention ratio. The average crude protein and protein retention ratios for membranes with MWCO of 5000 Da and 20,000 Da are shown in Table 4. It can be seen that most of the protein is retained by the ultrafiltration membranes with both MWCO of 5000 and 20,000 Da. We conclude that most of the detected raw protein in permeate is non-protein nitrogen, and has smaller MWCO than protein. These results are in agreement with the findings of Torang, Jonsson, and Zacchi (1999) who found that most of the hydrolytic proteins were retained by the hydrophilic membrane with MWCO of 20,000 Da and no protein was detected in the permeate when hydrophobic membrane with MWCO of 25000 Da was used. From our findings, it is recommended that membranes with MWCO of around 20,000 Da be used for the ultrafiltration of cheese whey fermentation broth. This is in line with the results that the membrane with the MWCO of 20,000 Da had a higher permeate flux when compared to that with a MWCO of 5000 Da and yet there was little difference between the protein retention ratios of the two membranes, 93.99% and 94.12% respectively.
Y. Li et al. / Journal of Food Engineering 75 (2006) 574–580 1.0
1.0
2 m/s
1 m/s
0.8
Protein retention ratio
Protein retention ratio
2 m/s
0.6 0.4 0.2
1 m/s
0.8
0.6
0.4
0.2
0.0
0.0
0
70
140
210
a
280
350
420
490
0
70
140
b
Pressure (kPa)
210
280
350
420
490
420
490
Pressure (kPa) 1.0
1.0
5,000 Da
20,000 Da
5,000 Da
20,000 Da
0.8
Protein retention ratio
0.8
Protein retention ratio
579
0.6
0.4
0.2
0.6 0.4 0.2 0.0
0.0 0
70
140
c
210
280
350
420
0
490
70
140
210
280
350
Pressure (kPa)
d
Pressure (kPa)
Fig. 4. Effect of transmembrane pressure and MWCO on the retention of crude protein: (a) MWCO: 20,000 Da, (b) MWCO: 5000 Da, (c) cross-flow velocity 1 m/s and (d) cross-flow velocity 2 m/s.
Table 3 Analysis of variance for protein retention Source
DF
SS
F-value
P>F
MWCO Velocity MWCO * velocity Pressure MWCO * pressure Velocity * pressure MWCO * velocity * pressure
1 1 1 4 4 4 4
0.3050 0.0001 0.0024 0.0005 0.0019 0.0044 0.0048
1368.71 0.17 10.57 0.55 2.16 4.98 5.42
<.0001 0.6842 0.0042 0.7017 0.1122 0.0065 0.0044
effect of cross-flow velocity and permeate flux on the crude protein and protein retention ratio was not significant. 3. Increasing the membrane MWCO from 5000 Da to 20,000 Da caused a significantly higher permeate flux and lower crude protein retention ratio, but the effect of membrane MWCO on the protein retention ratio was not significant.
Acknowledgement Table 4 Comparison of the average retention of protein and raw protein of ultrafiltration of cheese whey broth Constituent
Raw protein (total N * 6.38) Protein
Retention ratio (%) 5000 Da
20,000 Da
71.97 93.99
53.93 94.12
4. Conclusion It is concluded that the ultrafiltration method to separate protein and bacteria from cheese whey through a membrane can successfully be used. 1. Nearly all cells and proteins were retained by the ultrafiltration membrane with MWCO of 20,000 Das. 2. Increased transmembrane pressure and cross-flow velocity caused higher permeate flux, although the
Financial support from USDA-CSREES EvansAllen Program and USDA Capacity Building Program (contract number 2004-38814-15095) is greatly appreciated.
References Atkinson, B., & Mavituna, F. (1991). Biochemical engineering and biotechnology handbook pp. 1181–1182 (2nd ed.). New York, NY: Stockton Press. Barhate, R. S., Subramanian Nandini, K. E., & Hebbar, H. U. (2003). Processing of honey using polymeric microfiltration and ultrafiltration membranes. Journal of Food Engineering, 60, 49–54. Caric, M. D., Milanovic, S. D., Krstic, D. M., & Tekic, M. N. (1999). Fouling of inorganic membranes by adsorption of proteins. Journal of Membrane Science, 165, 83–88. Foss Tecator (1999). Application note. The determination of nitrogen according to kjeldahl using block digestion and steam distillation. 2400/24600 Kjetec Auto Sampler System. User Manual 10009165. Rev1.1.
