TOPIC AREA: Environmental and Natural Resources

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                          1890 JOINT RESEARCH AND EXTENSION CONFERENCE
                                           JUNE 19-22, 2005
                                      NEW ORLEANS, LOUISIANA

 Project Director(s) (PD) :                                                                       TOPIC AREA
                                                                                 □ Nutrition and Health
                                                                                 □ Small Farms
 PD:      Abloghasem Shahbazi            Institution: NC A&T State University    □ 4-H/Youth Development
                                                                                 □ Community and Economic
 CO-PD:                                  Institution:                            □ Development
                                                                                 □ Quality of Life for Families
 CO-PD:                                  Institution:                              Environment and Natural Resources
                                                                                 □ New and Emerging Issues
                                                                                                 FOCUS AREA
 Project Title:                                                                      Multidisciplinary - - research or extension
                                                                                 activity in which investigator
 Conversion of Cheese Whey into value-added products                             from two or more disciplines collaborate closely
                                                                                 □ Multi-Institutional - - research or extension
                                                                                 activity in which two or more investigators from
                                                                                 different institutions collaborate closely
                                ABSTRACT                                         □ Integrated - - initiative involving collaboration
                                                                                 between research and        extension personnel

                                        Lactic Acid Production from Cheese Whey
                                         Abloghasem Shahbazi, Yebo Li, Sekou Coulibaly
       1. Department of Natural Resources and Environmental Design, 2. Department of Human Environment and Family Science, 3.
                       Department of Mechanical and Chemical Engineering, North Carolina A&T State University
 Abstract: One goal of environmental and natural resources continues to be the development of alternative uses
 for agricultural by-products. Cheese whey is one by-product that can be used to produce lactic acid. A multi-
 disciplinary team composed of environmental engineers, microbiologists, process engineer, and chemical
 engineers is investigating producing lactic acid from cheese whey using Bifidobacterium longum (B. longum).

 The processes of lactic acid production include two key stages which are (a) fermentation and (b) product
 recovery. In this project, lactic acid was produced from cheese whey using both free cell and immobilized B.
 longum. After 48 hours of fermentation, nearly 100% of the lactose was converted and the lactic acid yield
 reached 0.73 g/g lactose utilized without any nutrient addition. Similar yield can only be obtained using L.
 helveticus with nutrient supplement. We have not found other report on the lactic acid production using B.
 longum by now.

 Ultrafiltration membrane was used to separate cells and protein from the above fermentation broth. About 94%
 of the proteins were retained by the ultrafiltration membrane with MWCO of 5,000 and 20,000 Daltons.
 Nanofiltration membrane was used to further separate lactic acid from lactose in the ultrafiltration permeate, 99-
 100% of lactose can be retained in the concentrate and 40-60% of lactic acid can be recovered in the permeate
 using a nanofiltration membrane of DS-5DK. Higher initial lactic acid caused significant higher permeate flux,
 lower lactose retention, and higher lactic acid recovery. Increased transmembrane pressure caused significant
 higher permeate flux, higher lactose retention, and lower lactic acid recovery.

 The above produced lactic acid can be further polymerized to produce biodegradable plastic. Compared to plastic
 produced from petroleum, the biodegradable plastic made from waste stream of food industry will create
 enormous impact on the local economy and environment.

APPROVED:
                                                                      or
                 Research Director                                         Extension Administrator
JUSTIFICATION/DESCRIPTION

       Whey is an important by-product from the cheese manufacturing industry. Typically, 100

grams of milk yield 10 grams of cheese and 90 grams of liquid whey. Disposal of liquid whey is

costly due to its high BOD and water content (1). 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 (2). Therefore,

there is an interest to utilize lactose from cheese whey in the production of value-added products

such as lactic acid. Lactic acid is a natural organic acid and has many applications in the

pharmaceutical, food, and chemical industries.

       In this study, researchers from bioenvironmental engineering, microbiology, process

engineering, and chemical engineering worked together to develop an appropriate technology

and prototype to produce biodegradable polymers using agricultural and food wastes (cheese

whey) that would have near-zero market cost. The current Cargill-Dow process uses food grade

starch as a substrate for lactic acid production. The starch must first be broken down to glucose

(dextrose) by saccharification before it could be used in producing lactic acid, while the cheese

whey can be directly fermented without any pretreatment. To further reduce the production cost,

the two major steps for biodegradable plastic production from cheese whey named fermentation,

and separation were studied and new process was developed.

