Indian Journal of Experimental Biology Vol. 37. March 1999. pp. 274-279

Effect of support materials on cephamycin C production by immobilized Streptomyces clavuligerus

V. Sridevi & Padilla Sridhar*

Department of Microbiology. OsmaI~i a Univers ity. Hyderabad SOO 007. India

Received 26 November 1997; Revised on 9 November 1998.

In order to know the eOcct of supports on cephamycin C producti on. under si mil ar experimental condit ions. S clavlIligerlls cells were immobilized with - sponge. 2% agar, 2% and 4% alginate support materials. 1\n experimental set of free cell was also maintained as control. Cephumycin C production by these immobili 7cd and free cells was estimated at 48. 96 and 120hr or fe rmentation . In all thc cases cephamycin C prodoction was round to be high at 120hr of fcnnentati on. Sponge was found to be a better support materi al than other supports used for immobili zat ion.

Broad spectrum acti v ity and res istance to 13-lactamases make ce phamycins more effec ti ve tn treating many cephal osporin res istant iso lates i.e. E. coli. Klebsiella. Proteus e tc. The extended spectrum of ceph amycifl s al so inc ludes such o rgani sms as Serratia. Protells. and Bacten 'o ides, whi ch are res istant to most cephalosporin s.

Cephamyc in C, a 13-l actam antibi oti c is used as an inte rmediate for semisynthe ti c ant ibi o tics such as cefo x itin , ce fm etazo le, and ce fotetan. Cefox itin and cefmetazol e are be ing used as therapeut ic agents. Many market forecasters see ceph a losporin s and cephamycin s taking over from peni c illins as the most important 13-lactam products of the future 1-, .

Production of antibiotics seems to be feas ible with immobilized cell s with va rio us supports like polyacrylamide, cal c ium a lginate, ca rrageenan, ce lite, as in the case o f the producti on o f bac itrac in4

,

patulin S, thienamycin6

. ni kkomyc in 7,R and penicillin

G9,

Morikawa et al. 10 suggested that the po ly­acrylamide gel po lymeri zation reagents individually inactivate the multienzyme systems in the myce lium . Such negative effects have al so been encounte red by Freeman and Aharo nowitz l l when entrapping viabl e Streptomyces ce ll for cephalosporin (cephamyc in ) production with polyacry lamide. However, no reports are avai lab le on sponge immobilization for cepha­mycin C production .

' Correspondent author

A n attempt has been made to study the production o f ceph amyc in C, at di f fe rent t ime interva ls i.e. 48. 96, and 120 hr o f fe rm en tation, by us ing free and immobili zed ce ll s of Streptomyces clavuligerus.

Streptomyces clavuligerus ce ll s ere immobili zed w ith three diffe rent supports - sponge, 2% aga r and 2 and 4% a lginate. Cephamyc in C prod ucti on by these immobili zed ce ll s and a lso by the free ce ll s is studied.

Materials and Methods Cultures

Cephamycin C producing organ ism: Cephamyc in C produ c ing strain of Streptomyces clavuligerus L.C 21 wa s obta ined from Prof. L.C Vin in g, Dalhousie Uni ve rs ity, Canada . The strain was improved for cephamyc in C producti on, by mutagenes is. T he improved mutant was des ignated as PS2, an d wa s used for immobili zation studi es .

Indicator organism: A s uper sens itive indi ca tor organi sm E. coli ESS I 2

, kindly suppli ed by Pro f. L.c. Vining, was used for cephamyc in C b ioassay.

Media used and cllltlire conditions Seed preparation l J

: S. clavuligerus PS2 culture was streaked on plates containing I % starch , 1% CSL (corn steep li quor) in 0.1 % CaCO" 0.44% K2HP0 4,

0.06% MgS04 and 0 .5% SFC(sunflower oil cake). Spore suspension was prepared from 5-day-old incu­

bated plates at 28°C, and was inoculated into 50 ml of seed medium of above composition at pH 7 in 25 0 ml

Erlenmeyer flasks and incubated for 48 hr at 28°C under shaking ( 150 rpm).

SRIDEVI & SRIDHAR: SUPPORT MATERIALS, CEPHAMYCIN & STREPTOMYCES CLA VULlGERUS 275

Actively grown seed inoculum (4%) was used for preparation of free and immobilized cell preparations.

Production medium 13. The immobilized and free

cells were inoculated in 25ml production media con­taining(%), starch 0.5; CSL 0.5; CaCO} 0.05 ; K2HP04

0.22 ; MgS04 0.03 and SFC 0 .25; at pH 6.5 and

fermentation was allowed at ISO rpm and at 28°C.