580
Y. Li et al. / Journal of Food Engineering 75 (2006) 574–580
Goldberg, E. (1997). Handbook of downstream processing (pp. 92–136). London; New York: Vlackie Academic and Professional. Hjorleifsdottir, S., Holst, O., & Mattiasson, B. (1991). Effects on product formation in lactococcus-lactis-65.1 in continuous culture with complete cell recycling. Bioprocess Engineering, 6(1–2), 29–34. Jeantet, R., Maubois, J. L., & Boyaval, P. (1996). Semicontinuous production of lactic acid in a bioreactor coupled with nanofiltration membranes. Enzyme and Microbial Technology, 19(8), 614–619. Marijana, D. C., Spasenija, D. M., Darko, M. K., & Miodrag, N. T. (2000). Fouling of inorganic membranes by adsorption of whey proteins. Journal of Membrane Science, 165, 83–88. AOAC International (1995). Official methods of analysis of AOAC international (17th ed.). S587.A3: AOAC Official Method 991.22, Protein Nitrogen Content of Milk. Arlington, VA: AOAC International. Persson, A., Jonsson, A., & Zacchi, G. (2001). Separation of lactic acid-producing bacteria from fermentation broth using a ceramic microfiltration membrane with constant permeate flow. Biotechnology and Bioengineering, 72(3), 269–277. Senthuran, A., Senthuran, V., Mattiasson, B., & Kaul, R. (1997). Lactic acid fermentation in a recycle batch reactor using immobi-
lized Lactobacillus casei. Biotechnology and Bioengineering, 55(6), 841–853. Siso, M. I. G. (1996). The biotechnological utilization of cheese whey: a review. Bioresource Technology, 57, 1–11. Shahbazi, A., Mims, M., Li, Y., Ibrahim, S. A., Vest, S., & Morris, A. (2005). Performance of immobilized Bifidobacteria in converting lactose to lactic acid. Applied Biochemistry and Biotechnology, 121– 124, 529–540. Suki, A., Fane, A. G., & Fell, C. J. D. (1984). Flux decline in protein ultrafiltration. Journal of Membrane Science, 21, 269–283. Tango, M. S. A., & Ghaly, A. E. (2002). A continuous lactic acid production system using an immobilized packed bed of Lactobacillus helveticus. Applied Microbiology and Biotechnology, 58, 712–720. Torang, A., Jonsson, A. S., & Zacchi, G. (1999). Separation of cells and proteins from fermentation broth in a shear-enhanced cross-flow ultrafiltration module as the first step in the refinement of lactic acid. Applied Biochemistry and Biotechnology, 76, 143– 157. Vigneswaran, S., & Kiat, W. Y. (1988). Detailed investigation of effects of operating parameters of ultrafiltration using laboratory-scale ultrafiltration unit. Desalination, 70, 299–316.