       Fermentation: Lactic acid can be produced by fermentation of sugar-containing

substrates such as cheese whey using Lactobacillus helveticus (3, 4), and Lactobacillus casei (5,

6) in most of the previous studies. Bifidobacterium longum is a bacteria that can both convert

lactose into lactic acid and also produce an anti-bacterial compound, which can boost the


                                                                                                    1
immune system in its host. Bifidobacterium longum produces high quality of L (+) lactic acid

instead of D (-) lactic acid (7). Most previous studies of B. longum have concentrated on

increasing B. longum cell production by cell immobilization and optimized pH (8, 9) for

application in the food and pharmaceutical industry. To date, there has been no report on using

B. longum to produce lactic acid from cheese whey.

       Separation: The biggest challenge in lactic acid production lies in the product recovery

and not in the fermentation step (10). The current chemical process uses neutralization with a

base followed by filtration, concentration, and acidification to recover the lactic acid. This is an

expensive method because it produces waste stream and is difficult to use in the large-scale

production of lactic acid. The waste stream of chemicals, e.g., gypsum has little or no value.

The common chemical separation processes, such as distillation, can not be used to concentrate

the acid because lactic acid has a high boiling point and polymerizes at elevated temperatures.

Compared with the traditional chemical separation, the membrane separation system developed

in this study has the following advantages: (1) generation of a minimal waste stream, (2) cells

and lactose that can be separated and recycled back to the fermentor to increase the lactic acid

yield, and (3) significant savings in time and production cost.

       Figure 1 presents a flow diagram that we followed in this study to produce value added

products from cheese whey. Based on our production model, three value added products whey

protein, cell biomass, and PLA polymers can be produced. Whey protein is obtained via the

ultrafiltration of cheese whey. Ultrafiltration of the fermentation broth can separate cells and

proteins. The cells can be recycled back to the fermentor and part of the cells can be harvested

for application in pharmaceutical and food industry. During the nanofiltration step, lactose can

be retained in the concentrate and recycled back to the fermentor. The permeate obtained from




                                                                                                       2
the nanofiltration is mainly composed of lactic acid and water. The water in the permeate of

nanofiltration can be separated with a reverse osmosis membrane. The obtained pure lactic acid

can be polymerized for PLA production. The application of fermentation, ultrafiltration and

nanofiltration techniques are reported in this paper.

OBJECTIVES

       The objectives of this study were two-fold: (1) Develop a suitable fermentation

technology for lactic acid and Bifidobacteria longum cell production from cheese whey; and (2)

Investigate the use of a membrane separation system to obtain cell biomass and lactic acid from

the fermentation broth.

                                                  Cheese whey


                                                  Ultrafiltration         Whey Protein



                                                  Fermentation
                                         Cells


                     Cells                        Ultrafiltration                      Stage 1
                                                                                   (Reported in this
                                                                                       paper)
                                                          Lactose+ lactic
                                                           acid +water

                                                  Nanofiltration
                                                                     Lactose
                                                            Lactic acid
                                                              +water

                                                  RO Filtration                Water


                                                            Lactic acid


                                                 Polymerization
                                                                                       Stage 2
                                                                                  (Under study now)
                                                  Polylacticacid
                                                      (PLA)




                             Figure 1. Conversion of cheese whey to value added products


                                                                                                       3
MATERIALS AND PROCEDURES

Cheese whey media

        Cheese whey media was prepared by dissolving 50 g of deproteinized cheese whey

powder (Davisco Foods International, Inc., Eden Prairie, MN, USA) into a liter of deionized (DI)

water and stirring for 5 minutes at ambient temperature. 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

minutes.

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 to 24 h under anaerobic conditions. This was used as a pre-culture to

initiate cell production of higher volume with a 1% inoculation into 100 ml fresh MRS broth,

incubated at 37°C for 24 h.

