Optimization of Fermentation Conditions for Bioactive Compounds Production by Enterococcus Faecium

Journal of A pplied Scienes Res earch, 5(10): 1445-1454, 2009
© 2009, INSInet Publication
Optimization of Fermentation Conditions for Bioactive Compounds Production by
Marine Bacterium Enterococcus Faecium
1
Wefky, S.H; 1Abou-Elela,G.M; 2El-Bestaw y, E.
1
Marine Microbiology laboratory, Environmental Division, National Institute of Oceanography
and Fisheries, Alexandria, Egypt.
2
Department of Environmental Studies, Institute of Graduate Studies and Research, University
of Alexandria, Alexandria, Egypt.
Abs tract: Thirty four bacterial s trains were is olated from s ea water, s ediments and algae s amples which
were collected from A lexandria beaches . A ll s trains were s creene d for their potentiality to produce
bioactive compounds by us in g well cut diffus ion technique agains t the following pathogens :
Staphylococcus aureus, Streptococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, Micrococcus
luteus and Candida albicans as indicator s trains . The mos t potent s train was identified at t h e mo lecular
level as Enterococcus faecium, while t he mos t s us ceptable s train was S.aureus. W ell cut diffus ion
technique w a s p e rfo rmed us ing different culture media (nutrient agar, Zobell agar and Luria Bertani), the
mos t s uitable medium was Luria Bertani with inhibition zone of 10 mm. Placket-Burman des ign was
applied to optimize the fermentation conditions and maximize the product iv ity. The optimized medium
was formulated as follows : (g/l): peptone, 15; yeas t extract, 2.5; concentrated s ea water ( >100%), adjus ted
t o p H 8 and inoculum s ize 1.5 ml, this medium gives inhibition zone of 16 mm when incubated a t 35 o C
for 48 h i.e inhibition zone was increas ed about 1.6 fold increas e. M utation techniques (phys ical and
chemical) were applied to increas e b io a ctive compound productivity but revers e effect was detected.
Immobilization us ing both entrapment (alginate) and ads orptio n (luffa and pumice) techniques were
applied. Only cells ads orped on pumice ga v e higher productivity and the inhibtion zone reached up to 17
mm.
Key words : Bioactive compounds – bacteria- inhibition zone- mutation –immobilization .
INTRODUCTION
Bacteria a nd other micro-organis ms are ubiquitous
in the marine environme n t. They are taxonomically
divers e, biologically active, a n d colonize all marine
habitats , from the d eep oceans to the s hallowes t
es tuaries [4 4 ]. It has been es timated that the ma jo rity of
bacteria in natural aquatic ecos ys tems are organize d in
biofilms [1 2 ]. In a biofilm, a microbia l community is
a t tached to a s urface and embedded in a s elf-produced
matrix compos ed of extracellular polymeric s ubs tances .
This s tructure provides the bacteria pres ent in the
biofilm with s everal advantages compared to thos e
liv ing as planktonic cells . Firs t, the bact e ria a re
maintained in the s electe d micro environment where
population s urviv a l d o e s n o t d e p e n d o n rapid
multiplication [3 0 ]. This is es pecially advantageous in
enviro n me nts where the bacteria are expos ed to
cons tant liquid movements , as , for example, in aquatic
environments . A dditionally, the bacterial cells pres ent
in a biofilm have an increas ed res is tance to des iccation,
grazing, and antimicrobial agents compared to their
p lanktonic counterparts [2 2 ,3 8 ]. A ls o, biofilms o ffe r
enhanced opportunities for interactions s uch as
horizontal gene trans fer and co-metabolis m [3 0 ,4 8 ].
The o ccurrence of large s cale of bioactive
compounds is not commo n to all living organis ms , but
res tricted to certain taxon o mic groups . Recent res earch
progres s es reported that many bioactive natural
produc t s from marine invertebrates have s triking
s imilarities to metabolites of their as s o c iated microorganis ms including bacteria [4 3 ]. Compared with
terre s t ria l o rganis ms , the s econdary metabolites
produced by marine organis ms have more novel and
unique s tructures o w ing to the complex living
circums tance and divers ity of s pecies , and the
bioactivities are much s tronger [4 6 ].
Competition among microbes for s pace and nutrient
in marine environment is a powerful s election pres s ure
that endows marine micro-organis ms to produce marine
natural products pos s es s in g medical and indus trial
values [3]. M any antimicrobial, antifouling s u bs tances
Corresponding Author: Dr. Gehan M . AbouElela, Associate professor, M arine M icrobiology laboratory, Environmental
Division, National Institute of Oceanography and Fisheries, Qyet Bay,ElAnfushy, Alexandria
Egypt. Tel: 0128636344
1445
J. Appl. Sci. Res., 5(10): 1445-1454, 2009
have been found among thes e kinds of bacteria due to
the s pecialized role they p la y in their res pective hos ts
[2 6 ]
. It is s ugges ted that the primary role of thes e
antibiotic s ubs tances c ould be related to ecological
competition [8].
Different applications of bacteria as probiotics have
been evaluated in vitro and in vivo for their poten t ial
to inhibit fis h and larvae in aquaculture [1 9 ] for e xa mp le
Carnobacterium [4 0 ] a n d Enterococcus s pp. [5 0 ,3 9 ]. Other
applicat io ns of bacteria including the production of
antifouling agents have been s tudied [1 1 ].