Separation of cells and proteins from fermentation broth using ultrafiltration Yebo Li a
a,*
, Abolghasem Shahbazi a, Charles T. Kadzere
b
Bioenvironmental Engineering, Department of Natural Resources and Environmental Design, North Carolina A&T State University, Greensboro, NC, USA b Department of Animal Sciences, North Carolina A & T State University, Greensboro, NC, USA Received 27 July 2004; received in revised form 9 April 2005; accepted 26 April 2005 Available online 6 July 2005
Abstract Ultrafiltration of fermented cheese whey broth was studied using a lab scale cross-flow membrane system. The molecular weight cut-offs (MWCO) of the used membranes were 5000 Da and 20,000 Da. The experiments were conducted at five levels of transmembrane pressure (70, 140, 210, 280, and 420 kPa), two temperatures (21 and 37 C) and two cross-flow velocities (1 m/s and 2 m/s). The permeate flow-rate decreased with time due to fouling of the membrane. Higher transmembrane pressure and cross- flow velocity caused higher permeate flux. Transmembrane pressure and cross-flow velocity had no significant effect (P > 0.1) on protein retention ratio. Higher MWCO of the membrane caused higher permeate flux and reduced the crude protein retention ratio. However, the MWCO of membrane had no significant effect on the protein retention ratio. 2005 Elsevier Ltd. All rights reserved. Keywords: Cheese whey; Fermentation; Lactic acid; Membrane; Protein; Ultrafiltration
1. Introduction Manufacturing of cheese produces large volumes of whey as a by-product, which must be disposed of. The United States generates nearly 1.2 billion tons of cheese whey per year (Shahbazi et al., 2005). It is estimated that as much as 40–50% of the whey produced is disposed of as sewage or as fertilizer applied to agricultural lands with the rest being used primarily as animal feed. Cheese whey contains about 4.5–5% lactose, 0.6–0.8% soluble proteins, 0.4–0.5% w/v lipids and varying concentrations of mineral salts (Siso, 1996). Therefore, there is an interest to utilize lactose from cheese whey in the production of value-added products. Lactic acid is one such * Corresponding author. Address: 123 Sockwell Hall, 1601 East Market Street, Greensboro, NC 27411, USA. Tel.: +1 336 334 7787; fax: +1 336 334 7270. E-mail address: [email protected] (Y. Li).
0260-8774/$ - see front matter 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2005.04.045
value-added product that is produced from processing cheese whey. Lactic acid is a natural organic acid and has many applications in the pharmaceutical, food, and chemical industries. It is used as an acidulant and as a preservative, and also as a substrate in the production of biodegradable plastics and other organic acids (Shahbazi et al., 2005; Tango & Ghaly, 2002). The processes of lactic acid production include two key stages which are (a) fermentation and (b) product recovery. The biggest challenge in lactic acid production lies in the recovery and not in the fermentation step (Atkinson & Mavituna, 1991). Usually, most of the separation of microorganisms from fermentation broth is performed by centrifugation. Recently, cross-flow microfiltration has been used to separate cells in continuous fermentation processes (Persson, Jonsson, & Zacchi, 2001). A successful lactic acid recovery approach has been that of continuous fermentation in a cell-recycled reactor where the cells are separated by a filtration
Y. Li et al. / Journal of Food Engineering 75 (2006) 574–580
unit and returned to the fermentor while the product is removed in the permeate (Jeantet, Maubois, & Boyaval, 1996; Senthuran, Senthuran, Mattiasson, & Kaul, 1997). The long term performance of membrane units at high cell densities is affected by the fouling of filtration membranes, which require extensive cleaning protocols (Hjorleifsdottir, Holst, & Mattiasson, 1991). Ultrafiltration is a pressure-driven membrane separation process that is increasingly being used in water and wastewater treatment processes, and in the pharmaceutical and liquid food industries. Ultrafiltration membranes are characterized by their nominal molecular weight cut-offs (MWCO). All the molecules larger than the MWCO of a particular membrane are retained while those molecules smaller than the MWCO can pass through the membrane (Atkinson & Mavituna, 1991). In cross-flow filtration, the direction of feed flow is tangential to the membrane surface. As a result, accumulation of filtered solids can be minimized by the sharing action of the flow (Goldberg, 1997). Ultrafiltration can remove dissolved macromolecules with MWCO between 1000 and 100,000 Da (Vigneswaran & Kiat, 1988). An important hurdle in the application of membrane technology in whey processing is the decline in permeate flux during the operation. The permeate flux decline during ultrafiltration of cheese whey is attributed to concentration polarization and membrane fouling (Caric, Milanovic, Krstic, & Tekic, 1999). The permeate flux declines from the beginning of filtration, initially falling rapidly and later leveling off to a rate that is dependent on media concentration, membrane MWCO and flow conditions including cross-flow velocity and transmembrane pressure (Marijana, Spasenija, Darko, & Miodrag, 2000). This study was part of a process to develop a method to produce lactic acid from cheese whey, which focused on cell and protein separation and recirculation, the first step in downstream processing of cheese whey. To produce lactic acid from cheese whey, the whey protein must be separated together with cells from the fermentation broth. The objective of these experiments was to study the performance of cross-flow ultrafiltration on protein and cell separation and to evaluate the effects of membrane MWCO, transmembrane pressure, and cross-flow velocity on the permeate flux, and on the retention of protein.