Fermentation

        Free cell fermentation was conducted in a stirred 2.0-liter bench top fermentor. The pH of

the broth was maintained at the designated value by neutralizing the acid with 5N 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 2 h during the

first 8 hours and every 12 h during the remaining fermentation process. The fermentation was

lasted for 48 h.




                                                                                                4
       A bioreactor with spiral-sheet polymeric membrane cartridge which is used as a support

matrix for cell immobilization was used to carry out the fermentation (Figure 2). After the

bacteria were immobilized, the MRS solution was drained off and fresh whey media was added

for fermentation. The bioreactor containing immobilized cells was connected to a stirred 2.0-liter

bench top fermentor to allow medium recirculation.




             Figure 2. Schematic diagram of the spiral sheet bioreactor and the fermentation system

Membrane Separation

       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 (Figure 3). 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


                                                                                                      5
computer that continually recorded time and permeate weight at 30 s intervals. Two types of

membranes (PES5 and PES20, Nadir Filtration GmbH, Wiesbaden, Germany) with MWCO of

5,000 and 20,000 Dalton were used in the ultrafiltration experiments. The membrane polymer

consisted of permanently hydrophilic polyethersulfone and polysulfone.

                                                  Concentrate
                                                    back to
                                                   fermentor                                                                                   PRO
                                                                                                                                                     SD




                                                                                       P ro fe s sio n a l W o rksta tio n 6 0 0 0




                                                                                Permeate

                     Tank


                                                  Recirculation      Membrane
                                                     pump              unit                                                          Balance


                   Fermentor

                            Figure 3. Schematic diagram of the membrane separation system

       In the nanofiltration system, the pump and ultrafiltration unit in the ultrafiltration system

was replaced with a high pressure pump (M03-S, Hydra-cell, Minneapolis, MN, USA) and

nanofiltration membrane unit (SEPA CF II, Osmonics, Minneapolis, MN, USA). The two tested

nano membranes (DS-5DK and DS-5HL, Osmonics, Minneapolis, MN, USA) in this study could

retain 98% of MgSO4 but had different levels of permeate flux. No MWCO information was

provided by the manufacturer.

       An alkali-acid treatment method was applied to the membrane system in the following

steps: (a) fully open the recirculation and permeate valves, (b) flush with tap water for 5 min, (c)

circulate 2 liters of 4% phosphoric acid for 10 min, (d) rinse with tap water for 5 min, (e)

circulate 2 liters of 0.1 N NaOH solution for 10 min, and (f) rinse with tap water for 5 min.




                                                                                                                                                          6
Analyses

       Lactose, lactic acid, and acetic acid were measured by 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

1ml/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 macro-Kjeldahl 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). When the protein nitrogen was determined,

the samples were precipitated using a trichloroacetic (TCA) solution before nitrogen analysis

(11). 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) lactose

conversion ratio. The conversion ratio was expressed as follows:

                                             initial lactose conc. - residual lactose conc.         (1)
                     Conversion ratio(%)                                                   100%
                                                          initial lactose conc.

The lactic acid yield was expressed as grams of lactic acid produced per gram of lactose used.

                                                                    lactic acid produced
                                    Lactic acid yield ( g / g )                                    (2)
                                                                         lactose used

The performance of membrane separation was evaluated by using three criteria: (a) permeate

flux, (b) lactose retention, and (c) lactic acid recovery. 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 volume
                             Permeate flux, J                          (l m 2 h 1 )              (3)
                                                   membrane area  time



                                                                                                     7
The component retention (%) was defined as:

                                                        C    
                                        Retention  1  LP   100
                                                     C                                          (4)
                                                        L0 



CL0 = concentration of component in feed stream, CLP= concentration of component in permeate.

The lactic acid recovery (%) was defined as:

                Lactic acid recovery 1  lactic acid retention ratio                             (5)

RESULTS AND CONCLUSIONS

Fermentation

       The lactose, lactic acid, and acetic acid concentrations obtained during the 48 hours of

fermentation with free cell and immobilized B. longum are shown in Table 1 and 2, respectively.

The values in the tables represent the averaged values of two runs. The results show that about

96.7% and 91.7% of the lactose was utilized and that 0.73 and 0.67 g lactic acid was produced

from one gram of lactose used at pH 5.5 and 6.5 when the free cells of B. longum were used .