Preparation offree and immobilized cells

Preparation of free cell samples: The above 4% seed culture was inoculated into 25 ml of production medium.

Immobilization by sponge adsorption method 13:

Seed culture (I ml) was 'mixed with 2S0mg of sponge pieces of uni form size ( lOx I Omm) and allowed for absorption in a vial. Pre autoclaved sodi um alginate (2 ml of 0.25%) was added on to the sponge - seed inoculum mixtu re. and allowed for absorption for 2min . Sterile CaCI2 (5 ml of 0.05 i\{) was poured for entrapping the cells in sponge. After' 30 min of curing the immobi li ze.d sponge pieces were transferred to 25 ml of production medi um,

Alginate entrapment method I4,15: Seed inoculu m (I

ml) was mixed wi th I ml of 2% alginate and in another set I ml of inoculum was mixed with Iml of

400

----

4% alginate and the beads were aseptically prepared by adding the mixture using I ml syringe in Sml of 0,5 M CaCI 2. After 30 min of curing, the 2 and 4% alginate immobilized beads(bead diameter: 0.28 mm) were separately inoculated into 2Sml of production media for cephamycin C production.

Immobilization of cells in agar /4,15: Seed

inoculum( 4 ml) mixed with 20 ml of 2% agar was

maintained at 40°C; and the mixture was poured in to petri plate, cut into 4 equal blocks and I block was cut into 2 equal parts, and these 2 pieces of agar blocks were transferred to production medium.

The immobilized and free cell experimental sets were taken in triplicates for the analysi s of resul ts .

Bioassay of cephamycin C-Production of cepha­myc in C by free and immobi lized cells was determined by agar plate diffusion method l6

, usin g super sensitive stra in - E. coli ESS I2

, as indicator organism. Cephamycin C potency in fermentat ion broth was biologically determined by a standard agar plate diffusion method as described by Kavanagh l6

.

Aliq uots of the fermentation broth (10 fo ld diluted) were loaded into wells bored in agar plates which were overlaid with E. coli ESS . The diameter of clear zone of inhibition was measured after 18hr of

1.6

E [J]48hr D 96hr El120hr 1.4 -2: r

I-

~Cel\ leakage at 120hr ~Free ::ell biomass

e 300 r 1.2 0 0

CD 1 '0

..... [ rn

s-..... e CD Q) Q)

0,8 '7' Q)

<.C

u 200 e 0 ~ U CD

0.6 ----<.C --.... 0.4

() ~ c

'0 l >- 100 E ell

L .r:. 0.2 c.. ~ Q)

() f-

0 i

Free cells 2% Agar 2% Alginate 4°/-:.: Alginate . Sponge 0

Type of cells. Fig. 1 -!'~oduc t i0I1 of cephamycin C b) free and immobi lized cell of S ciavllligerlis

276 IN DIAN J EXP BIOL, MARCH 1999

incubation at 37°C. The cephamycin C production by free and immobilized cell:; were calculated fTom a standard calibration curve.

Cell leakage--After 120hr of fermentation, in all immobilized cell . samples, cell leakage in the fermentation broth was measured by dry weight method . In free cell samples biomass was estimated by dry weight method .

Effectiveness jactors--Effectiveness factors 14 for im mobilized cells of S.clavuligerus w ith different supports i.e . sponge, 2% alg inate, 4% alginate and

2% agar at 48, 96 and 120hr of ferme ntation were calculated as the ratio cephamycin C product ion with free and immobilized cell s.

Statistical analysis Analysis of variance: Analys is of var iance consists

of class ifying and cross class ifying stat istical results and testing whether the means of a specified class ification differ significantly. T hi s allows the analys is of total variation of a data set into components which may be attributed to various sources(i.e. different support materials employed for im mobilization of cells for cephamycin C production) or causes of variation(i .e . cephamycin C prod uctions at different fermentation time interval s) .

Descriptive statistica l analysis: Descriptive statistical analysis reveals the arithmetic mean, standard deviation, sample variance, coeffic ient of variation, std. error of mean , minimum and maximum production, sum of antibiotic production va lues of

free cells and immobil ized cells of di fferent supports, at different fermentation time intervals .