2. Materials and methods 2.1. Cheese whey media Cheese whey media was prepared by dissolving 50 g of deproteinized cheese whey powder (Davisco Foods International, Inc., Eden Prairie, MN) per liter of deionized (DI) water and stirring for 5 min at ambient tem-
575
perature. The composition of the deproteinized cheese whey powder was as follows: crude protein (total nitrogen · 6.38) 6.8%, crude fat 0.8%, lactose 78.6%, ash 9.4%, and moisture 4.4%. The solutions were autoclaved at 103 C for 10 min. 2.2. Microorganism and culture media Bifidobacteria longum was obtained from the National Collection of Food Bacteria (NCFB 2259). Stock culture of this strain was maintained in 50% glycerol and Man Rogosa Sharpe (MRS) broth media at 80 C. Active cultures were propagated in 10 ml MRS broth at a temperature of 37 C for 18–24 h under anaerobic conditions. This was used as a preculture to initiate cell production of higher volume with a 1% inoculation into 100 ml fresh MRS broth, incubated at 37 C for 24 h. 2.3. Fermentation Fermentation was conducted in a stirred 5.0-L bench top fermentor. The pH of the broth was maintained at 6.5 by neutralizing the acid with 5 N ammonium hydroxide during fermentation. The agitation speed of the fermentor was maintained at 150 rpm, while the temperature was maintained at 37 C. Samples were withdrawn every 6 h intervals and analyzed for lactose, lactic acid, and acetic acid. After a fermentation period of 48 h, the broth was pumped into the membrane unit for separation. 2.4. Membrane system The ultrafiltration membrane system consisted of a recirculation pump, cross-flow ultrafiltration module (OPTISEP, North Carolina SRT, Inc., Cary, NC), and an online permeate weighting unit (Fig. 1). The media was fed from the fermentor at constant velocities via the recirculation pump. The concentrate was recycled to the fermentor while permeate was collected in a reservoir placed on an electronic balance. The balance was interfaced via RS232 to a computer that continually recorded time and permeate weight at 30 s intervals. The transmembrane pressure and cross-flow velocity were adjusted by a manual valve and pump controller. The pressure was measured by a standard pressure gauge. The selected cross-flow velocities were 1 and 2 m/s. The time required to pass 1 L of liquid through the membrane unit was 16.6 s and 8.3 s at membrane cross-flow velocities of 1 m/s and 2 m/s, respectively. The time required to circulate 1 L of liquid through the membrane system was set by adjusting the flow-rate of the pump. The pressure levels used in the unit were 70, 140, 210, 280, and 420 kPa. Membranes (PES5 and PES20, Nadir Filtration GmbH, Wiesbaden, Germany) of MWCO of 5000 and
576
Y. Li et al. / Journal of Food Engineering 75 (2006) 574–580 Concentrate back to fermentor
SD
P rofessionalW orkstation 6000
Permeate Tank Recirculation pump
Membrane unit
Balance
Fermentor
Fig. 1. Schematic diagram of the ultrafiltration membrane separation system.
