VIABILITY OF CALCIUM ALGINATE ENTRAPPED LACTOBACILLUS CASEI DURING CONTINUOUS LACTIC ACID

Lnctobacillz~s casei cells harvested in log phase (20h) were entrapped in calcium alginate (10.0g 1-I). When the beads were packed in a tl~ermostated (42'C) column (30.1) x 2.0 cm), the optimum flow rate for mnximum lactic acid yield (74.0%) was 18.0 mill-l. At this flow rate, the productivity obtained was 21.2g 1.' 11-l. With increase in the flow rate, viable cells in the beads were decreased. However the cell numher was more than those present initially. When different concentrations of glucose in the range of 50.0 to 250.0g 1.' was passed at 18.0ml ll-l, maximum lactic acid yielcl(81.(if%) was obtained at the concentration of 125.0g 1-l. With an increase u glucose concentration from 50.0 to 100.0g 1-', the viable cells in the beads were increased and further increase in glucose concentration in the feed decreased the viahle cell numher. When the nu t~ ien t medium containing 125.0g 1-I glucose was passecl contiruou'sly, steady state was observed after 3 days and continued for 16 days. l11e viable cell number i n the beads was constant for 16 days. Key Word: Flow rate, immobilization, lactic acid, La.cfobacil.1us casei, viable cell count,.


INTRODUCTION
Lactic acid production from bacteria is more attractive than chemical methods.One of the mayx hindrances with the fermentation technique is product inhibition with decrease in PHI-^.In conventional methods, lactic acid was removed from the fermentation broth as calcium lactate4.Later studies revealed t h a t use of immobilized cells5-" or e l e c t r o d i a l y ~i s ~~-~~ or extractive b i o c o n v e r s i o n ~~~-~~ reduce product inhibition with increase in productivity.However viability of the entrapped cells in different polymers is another hindrance for their u ~e " ~~-l ?
In this paper we present some studies of the viability of Lactobacillus casei entrapped in calcium alginate during continuous operations under different conditions.

Organism. Lactobacillus casei DSM 20021 Ana,lytical methods
Glucose and lactic acid were measured by HPLC (Shimadzu, Auto injector).Sugar Pak'"', (Waters, Millipore, USA) stainless steel column (300 x 6.5, microparticulated sillca gel) was used and the mobile phase was deionized water.Reference time for glucose was 8.6min.Samples collected at different time intervals were prepared for lactic acid estimation by filtering through a 0.45mp Milipore filter.Viable cell count of the immobilized L.casei was determined by dissolving 2.0g (wet weight) of entrapped cells in 30.0 ml of 0.1M phosphate buffer (pH 6.5) and growing the cells in MRS agar plate (MRS broth, 50.0gI-' and agar, 2 0 .0 ~ 1-I).

Cultivation a7ad harvesting of L. casei for imrnobtlim,tion
The inoculum was prepared in MRS broth (52.0g 1-I, 50.0ml) a t pH 6.5 by inoculating with 100p1 L. casei from frozen sample and incubating at 42°C for 14 h (300 rpm).Nutrient medium (2.51) in a fermenter (working volume 5.01) was inoculated with 2.5ml of inoculum and incubated at 42°C while mixing.The pH was maintained by titrating with sterile 8N NaOH.Nutrient medium a t pH 6.5 contained the following in gF1, glucose 150.0, yeast extract, 10.0; I<2HP0,, 0.5; KH,PO,, 0.5 and sodium citrate, 1.0 and salt solution, 1.0 ml.The salt solution contaj.ned in gl-I MgS0,.7H20,50.0;MnSO,.H,O, 3.1; Fe S0,.7H,O, 2.0 and ascorbic acid, 5.0.L. casei cells were harvested at 20h by centrifuging (12000 rpm) in a refrigerated centrifuge at 10% for 15min.Cells washed three times with saline were used for immobilization.L. casei harvested a t 20h (in log phase for immobilization had the optical density of 12.8 (620nm1, dry weight of 6.9g 1.' and the viable cel.1 count of 9.0 x lo1".Inrnzobilizatio7z of L. casei.L. ca.set cells (10.0g wet weight) suspended in 30.0ml of sterile sod.ium alginate solutio~i (10.0g 1.' ) were extruded by syringe through a need.le(0.7 mm diameter) into 10.0%(wlw) glucose -0.05h/I CaC1, (1.0 1) solution and the beads were left in the solution for 2.0 h.Beads were washed with 0.05M maleate -0.05M CaC1, buffer (pH 6.5).Immobilized cells in the beads were incubated with nutrient medium for 72h ancl washed with sterile 0.05M CaC1, solution.Beads were packed jn a jacketed.column (30.0 x 2.0 cm) for further studies.Temperature of the colunln was maintained by circulating hot water from a water bath at 42°C.