The production of acetic acid was negligible in comparison to that of lactic acid production.

When the bioreactor with immobilized cells of B. longum was used, about 68.5% of the lactose

was converted and that 0.51g lactic acid was produced from one gram of lactose used at pH 6.5.

The fermentation with free cell of B. longum performed better in this study. Details about the

lactic acid production from cheese whey can be found in an article published by the first author

and colleagues (12).

       The lactose conversion ratio and lactic acid yield are similar to results of other lactic acid

producing bacteria such as L. helveticus. Tango and Ghaly (3) 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. Most of the previous works were




                                                                                                    8
focused on obtaining high lactose conversion ratios and lactic acid yields. These experiments

were carried out with immobilized cells and nutrient supplementation. In this study, free cells of

B. longum were grown with no nutrient supplements, which would significantly reduce the cost

of lactic acid production and be more compatible with the current fermentation facilities.

       Table 1. Lactic acid production from cheese whey using free cell of B. longum (pH 5.5 and 6.5)

                        Lactose          Lactic acid          Acetic acid
                                                                             Conversion ratio      Yield (g lactic
                     concentration      concentration        concentration
  Time (hrs)                                                                      (%)              acid /g lactose)
                         (g/L)              (g/L)                (g/L)
      pH             5.5        6.5     5.5        6.5       5.5       6.5    5.5       6.5         5.5        6.5
       0            37.3       39.7     2.2        1.4       0.4        0
       2            34.8       38.6     3.8        2.2       0.6       0.4    6.7       3.0        0.63        0.71
       4            32.4       36.6     5.5        3.1       0.7       0.4   13.2       7.8        0.67        0.55
       6            29.6       34/9     7.0        3.9       0.7       0.6   20.6      12.2        0.63        0.52
       8            27.2       33.6     8.2        4.7       0.7       0.6   27.1      15.3        0.59        0.55
      12            24.5       30.6    10.7        6.7       0.7       0.7   34.3      23.0        0.66        0.58
      24            14.6       23.2    19.1       12.0       0.7       0.8   60.8      41.6        0.74        0.65
      36             6.6       12.2    24.9       20.0       0.7       0.9   83.0      69.4        0.73        0.66
      48             1.2        3.3    28.6       25.9       0.7       1.0   96.7      91.7        0.73        0.67


Table 2. The lactose conversion ratio and lactic acid yield using immobilized B. longum in bioreactor (pH 6.5)

                               Lactose        Lactic acid        Lactose                        Yield (g
                                                                             Conversion
                             concentration   concentration       utilized                     lactic acid /g
           Time (hrs)                                                         ratio (%)
                                 (g/L)           (g/L)            (g/L)                          lactose)
               0                 52.6             0.6
               12                43.1             7.0               9.5         18.1              0.67
               18                37.3             9.6              15.3         29.1              0.59
               24                32.7            11.8              19.9         37.9              0.56
               36                23.8            15.9              28.9         54.9              0.53
               42                20.2            18.1              32.5         61.7              0.54
               48                16.6            19.2              36.1         68.5              0.51


Membrane Separation

        Ultrafiltration Figure 4 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. Each separation test lasted 2 h and the permeate flux was calculated based on the

permeate volume collected in the 2h test. The permeate flux values in Figure 4 are the average of

two replicate tests. It can be discerned that increased transmembrane pressure caused an increase



                                                                                                                      9
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 Vigneswran and Kiat (13) who obtained the optimum

pressure for maximum permeate flux during the ultrafiltration of polyvinyl alcohol solution at

different concentrations.



                         35                                                                                    35
                                                                                                                        Velocity 2m/s
                         30                                                                                    30




                                                                                       Permeate Flux (L/m2h)
Permeate Flux (L/m2h)




                         25       Velocity 2m/s                                                                25

                         20                                                                                    20

                         15                                                                                    15

                         10                                                                                    10
                                                                                                                                                Velocity 1m/s
                         5                                Velocity 1m/s                                        5

                         0                                                                                     0
                              0      100       200        300      400       500                                    0       100       200      300      400        500

                                              Pressure (KPa)                                                                         Pressure (kPa)
                                            (a) MWCO: 5,000 Dalton                                                          (b) MWCO: 20,000 Dalton