Results Treatment combinations

From the preliminary work l] the concentrati ons of support materials(sponge weight: 250 mg; alginate:2% and 4%; agar:2%) and experimental conditions (i.e . production medium composition; fe rmentation temperature: 28°C; fermantation ti me : 120 hr; shaking condition: 150 rpm ; pH:6.5) were optimi zed . In o rder to evaluate the best support material for ccphamyc in C production, S. clavuligerus ce lls ""ere immobilized with th ree di f­ferent supports - sponge, agar and a lg inate. A set of free cells was also taken under s imi lar experimental condit ions as control. During fer mentation, the cephamyc in C production by these immobilized and free cells was estimated by bioassay at 48, 96 and 120 hr time intervals and zone of inhibition measured(mm). Corresponding cephamycin C COl1-

centra-tions were obtained from standard cephamycin C calibration curve and presented in Fig. I .

Since antim icrob ial activity l6 (or) antibiotic potency is proportional to antibiotic concentration, the diameter of clear zone of inhibit ion was taken for statistical ana lysis.

Cephamycin C production in treatment combinations In all treatment combinat ions cephamycin

C production was gradually increased from 48 to 120

Tab lc I- Descriptive stati stical analys is of cephamycin C production by free and immobilized cells at time lIl terval s of 48, 96 and 120hr fermcntation .

Immobilized cells of Free Agar 2% 4% Sponge. Cells Algi nate Alginate

Arithmetic mean df zone of inhibition : (mm). 20 .833 12.833 17.660 13 .000 13 .666 Cephamycin C production : (I-lg/ml). 272 86 158 98 115

Sample standard deviation : 2.466 I 1.130 5.008 11 .357 11 .930 Sample vari ance : 6 .083 124.083 25 .083 129.000 142.333

CoeffiCient of variation: 1.838% 86.799% 28 .34% 87.367% 87.295%

Std. Error of mean : 1.424 6.430 2.89 1 6.557 6.887

Minimum zone of inhibit ion : (mm) 18.000 0 .000 12.000 0.000 0 .000 Ma.ximum zone of inhibition :(mm) 22 .500 20.00 21.500 21.000 22000

Sum of produ ction (at 48, 96, 120hr): 62.499 38.490 53 .000 39.000 41 .000 Sum of squares : 1314.250 742. 249 986.500 765 .000 845.000

Deviation sum of sguares: 12.1 66 248.166 50. 166 258 .000 284 .660

SRIDEVI & SRIDHAR: SUPPORT MATERIALS, CEPHAMYCIN & STREPTOMYCES CLA VULIGERlJ.S 277

hr of fermentation . At 48 hr of fermentation, among all treatment combinations, only 2% alginate

immobilized cell system (80 Ilg/ml) and free cell

systems (165 Ilg/ml) exhibited production. In agar immobilized cell system, the production was 175 and

250 Ilg/ml at 96 and 120 hr of fermentation . With 2% alginate immobilized cell system, approximately a

gradual increase of 100 Ilg/ml in production was

observed 48(80 Ilg/ml) to 96hr( 185 Ilg/ml) and 96 to

120hr(280 Ilg/ml ) of fermentation . From 96 to 120 hr of fermentation , 1.66 and 1.77 fo ld increase in production was observed with 4% alginate (165 to

275 Ilg/ml) and sponge( 180 to 320 ~lg/ml ) immo­bilized cell systems respectively.

Statistical analysis--J.n order to know the effect of alginate, sponge and agar support materia l on cephamycin C producti on at different time in te rval s during fe rmen tat ion the descriptive stati sti cal analys is was performed and compared. The data of the descriptive statistica l ana lysis was illustrated in Table

Table 2- Randomi zed block ANOV A of cephamycin C production (in terms of zone of inhibi tion) of free and

immobi lized cells ofS clovuligeru

(Estimates of production means)

Immobil ized cell samp le Mean Cephamycin C (mm) conc.

(J.lg/ml)

Free cells 20.16 222 Agar immobili zed ccl ls 12.R3 R6 2% alginate immohi lized cell s 17.66 ISR 4% alginate immobil ilcd cell s 13.00 9R Sponge immobil ized ce ll s 13.66 115 48hr fermentation samples of 6.00 25 free and immobilized cell s 96hr fermentation samples of 19.00 189 free and immobilized cells 120hr fermen tation sam ples of 2 1.4-l 285 free and immob ili zed cell s

Source of Degrl'es SUIll of Mean F-Ratio Proba-vari ation . of squares square hili ty

freedom

!3etween 4 129.56 7 32.392 I.5 7-l 0.270R

supports of Immobili zed cells Within time 2 686.S33 243 .267 16.68 IAOOe-intervals 03 Errors R 164 .63 20.579 Totals l-l 9XO.733

I. From the data of Table I the following specific inferences can be drawn :

• Higher cephamycin C production mean value (20.83 mm zone of inhibition) was observed with free cells. Lowest coefficient of variation value (\.838%) of free cells indicating the higher homogenei ty in production at different fermentation time intervals. Highest total sum of production (20 .83mm zone of inhibition at 48, 96 and 120hr o f fermentation) was observed with free

cell s.