Oj~tinzization of conditions for co?zti7zuous lactic acid procluctto~~
Effect of flow rate: Nutrient medium was pumped at flow rates ranging from 6.0 to 36.0ml h-I into the column.For each flow rate the column was repacked with freshly entrapped cells.Residual glucose and lactic acid in the spent medium were estimated.Viable cells in the beads were determined.
Effect ofglucos.econcer7.tration:Nutrient medium containing varying concentration of glucose (from 50.0 to 250.0g 1.') was pumped a t optimum flow rate into the c o l u m i ~. ~ The experiment was continued as above.
Con.tin,uous fermentation of glucose to lactic acid: Nutrient medium containing optimized.amount of glucose was pumped into the column a t the optimum flow rate.Fractions were collected a t different time intervals and analysed for lactic acid.

RESULTS
Optimum conditions for continuous lactic acid production Glucose was completely fermented when the flow rate was increased up to 12.0ml h-'.But lactic acid yield has decreased with an increase in flow rate (Figures 1 and 2).Lactic acid productivity was increased from 8.8 to 21gk1h-' when the flow rate was increased from 6.0 to 24.0ml h-l.Viable cells entrapped in the beads before passing the medium was 23.0 x 107cells g1 we't bead.With an increase in flow rate from 6.0 to 36.0ml h-I the viable cell count in the bead was higher than what was present initially (Figure 1).Even though the productivity (21.5g 1-lh-I) was highest at the flow rate of 24.0ml h-l, 18.0ml h-I was selected (where the productivity was 21.2g 1-lh-I), because at this flow rate lactic acid formed was about 18.0%) more than that at 20.0ml h-'.
Optimum concentration of glucose for maximum lactic acid production was 125g I -I a t the optimum flow rate (18.0ml h-I, Figure .3).When the glucose.concentration was increased from 50.0 to 250.0g l'l, maximum productivity of 22.3g 1-Ih-' was obtained a t the glucose concentration of 150.0g 1-I (Figure 4).Above 125g 1.' glucose in the medium, the residual glucose also increased (Figure 3).The viable cell count in the freshly entrapped beads was 23.0 x lo7 cells g1 wet bead.Wit11 increase in glucose concentration, proliferation of the cells has taken place and, the cell number has increased up to 9 1'.8 x lo1'$ while the glucose concentration was 100.0g 1.'.But with further increase of glucose concentration, the cell multiplication has decreased (Figure .3).

Continuous fermentation of glucose by immobilized L. casei
The steady state of lactic acid production was observed after 3 days and was continued for 16 days witl~out problem.But after the ITt'' day, lactic acid production started t o decline.The cell number in the beads showed increase in number initially but after the third d.ay the j.ncrease was not significant with an increase in the cell Sealcage into the medium.However by the lot" day it was observed that the cell leakage was pronounced and the disintergration of the beads had started.By the 26t1' day leakage of the cells was obvio.usdue to breaking of the beads.The process was terminated by the 32"" day (Figure 5).

FIOW Rate (mi h-')
Figure 1 : Effect of flow rate 011 ( 0 ) lactic acid production and ( a ) residual glucose in the medium and ( A ) viable cells in the beads when nutrient medium was passed a t pH 6.5 and 42°C into a column packed with calciumn alginate entrapped Lactobacillz~s casei.

Flow Rate (ml Y')
Figure 2: Effect of flow rate on lactic acid (U ) yield and (B) productivity when nutrient medium was passed a t pH 6.5 and 42°C into a column packed with calcium alginate entrapped Lactobacillus casei.

Glucose (g r')
Figure 3: Effect of glucose conceiltration in nutrient medium on (0 ) lactic acid production,( e ) residual glucose in the medium and ( A ) viable cells in the beads when nutrient medium was passed a t optimum flow rate (18.0 ml h-l), pH 6.