20,000 Da were used in these experiments. The surface area of the membrane was 0.02 m2. The membrane polymer consisted of permanently hydrophilic polyethersulfone and polysulfone. After each run, the membrane was cleaned by an alkali-acid treatment method in the following steps: (a) fully open the recirculation and permeate valves, (b) flush with tap water for 5 min, (c) circulate 2 L of 4% phosphoric acid for 15 min, (d) rinse with tap water for 5 min, (e) circulate 2 L of 0.1 N NaOH solution for 15 min, and (f) rinse with 5 L of DI water. 2.5. Analyses Lactose, lactic acid, and acetic acid were determined by a high-performance liquid chromatography (Waters, Milford, MA) with a KC-811 ion exclusion column and a Waters 410 differential refractometer detector. The mobile phase was 0.1% H3PO4 solution at a flow-rate of 1 ml/min. The temperatures of the detector and of the column were maintained at 35 C and 60 C respectively. The total nitrogen was analyzed using the macroKjeldahl method. Samples were digested using a block digestion (FOSS Tecator, Sweden) and analyzed for nitrogen on a Tecator Kjeltec auto 2400 analyzer (FOSS Tecator, Sweden) as described in Foss Tecator Application Note, AN300 (Foss Tecator, 1999). When the protein nitrogen was determined, the samples were precipitated using a trichloroacetic (TCA) solution before nitrogen analysis (AOAC, 1995). The digestion and analysis procedure for crude protein was the same as that for total nitrogen analysis. The lactic acid productivity was evaluated by (a) lactic acid yield and (b) conversion ratio. The conversion ratio was expressed as follows: Conversion ratio ð%Þ initial lactose conc: residual lactose conc. ¼ 100% initial lactose conc. ð1Þ
The lactic acid yield was expressed as grams of lactic acid produced per gram of lactose used. Lactic acid yield ðg=gÞ ¼
lactic acid produced lactose used
ð2Þ
The membrane separation of cheese whey was evaluated by using two criteria: (a) permeate flux and (b) protein retention. The permeate flux was calculated by measuring the quantity of permeate collected during a certain time and dividing it by the effective membrane area for filtration. Permeate flux; J permeate volume ¼ ðL m2 h1 Þ membrane area time The protein retention ratio was defined as CP Retention ratio ð%Þ; R ¼ 1 100 CF
ð3Þ
ð4Þ
CF is the concentration of protein in feed stream; CP is the concentration of protein in permeate.
3. Results and discussion 3.1. Fermentation The fermentation time of cheese whey in the production of lactic acid by B. longum was 48 h. The lactose, lactic acid, and acetic acid concentrations shown in Table 1 are average values from three runs. The lactose, lactic acid, and acetic acid concentration in the fermentation broth after 48 h was 2.2, 25.6 and 1.0 g/L, respectively (Table 1). The standard error of the lactose and lactic acid concentration are also shown in Table 1. The results in Table 1 shows that about 94.5% of the lactose was converted and that 0.65 g lactic acid was produced from one gram of lactose used. The production of acetic acid is trivial in comparison to that of lactic
Y. Li et al. / Journal of Food Engineering 75 (2006) 574–580
577
Table 1 The lactose conversion ratio, lactic acid yield and production efficiency using immobilized B. longerm in bioreactor Time (h)
Lactose concentration (g/L)
Lactic acid concentration (g/L)
Acetic acid concentration (g/L)
Conversion ratio (%)
Yield (g lactic acid/g lactose)
0 2 4 6 8 12 24 36 48
39.6±0.2 37.7±0.9 34.6±2.1 32.9±2.1 33.6±1.4 29.7±1.4 22.9±1.7 11.2±1.0 2.2±2.1
1.2±0.1 1.9±0.3 2.8±0.3 3.6±0.4 4.7±0.8 5.8±1.1 10.4±2.1 18.6±1.1 25.6±1.0
0.0 0.4 0.4 0.6 0.6 0.7 0.8 0.9 1.0
4.9 12.7 17.0 15.2 25.1 42.3 71.8 94.5
0.36 0.32 0.35 0.58 0.46 0.54 0.61 0.65
acid production. The lactose conversion ratio is similar to that of other lactic acid producing bacteria such as Lactobacillus helveticus while the lactic acid yield is lower than that of L. helveticus. Tango and Ghaly (2002) obtained a lactose utilization value of 92–95% and a lactic acid yield of 0.86 g lactic acid/g lactose when using immobilized L. helveticus with nutrient supplement at 36 h of fermentation. The current results are better than our previous findings in which we obtained a lactose conversion ratio of 69% and a lactic acid yield of 0.51 g lactic acid/g lactose utilized (Shahbazi et al., 2005). As there was no nutrient supplement used in the current experiments, we postulate that with optimized fermentation conditions which will include the pH and a nutrient supplement, the lactic acid yield could be further improved. 3.2. Permeate flux The performance of two membranes with MWCO of 5000 (PES 5) and 20,000 (PES20) was tested with different cross-flow velocity and transmembrane pressure. Fig. 2 shows the changes in permeate flux over time when a membrane with MWCO of 20,000 Da was used under pressure of 280 kPa at 21 C and 37 C. The fer-
80
Permeate flux (L/m2h)
70
2m/s, 37 oC
60
1m/s, 37 oC
50 40
2m/s, 21 oC 30 20
1m/s, 21 oC
10 0
20
40
60
80
100
Time (min)
Fig. 2. Change of permeate flux over time.