• Among immobilized cell s of different support materia ls 2% algi nate showed hi gher cephamyc in C product ion mean va lue (17 .66mm zone of inhibition ), with lowest coefficient of variation value (28.34% ). In case of immobilized cell s of diffe rent support materials, 2% alginate showed hi ghest sum ofJ3F0duction (53 mm zone of inhibiti on) than other immobilized ce ll s. Lowest sum of production (38A9mm total zone of inhibition at 48, 96 and 120 hr of fermentation) was observed with 2% agar immobil ized ce ll s.

Analysis of I'oriunce--J. n order to find out whether there was a sign ifican t difference in production among free and differellt immobilized ce ll systems, appropriate stati st ical treatment 17 was done by randomized bl ock ANOYA by using Microstat computer package (Table 2). Based on the probability va lues, the cephamycin C production variance ratios between different sllpports of im'mobil ized ce ll s was J .574, which was significant at a 0 .2708 probability va lue. Within fermentation time interva ls, the F-ratio val ue bei ng J 6 .68, which was s ign ificant even at a

lower probab ili ty va lue ( I AOOe-03) . So, there were more statistically s ignificant variations Il1 the prod uction was fo und within fermen tation ti me interva ls i.e . at 48 , 96 and J 20hr than the d ifferent

Tabk 3-Effectiveness i11cto rs for immob ilized cell s of S . clo vuligerus

Cephamyc in C producti on rati o of free and immobilized cell s

Fermentation Immobi lized cell systems of time (hr) 2% 2% 4% Sponge

Agar Al ginate Alginate

48 hr 0 OA8 0 0 96 hr 0.70 0.74 0.66 0.72 120 hr 0.694 0.77 0.763 O.8R

278 INDI AN J EX P BIOL, MARCH 1999

supports employed i.e. agar, a lg inate of 2% and 4% and sponge.

Cell leakage-At 120hr of fe rmentation, ce ll leakage in fe rmentati on broth of immobilized cell s and biomass we ight of free ce ll s was measured (Fig. I). Very hi gh cell leakage va lues were observed in agar (0.5 gil) and 2% a lginate ce ll systems (0 .425 gil). Sponge immobi lized ce ll systems exhibited lower ce ll leakage than a ll immobil ized systems ( i. e. a lg inate of 2 and 4%, and agar) under s imilar ex peri menta l conditions.

Effectiveness fac tor- T he effecti veness fac to r l4 o f im mobilized cell s w ith di f ferent supports were calculated as the ratio of free and immobil ized ce ll s at different fe rmentati on time interva ls and the da ta was dep icted in Tabl e 3. At 48 and 96 hr of fermentation, the calcul ated effectiveness fac to rs 0.48 and 0.74 wit h 2% a lg inate immobili zed ce ll s, were found to be higher than other immobili zed cell systems. But at 120 hr of fermentation, sponge immob il ized ce ll systems ex hibited highest e ffecti veness factor i.e. 0 .88 .

Discussion Inf luence offermentation time on cephamycin C

production by free and immobilized cells- The antibiotic fermentation was initiated at 48hr of fe rmentation in free and immobilized cel ls. In all th e cases, there was gradual increase in prod uction fro m 48 to 120 hr, and then decreased. The comparati ve cephamycin C production at var ious fe rmentation time interva ls i.e. 48, 96 and 120 hr was accounted as fo llows:

At 48 hr of fermentat ion, production was not seen with 2% agar, 4% alg inate and sponge immobili zed ce ll s. However, producti on was observed w ith 2% alg inate (80 J.lg/ml) wh ich was 2 fo lds less than free ce ll system. At 96 hr of fermentation, a ll the immobilized ce ll systems showed production, a lthough with variations. Among a ll these, 2% a lg i­

nate system exhib ited more producti on ( 185 ~lglml ) . It is interesting to menti on that at 120 hr of fe rmentation, sponge immobilized ce ll system showed more production than a lg inate and agar.