                         35                                                                                    35
                         30
                                       20,000 Dalton                                                           30       20,000 Dalton
 Permeate Flux (L/m h)




                                                                                   Permeate flux (Lm h)
                   2




                                                                                                    2




                         25                                                                                    25
                         20                                                                                    20
                         15                                                                                    15
                         10                                                                                    10                             5,000 Dalton
                          5                                                                                     5
                                                        5,000 Dalton
                          0                                                                                     0
                              0       100         200      300         400   500                                    0       100         200    300       400       500
                                               Pressure (kPa)                                                                        Pressure (KPa)
                                     (c) Cross flow velocity: 1 m/s                                                               (d) Cross flow velocity: 2 m/s

                                  Figure 4. Effect of transmembrane pressure, cross flow velocity, and membrane cutoff on permeate flux

                              Results in Figure 4 also indicate that higher cross flow velocity caused higher permeate

flux for the membrane with MWCO of both 5,000 and 20,000 Dalton. At the same cross flow




                                                                                                                                                                         10
velocity, the membrane with MWCO of 20,000 Dalton had a higher permeate flux than that with

MWCO of 5,000 Dalton. The analysis of variance performed on the permeate flux data using a

statistical package from the SAS System (SAS Institute, Cary, NC) showed that pressure and

cross flow velocity had significant (P < 0.0001) effects on the permeate flux. Most of the

interactions between the parameters were not significant.

       The average crude protein (total nitrogen) retention ratios for membranes with MWCO of

5,000 Dalton and 20,000 Dalton were 72.0 and 53.9%, respectively. The average protein

retention ratio was 94.0% for both of the two membranes with MWCO of 5,000 Dalton and

20,000 Dalton. It can be concluded that most of the protein is retained by the ultrafiltration

membranes with both MWCO of 5,000 and 20,000 Dalton. We conclude that most of the

detected raw protein in permeate is non-protein nitrogen, which has smaller MWCO than

protein.

Nanofiltration

       Figures 5a, and 5b show that permeate flux increased with the increase of transmembrane

pressure. Higher permeate flux could be obtained at higher initial lactic acid concentrations.

When the initial lactic acid concentration was increased from 18.6 g/L to 27.0 g/L, the permeate

flux increased about 30%, 26%, and 14% at pressure 1.4, 2.1 ad 2.8 MPa, respectively for

membrane of DS-5DK. Among the two tested membrane of DS-5DK and DS-5HL, higher

permeate flux levels were obtained with membrane of DS-5HL (Figure 5b). The analysis of

variance performed on the permeate flux data showed that membrane, pressure and initial lactic

acid concentration has significant (P < 0.0001) effects on the permeate flux. The interaction

between these parameters were not significant (P=0.045 and 0.11, respectively).




                                                                                                 11
       Figure 5c and 5d show that lactose retention increased with the increase of

transmembrane pressure. Lower lactose retention was obtained at higher initial lactic acid

concentration. When the DS-5DK membrane was used, 100% retention of lactose was obtained

at initial lactic acid concentration of 18.6 g/L for all tested transmembrane pressures. When the

initial lactic acid concentration was increased to 27.0 g/l, lactose retention rates of 94.7, 96.8,

and 99.5% were obtained at pressure levels of 1.4, 2.1, and 2.8 MPa, respectively. This indicates

that at higher initial lactic acid concentrations, higher lactose retention can be obtained by

increasing transmembrane pressure. When the DS-5HL membrane was used to separate media

with initial lactic acid concentration of 18.6 g/L, with the same pressure levels as for the DS-

5DK membrane, lactose retention rates were 82.2, 87.3, and 90.7%, respectively. At most of the

test conditions, lactose retention of DS-5HL was lower than 91%, while the lactose retention for

membrane of DS-5DK reached about 99-100%. These results indicate that in comparison with

the DS-5HL membrane, the DS-5DK membrane should be used for separating lactose from lactic

acid in nanofiltration process.