Figure 5: Continuous lactic acici p r o d u c t i o n by calcium alginate ent r a p p e d
Lactoliacillz~s casei when n u t r i e n t medium containing 125 g 1-' glucose w a s fed a t 18.0 ml h-I i n t o t h e column, pH 6.5 and 42°C.

DISCUSSION
The .increase in flow rate decreased glucose consumption.Reduction in lactic acid yieltf was due to the reduction in contact time between the cells and the sul~strate and 1e;rlrage of the cells from the beads.Boyaval and Gouletl" have reported that the dc:calcification of the cal.cj.um alginate by 1.actic acid.was prono~mced at low diluhion rates and the level of the leakage of the cells was also high in such cases.Thus, it is better t o change to a flow rate, m71lich gives higher prod~lct,ivit,y and product.yield to avoid decal.cification of the calcium alginate beads.Lactohactllus h e l a e t i c ~~. ~ entrapped 111 k-carrageenan-locust bean gun1 gel at a dilutjon rate of 1.2 1 h-lgave a productivity of 28.5g 1:lh-l ", which is comparal11.e to our results.The viable.: cell comlt in the immobil.izedpreparation was increased even when the flow rate was increased.Thus t,he cell lnultiplication more than compensated for the cell leakage.This also indicated that the sodium alginate did not show toxicity to L. ca,.wi.However the inhibitory effect of free chitosan was demon~t~rated.for lactic acj.d bacteria growtl~."-'"Increase ill glucose concentration beyond 100.0g1.' has decreased the cell ~nul tipl.icnt,ion.This is due t,o the osmotic effect of high sugar concentratiori and 'l.clctic acid j.nhjbjtjon on cell'rnet,abolism.' Dj.ffusjona1 1.imitations for the s ~b s t r a t e and product in the microenvi~onment would have been prominent wit11 increased cell number in the beads.All these factors would have contributed to the decreased cell viabi1it.vwith increased glucose concentratjon.
The main defect in our experiment was that in the feed medium no calcium was included.'"-l"Calcium ions which stabilize calcium alginate would have been taken away by lactate ions produced during f e r ~n e n t a t i o n .~~W a d a et a1.21 have reported the weakening of the gel network during growth of the cells.L .he1veticu.sentrapped in calcium alginate has shown a productivity of 8.0g 1-I h-l with 5.0% conversjon of lactose (5.0g 1-I) where the flow rate of the substrate was 400ml h-'.Aft.er a weeli's operation, clogging in the packed bed was observed.17Stenroos et a.l." reported that calcium alginate entrapped L. delbrueckii has yiel.ded97.0% of lactic acid and the biocatalyst with a half life of 50.0 days.In our experiment the lactic acid yield was 92.0%.In the experiment of Stenroos et aLZ2 the steady state was reached after one week and in our case it was faster (3 days).With L. Z~l~lgaricus immobilized in calcium alginate, 50-6091 lactic acid was obt,ained and the process continued for 25 days while reaching the steady state after 10 days ."In 15-carrageenan-locust bean gum gel beads four different strains of lnesophilic lactic acid bacteria were entrapped and the productivities were high for eight weelis."However microscopic observation showed a progressive destructi.on of the beads and.release of viable cells from peripheral cavities of the gels .'5L. casei cells entrapped in calcium alginate yielded maximum lactic acid at 1S.Oml 11.' flow rate with a productivity of 21.2g 1.' h-l.With increase in the flow rate, viable cel1.s in the beads were decreased.However the cell number was more than those present initially.Maximum lactic acid yield (81.6%) was obtained a t the glucose concentration of 125.0g 1-I.The viable cells in the beads increased with increase in glucose concentration.When the nutrient medium containing 125g 1.' glucose was passed continuously, steady state was reached after 3 days and co~ltinued for 16 d.aysand. the viable cell.number in the beads was constant for 16 days.
5 and 42°C into a column packed with calcium alginate entrapped L a c t o b a c i l l r ~s casei.

Figure 4 :
Figure 4: Effect of glucose collcelltration present in nutrient medium on lactic acid (U) yield and (B) productivity when nutrient medium was fed a t 18.0 nll h-'.pH 6.5 and 42°C into a colurnil packed with calcium alginate entrapped Lactobacillus ca.sei.