120
mentation broth was maintained at pH 6.5 during separation. The permeate flux values shown in Fig. 2 are average values of duplicate tests. The standard errors of the permeate flux for the four tests at 2 m/s, 37 C; 1 m/s, 37 C; 2 m/s, 21 C; and 1 m/s, 21 C were 4.56, 3.43, 1.81, and 0.50, respectively. It is evident from Fig. 2, that an increase of cross-flow velocity caused a higher permeate flux. The decrease in permeate flux over time is a result of fouling of the membranes. These results show that the permeate flux of cheese whey broth was higher at a higher temperature (Fig. 2). Increasing the cross-flow velocity also resulted in an increase of the permeate flow-rate. These results are in agreement with the findings of Suki, Fane, and Fell (1984) who reported a permeate flux decrease in protein ultrafiltration and also with Barhate, Subramanian, Nandini, and Hebbar (2003) who demonstrated that the permeate flux decreased during the processing of honey using polymeric ultrafiltration membranes. The results are also consistent with the findings of Vigneswaran and Kiat (1988) who showed that increased temperature and larger MWCO caused higher permeate flux during ultrafiltration of polyvinyl alcohol. Fig. 3 shows the effects of transmembrane pressure, cross-flow velocity and MWCO on the permeate flux at 21 C. The fermentation broth was obtained by fermentation for 48 h at pH 6.5. Each separation test lasted 2 h and the permeate flux was calculated based on the permeate volume collected in the 2-h test. The permeate flux values in Fig. 3 are the average of two duplicate tests. It can be discerned that increased transmembrane pressure caused an increase of the permeate flux. Beyond a certain pressure, the increase in permeate flux with pressure was negligible which indicates that there is an optimum pressure to obtain the maximum permeate flux. Similar results were also reported by Vigneswaran and Kiat (1988) who obtained the optimum pressure for maximum permeate flux during the ultrafiltration of polyvinyl alcohol solution at different concentrations. Results in Fig. 3 indicate that higher cross-flow velocity caused higher permeate flux for the membrane with
578
Y. Li et al. / Journal of Food Engineering 75 (2006) 574–580 35
30
Permeate Flux (L/m2h)
Permeate Flux (L/m2h)
35
Cross flow velocity 2 m/s
25 20 15 10 5
Cross flow velocity 1 m/s
25 20 15 10 Cross flow velocity 1 m/s
5
0
0 0
100
a
200
300
400
500
0
100
b
Pressure (kPa)
200
300
400
500
Pressure (kPa) 35
30
Permeate Flux (L/m2h)
35
Permeate Flux (L/m2h)
Cross flow velocity 2 m/s
30
20,000 Da
25 20 15 10 5
30
20,000 Da
25 20 15 10
5,000 Da
5
5,000 Da
0
0 0
100
c
200
300
400
0
500
d
Pressure (kPa)
100
200
300
400
500
Pressure (kPa)
Fig. 3. Effect of transmembrane pressure, cross-flow velocity, and membrane cutoff on permeate flux: (a) MWCO: 5000 Da, (b) MWCO: 20,000 Da, (c) cross-flow velocity: 1 m/s and (d) cross-flow velocity: 2 m/s.
Table 2 Analysis of variance of the permeate flux Source
DF
SS
F-value
P>F
MWCO Velocity MWCO * velocity Pressure MWCO * pressure Velocity * pressure MWCO * velocity * pressure
1 1 1 4 4 4 4
526.1 88.8 54.2 1560.5 29.5 17.2 10.4
149.2 25.2 15.4 110.6 2.1 1.2 0.7
<0.0001 <0.0001 0.0009 <0.0001 0.1223 0.3360 0.5780
DF: Degree of freedom, SS: sum of squares.