       Increased retention of lactic acid corresponded positively with increased lactose retention

(Figure 5e and 5f). Increases of transmembrane pressure were associated with lower levels of

lactic acid recovery in permeate. Higher lactic acid recovery was obtained at higher initial lactic

acid concentration. When the initial lactic acid concentration increased from 18.6 g/L to 27.0 g/l,

the lactic acid recovery increased from 54.4, 43.9, and 36.6 % to 76.9, 69.3 and 63.5 at pressure

of 1.4, 2.1 and 2.8 MPa, respectively for membrane of DS-5DK. Considering the effects of

increased initial lactic acid concentration on the permeate flux and lactose concentration, both of

the increased permeate flux and lactic acid retention are desired while the decreasing of lactose

retention need to be compensated by optimized parameter such as increased pressure.




                                                                                                      12
                                       100                                                                                                                                           140
                                                              18.6 g/L Lactic Acid                                                                                                                        Membrane DS-5DK
                                        90                    27.0 g/L Lactic Acid
                                                                                                                                                                                     120                  Membrane DS-5HL
                                        80
Flux (Lm-2hr-1)




                                                                                                                                                                                     100




                                                                                                                                                          Flux (Lm-2hr-1)
                                        70

                                        60                                                                                                                                            80

                                        50
                                                                                                                                                                                      60
                                        40

                                        30                                                                                                                                            40

                                        20
                                                                                                                                                                                      20
                                             1.2       1.4      1.6     1.8       2.0     2.2         2.4         2.6         2.8             3.0
                                                                                                                                                                                           1.2      1.4      1.6     1.8         2.0         2.2         2.4     2.6     2.8     3.0
                                                                               Pressure (MPa)
                                                                                                                                                     a.                                                                    Pressure (MPa)                                              b.
                                                      Permeate flux (membrane: DS-5DK)                                                                                                           Permeate flux (lactic acid conc. 18.6 g/L)


                                      120                                                                                                                                           120


                                      100                                                                                                                                           100
                                                                                                                                                          Lactose retention (%)
Lactose retention (%)




                                       80                                                                                                                                            80


                                       60                                                                                                                                            60


                                       40                                                                                                                                            40


                                                              18.6 g/L Lactic acid                                                                                                                         Membrane:DS-5HL
                                       20                                                                                                                                            20
                                                              27.0 g/L Lactic acid                                                                                                                         Membrane: DS-5DK

                                        0                                                                                                                                             0
                                            1.2       1.4     1.6      1.8      2.0     2.2         2.4         2.6         2.8         3.0                                               1.2     1.4      1.6     1.8     2.0         2.2         2.4     2.6     2.8     3.0

                                                                             Pressure (MPa)                                                                                                                              Pressure (MPa)
                                                  c. Lactose retention (membrane: DS-5DK)                                                                                                 d. Lactose retention (lactic acid conc. 18.6 g/L)


                                       100                                                                                                                                          100



                                                                                                                                                                                     80
                                        80
                                                                                                                                                          Lactic acid recovery(%)
            Lactic acid recovery(%)




                                                                                                                                                                                     60
                                        60


                                                                                                                                                                                     40
                                        40


                                                                                                                                                                                     20                   Membrane DS-5DK
                                        20                      18.6 g/L Lactic acid                                                                                                                      Membrane DS-5HL
                                                                27.0 g/L Lactic acid
                                                                                                                                                                                      0
                                            0                                                                                                                                             1.2     1.4      1.6     1.8     2.0         2.2         2.4     2.6     2.8     3.0
                                                1.2     1.4      1.6     1.8      2.0         2.2         2.4         2.6         2.8          3.0
                                                                                                                                                                                                                         Pressure (MPa)
                                                                               Pressure (MPa)
                                             e. Lactic acid recovery (membrane: DS-5DK)                                                                                               f.Lactic acid recovery (lactic acid conc. 18.6 g/L)
                           Figure 5 Effects of pressure, membrane, and initial lactic acid concentration on permeate flux, lactose retention, and
                                                                 lactic acid recovery of nanofiltration




                                                                                                                                                                                                                                                                           13
OUTCOME AND IMPACT

1. B. longum has been demonstrated to be promising bacteria for lactic acid production from

   cheese whey. At pH 5.5, about 96.7% and 91.7% of the lactose was converted and that 0.73

   and 0.67 g lactic acid was produced from one gram of lactose using free cells of B. longum

   and without nutrient supplement. Such high conversion ratios and lactic acid yield could only

   be obtained with nutrient supplement and immobilized cells for L. helveticus. Based on our

   review of the literature we have not found any other study to use B. longum for lactic acid

   production from cheese whey.