Evaluation of best support material with re.\p ec;t to cell leakage and effectiveness fac tor- T he res id ua l starch in the production medium was est imated at 48hr to determine the substrate utili zation by

immobilized systems w ith various supports. To our

surprIse, free and immobilized ce ll s of different supports showed a lmost no difference in the pattern of utili zation of substrate . The best support material and preferred fermentation time interval were eval uated w ith respect to ce ll leakage and effectiveness factor. T he significance of this measurement of ce ll leakage by immobilized cells, reflect on the effi c iency of immob ilization process . T he lower the leakage the better is the immobilization process and better support . The red uced cell leakage fac ilitates enhance separat ion of product, reduces diffus iona l and mass transfer limitations cloggi ng problems during fermentation.

Very high ce ll leakage va lues were observed in agar (0.5 gi L) and 2% a lg inate ce ll systems (0.425 giL) (F ig. I) . . A lthough, the results of descripti ve statistica l ana lys is (Table I) revealed that 2% a lginate system had the hi ghest sum of production than other immobili zed cell s may be because of its high ce ll leakage, it was not cons idered as idea l support materia l, in whi ch the cephamycin C production was governed by both free and immobili zed cell s . Sponge immobili zed ce ll systems exhibited lower ce ll leakage(0. 12 giL) than 4% a lg inate(0 .26 giL) under s imilar experimental conditions.

Immobilized ce ll systems w ith vari ous supports ca n be made an a lternati ve to the established antibioti c fe rmentation s, only when they offer di stinct d 11 . . h I a vantages ' over eXlstlI1g processes, suc as ow

leakage of ce ll s and high biocatalytic activi ty . T he ratio o f producti ons of free ce ll s and

immobilized ce ll s which can be considered as an effectiveness factor of immobilized systems are shown in Table 3 . Of a ll immobilized cell systems sponge system at 120 hr of fe rmentation showed highest e ffectiveness factor (0 .88) . Thus, the present in vestigations suggest that - .sp onge support material was better than other supports tested for immobilization of S. clavuligerus ce ll s for production of cephamycin C.

Acknowledgement T hi s work was fin anc ia lly supported by the

Department of Biotechnology, Govt. of India to PS.

References I Stapley E 0 & 13 irn baun. ~-Iactam antibiotics (Academic

Press, New York ) 198 1. 327. 2 Nagarajan R, Boeck L D. Gorman ,\11 , Il amill R L, Higgens C

E. Iloehn M M, Stark W M & Wh itney. J G. JAm Chem Soc. 93 , (197 1) 2308.

SRIDEVI & SRIDHAR: SUPPORT MATERIALS. CEPHAMYCIN & STREPTOMYCES CIA VULIGERUS 279

3 Omstead DR, Hunt G R, & Buckland B C. in Comprehensive biotechnology, Vol. 3. edi ted by M Moo Young (Pergamon Press) 1986, 187.

4 Morikawa Y, Karube I, & Suzuki S. Biotechnol Bioeng. 22. (1980) 10 I 5.

5 Foster B C, Coutts R T, Pasuttto F M & Dossetor J B. Biotechnol Lett, S, (1983) 693 .

6 Arcuri E J, Nichols J R, Brix T S, Santamarina V G. Buckland B C & Drew S W. Biotechnol Bioeng, 25, ( 1983) 2399.

7 Berk D, Behie L A, Jones A. Lesser B H & Gaucher M, Can J Chem Eng, 62, ( 1984a) 1 12.

8 Berk D, Behie L A, Jones A, Lesser B H & Gaucher M, Can J Chem Eng, 62, (1984b) 120.

9 Deo Y M & Gaucher G M, Appl Microbiol Biotechnol, 15 . (1985) 220. .

10 Morikawa Y, Karubc I, & Suzuki S, Antimicrob Agents Chemothe. 15, (1979) 23 .

II Freeman A & Aharonowitz Y. Biotechnol Bioeng. 23. ( 198 1) 2747.

12 Sai laja Rani G, Sarada I, Sarcar A. & Sridhar P, Indian J £-rp Bioi, 34. (1996) 816.

13 Sridcvi V, in : Production of cephamycin C from immobili zed Streptomyces c1avlIligerus (Ph.D thesis). Osmania Universi ty. ( 1998) 100.

14 D' Souza, Whole cell and enzyme immobilization techniq ues. Short term course, DBT 1993, 21.

15 Phi lips C R & Poon Y C, Immobilization of cells , 1988. 68. 16 Kavanagh F(edr.). Analytical microbiology V.1. (Academic

Press, New York) 1963, 313 . 17 Khan & Khanum, Fundamentals of Biostatistics, 1993, 248.