MWCO of both 5000 and 20,000 Da. At the same crossflow velocity, the membrane with MWCO of 20,000 Da had a higher permeate flux than that with MWCO of 5000 Da. Table 2 shows the results of the analysis of variance performed on the permeate flux data using a statistical package from the SAS System (SAS Institute, Cary, NC). The MWCO, pressure and cross-flow velocity showed significant (P < 0.0001) effects on the permeate flux. Most of the interactions between the parameters were not significant. 3.3. Protein retention Fig. 4 shows the effect of transmembrane pressure and cross-flow velocity on the crude protein retention ratio. Table 3 shows the results of the analysis of variance performed on the data of crude protein retention ratio. Only the MWCO had a significant (P < 0.0001) ef-
fect on the crude protein retention ratio flux. The higher MWCO caused a significantly lower protein retention ratio. The effect of pressure and cross-flow velocity had no significant (P > 0.5) effect on the crude protein retention ratio. The interactions between these parameters had no significant effects (P > 0.004) on the crude protein retention ratio. The average crude protein and protein retention ratios for membranes with MWCO of 5000 Da and 20,000 Da are shown in Table 4. It can be seen that most of the protein is retained by the ultrafiltration membranes with both MWCO of 5000 and 20,000 Da. We conclude that most of the detected raw protein in permeate is non-protein nitrogen, and has smaller MWCO than protein. These results are in agreement with the findings of Torang, Jonsson, and Zacchi (1999) who found that most of the hydrolytic proteins were retained by the hydrophilic membrane with MWCO of 20,000 Da and no protein was detected in the permeate when hydrophobic membrane with MWCO of 25000 Da was used. From our findings, it is recommended that membranes with MWCO of around 20,000 Da be used for the ultrafiltration of cheese whey fermentation broth. This is in line with the results that the membrane with the MWCO of 20,000 Da had a higher permeate flux when compared to that with a MWCO of 5000 Da and yet there was little difference between the protein retention ratios of the two membranes, 93.99% and 94.12% respectively.
Y. Li et al. / Journal of Food Engineering 75 (2006) 574–580 1.0
1.0
2 m/s
1 m/s
0.8
Protein retention ratio
Protein retention ratio
2 m/s
0.6 0.4 0.2
1 m/s
0.8
0.6
0.4
0.2
0.0
0.0
0
70
140
210
a
280
350
420
490
0
70
140
b
Pressure (kPa)
210
280
350
420
490
420
490
Pressure (kPa) 1.0
1.0
5,000 Da
20,000 Da
5,000 Da
20,000 Da
0.8
Protein retention ratio
0.8
Protein retention ratio
579
0.6
0.4
0.2
0.6 0.4 0.2 0.0
0.0 0
70
140
c
210
280
350
420
0
490
70
140
210
280
350
Pressure (kPa)
d
Pressure (kPa)
Fig. 4. Effect of transmembrane pressure and MWCO on the retention of crude protein: (a) MWCO: 20,000 Da, (b) MWCO: 5000 Da, (c) cross-flow velocity 1 m/s and (d) cross-flow velocity 2 m/s.
Table 3 Analysis of variance for protein retention Source
DF
SS
F-value
P>F
MWCO Velocity MWCO * velocity Pressure MWCO * pressure Velocity * pressure MWCO * velocity * pressure
1 1 1 4 4 4 4
0.3050 0.0001 0.0024 0.0005 0.0019 0.0044 0.0048
1368.71 0.17 10.57 0.55 2.16 4.98 5.42
<.0001 0.6842 0.0042 0.7017 0.1122 0.0065 0.0044
effect of cross-flow velocity and permeate flux on the crude protein and protein retention ratio was not significant. 3. Increasing the membrane MWCO from 5000 Da to 20,000 Da caused a significantly higher permeate flux and lower crude protein retention ratio, but the effect of membrane MWCO on the protein retention ratio was not significant.