2. Ultrafiltration can be successfully used to separate protein and bacteria cells from cheese

   whey fermentation broth. Nearly all cells and proteins were retained by the ultrafiltration

   membrane with MWCO of 20,000 Daltons. Increased transmembrane pressure and cross

   flow velocity caused higher permeate flux. Increasing the membrane MWCO from 5,000

   Dalton to 20,000 Dalton caused a significantly higher permeate flux and lower crude protein

   retention ratio.

3. Nanofiltration can be successfully used to separate lactose and lactic acid. Nearly all the

   lactose (99-100%) was retained using a DS-5DK membrane at both of the tested initial lactic

   acid concentration of 18.6 g/L and 27.0 g/L. To obtain 100% of lactose retention,

   transmembrane pressure higher than 2.8 MPa needs to be applied when the initial lactic acid

   concentration reached 27.0 g/L. For the tests when 99-100% of lactose was retained in the

   concentrate, the highest lactic acid recovery in the permeate reached 63.5%.

4. The developed fermentation and separation technologies for lactic acid production from

   cheese whey can have a significant impact on the environment and domestic economy.

   Converting the zero value cheese whey to lactic acid and further to biodegradable plastic has


                                                                                                 14
    the potential for generating new business opportunities and income for the dairy industry. By

    replacing some of the plastic made from petroleum with biodegradable plastic made from

    cheese whey, this could undoubtedly generate a significant impact on the environment,

    especially in the area of “white” pollution resulting from our enormous use of plastic.

5. It has been successfully demonstrated that B. longum cell biomass and antimicrobial compounds can

    be produced and separated during the fermentation of cheese whey in the presence of B. longum. As

    these anti-bacteria compounds can boost the immune system in its host, it has a huge market in the

    food and pharmaceutical industry.

6. This project has successfully created multidisciplinary collaboration among researchers from food

    science and nutrition, bioenvironmental engineering, and chemical engineering programs. It is being

    used as a model in creating interdisciplinary research and academic program at NC A&T S U.


REFERENCES

1. GE Water & Process Technologies. http://www.gewater.com/library/tp/232_Ultrafiltration-
    121_Whey.jsp. Accessed on May 20, 2005.
2. Siso, M. I. G. (1996), Bioresour. Technol. 57:1-11.
3. Tango, M. S. A., and Ghaly, A. E. (2002), Appl. Microbio. Biotechnol. 58:712-720.
4. Roy, D., Goulet, J., and LeDuy, A. (1986), Appl. Microbio. Biotechnol. 24:206-213.
5. Bruno-Barcena, J. M., Ragout, A. L., Cordoba, P.R., and Sineriz, F.(1999), Appl. Microbio.
    Biotechnol., 51:316-324.
6. Roukas, T., and Kotzekidou, P. (1998), Enzyme Microb. Technol. 22: 199-204.
7. Gomes, A. M.P and Malcata, F. X. (1999), Trends in Food Sci. and Technol. 10: 139-157.
8. Doleyres, Y., Paquin, C., LeRoy, M. and Lacroix, C. (2002), Appl. Microbiol. Biotechnol. 60:168-
    173.
9. Song, S. H., Kim, T. B., Oh, H. I., and Oh, D. K. (2003), World J. Microbiol. & Biotechnol. 19: 721-
    731.
10. Atkinson, B., and F. Mavituna. (1991), Biochemical Engineering and Biotechnology Handbook.
    1181-1182. 2nd ed. New York, N.Y.: Stockton Press.
11. Official Methods of Analysis of AOAC International, 17th ed. 1995. S587.A3:Arlington, VA: AOAC
    International.
12. Shahbazi, A., Mims, M. R., Li, Y., Shirley, V., Ibrahim, S. A., and Morris, A. (2005). Appl. Biochem.
    Biotechnol., 121-124:529-540.
13. Vigneswaran, S., and W. Y. Kiat. (1988), Desalination 70: 299-316.




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