Acknowledgement Table 4 Comparison of the average retention of protein and raw protein of ultrafiltration of cheese whey broth Constituent
Raw protein (total N * 6.38) Protein
Retention ratio (%) 5000 Da
20,000 Da
71.97 93.99
53.93 94.12
4. Conclusion It is concluded that the ultrafiltration method to separate protein and bacteria from cheese whey through a membrane can successfully be used. 1. Nearly all cells and proteins were retained by the ultrafiltration membrane with MWCO of 20,000 Das. 2. Increased transmembrane pressure and cross-flow velocity caused higher permeate flux, although the
Financial support from USDA-CSREES EvansAllen Program and USDA Capacity Building Program (contract number 2004-38814-15095) is greatly appreciated.
References Atkinson, B., & Mavituna, F. (1991). Biochemical engineering and biotechnology handbook pp. 1181–1182 (2nd ed.). New York, NY: Stockton Press. Barhate, R. S., Subramanian Nandini, K. E., & Hebbar, H. U. (2003). Processing of honey using polymeric microfiltration and ultrafiltration membranes. Journal of Food Engineering, 60, 49–54. Caric, M. D., Milanovic, S. D., Krstic, D. M., & Tekic, M. N. (1999). Fouling of inorganic membranes by adsorption of proteins. Journal of Membrane Science, 165, 83–88. Foss Tecator (1999). Application note. The determination of nitrogen according to kjeldahl using block digestion and steam distillation. 2400/24600 Kjetec Auto Sampler System. User Manual 10009165. Rev1.1.
580
Y. Li et al. / Journal of Food Engineering 75 (2006) 574–580
Goldberg, E. (1997). Handbook of downstream processing (pp. 92–136). London; New York: Vlackie Academic and Professional. Hjorleifsdottir, S., Holst, O., & Mattiasson, B. (1991). Effects on product formation in lactococcus-lactis-65.1 in continuous culture with complete cell recycling. Bioprocess Engineering, 6(1–2), 29–34. Jeantet, R., Maubois, J. L., & Boyaval, P. (1996). Semicontinuous production of lactic acid in a bioreactor coupled with nanofiltration membranes. Enzyme and Microbial Technology, 19(8), 614–619. Marijana, D. C., Spasenija, D. M., Darko, M. K., & Miodrag, N. T. (2000). Fouling of inorganic membranes by adsorption of whey proteins. Journal of Membrane Science, 165, 83–88. AOAC International (1995). Official methods of analysis of AOAC international (17th ed.). S587.A3: AOAC Official Method 991.22, Protein Nitrogen Content of Milk. Arlington, VA: AOAC International. Persson, A., Jonsson, A., & Zacchi, G. (2001). Separation of lactic acid-producing bacteria from fermentation broth using a ceramic microfiltration membrane with constant permeate flow. Biotechnology and Bioengineering, 72(3), 269–277. Senthuran, A., Senthuran, V., Mattiasson, B., & Kaul, R. (1997). Lactic acid fermentation in a recycle batch reactor using immobi-
lized Lactobacillus casei. Biotechnology and Bioengineering, 55(6), 841–853. Siso, M. I. G. (1996). The biotechnological utilization of cheese whey: a review. Bioresource Technology, 57, 1–11. Shahbazi, A., Mims, M., Li, Y., Ibrahim, S. A., Vest, S., & Morris, A. (2005). Performance of immobilized Bifidobacteria in converting lactose to lactic acid. Applied Biochemistry and Biotechnology, 121– 124, 529–540. Suki, A., Fane, A. G., & Fell, C. J. D. (1984). Flux decline in protein ultrafiltration. Journal of Membrane Science, 21, 269–283. Tango, M. S. A., & Ghaly, A. E. (2002). A continuous lactic acid production system using an immobilized packed bed of Lactobacillus helveticus. Applied Microbiology and Biotechnology, 58, 712–720. Torang, A., Jonsson, A. S., & Zacchi, G. (1999). Separation of cells and proteins from fermentation broth in a shear-enhanced cross-flow ultrafiltration module as the first step in the refinement of lactic acid. Applied Biochemistry and Biotechnology, 76, 143– 157. Vigneswaran, S., & Kiat, W. Y. (1988). Detailed investigation of effects of operating parameters of ultrafiltration using laboratory-scale ultrafiltration unit. Desalination, 70, 299–316.