The nutritio nal and environmental conditions have
a great influence on production of the antimicrobial
s ubs tances [3 4 ]. In order to develop an e ffic ient
production of antimicrobial s ubs tances , knowledge
regarding the e nvironmental factors affecting this
proces s needs to be well identified. Experimental
des igns are excellent tech n iq ues for optimization of
culture conditions to achieve optimal production [1 4 ,1 5 ].
The aim of this work was is olation o f s ome marine
bacteria capable of producing bioactive compounds and
optimizing the fermentation conditions for maximum
production.
MATERIALS AND METHODS
Organis ms and Maintainance: Thirty four bact erial
s trains were is olated from s ea water, s ediments and
algae s amples which were collected from A lexandria
beaches on nutrient agar medium. A ll s trains were
s creened for their potentiality to produce bioactive
compounds agains t the pathogen Staphylococcus aureus
(A TCC 6538), Streptococcus faecalis (A TCC 8043),
Pseudomonas aeruginosa (A TCC 8739), Escherichia
coli (A TCC 8739), M i c r o coccus luteus (A TCC 10240)
and Candida albicans were us ed as indicat or s trains .
Thes e indicators were kindly provid e d by Dr.W efky,
S.H. (National Ins titute of Oceanography, and Fis heries ,
A lex.Egypt). The mos t promis ing s train was chos en
and identified according to the s tandard procedures
d e s c ribed by [4 7 ,4 9 ,5 2 ]. M oreover it was s ubjected t o
molecular identification.
A n ta g o n i s t i c A c t i o n A g a i n s t I n d i c ator
Microorganis ms : The well-cut diffus ion technique was
us ed to tes t the abilit y of the bacterial is olates to
inhibit the growth of indicator bacteria a n d y eas t, 50µl
was a d d in each well, A fter incubation period, the
radius of clear zone around each well (Y) and the
radius of the well (X) were linearly meas ured in mm,
where dividing Y2 over X2 determines an abs olu t e u n it
(A U) for the c lear zone. The abs olute unit of each
antagonis tic is olate, which indicates a pos itive res ult in
the antagonis tic action, was calculated according to the
following equation [1 6 ]: A U= Y2 /X2
1446
A ll experiments were done in triplicates and the
average was calculated.
Molecular Characterization:
Is olation of Bacteri al D N A : The genomic DNA of the
mos t promis ing producer S14 was is olated from
overnight cultures accordin g to the method des cribed
by Sambrook et al. 1989. Cells were collec t e d by
centrifugation and re-s us pended in 500 µl TEN buffer.
A fter incu b a t ion at 37°C for 30 min, 30 µl of 10%
Sodium Dodecyl Sulpha te SDS were added and the
tubes were inverted gently s e veral times till complete
lys es . A n aliquot of 5 mg/ml of proteinas e K added
and the t ubes were incubated at 37°C for one hour.
A fter incubation the s o lu t ion was phenol-extracted
s eve ra l times to remove the protein and once with
chloroform to remove the phenol traces . The DNA was
pre c ipitated us ing 0.8 volume of is opropanol and
was hed w it h 70% ethanol. The DNA was dried and
dis s o lv ed in 10 mM Tris HCl, pH 8 and s tored at
20°C.
Amplification of 16S rRNA Gene: The 16S rDNA
w a s amplified by polymeras e chain reaction (PCR)
us ing the primers 16F27 a n d 16R1492. A pproximately
a 1500-bas e pair fragment of 16S rDNA region was
amplified according to the Escherichia c o li genomic
DNA s equenc e. A mplicons were obtained with a PCR
cycling program of 94°C for 1 min followed by 30
cycles of denaturating at 94°C for 1 min, annealing at
55°C for 1 min and polymerization at 72 for 2 min. A t
the end of thermocycling, The PCR re a ction mixture
was incubated at 72°C for 10 minutes . A s des cribed by
[4]
. amplicons were vis ualized by e lectrophoretic
s eparat io n o n 1% agaros e gels s tained with ethidium
b ro mid e . P CR fragments were p u rifie d fro m
amplification rea c t io n s w it h Q IA q u ic k P CR
purification reagents (Q IA EN) according to the kit
manual.
S equencin g of PCR-DNA Product: DNA s equence
was obtain e d u s ing DNA s equencer (A BI 310). The
PCR product w a s s equenced us ing the s ame PCR
primers and other internal primers to co n firm the
s equence.
S equence S imilarities and Phylogenetic Analys is :
Blas t program (www.ncbi.nlm.nih.gov/blas t) was us ed
t o as s es the DNA s imilarities . M ultiple s equ e n c e
alignment and molecular phylogeny were performed
us ing BioEdit s oftware [2 3 ] . The phylogenetic tree was
dis played us ing the TREEVIEW program [4 1 ].
Effect of Culture Medium Type on Production of
the Bioactive Compoun ds : The s elected bacterial
J. Appl. Sci. Res., 5(10): 1445-1454, 2009
s train was grown us in g nutrient agar, ZoBell and Luria
Bertani (LB) media (prepared with s ea water) at 30°C
for 24 hrs . Culture cell free s upernatant was tes ted
agains t the previous ly chos en indicator organis ms us ing
agar well diffus ion technique aiming to obtain the
highes t productivity [1].
Evaluation of Nutritional Factors : The PlackettBurman de s ig n [4 2 ,5 3 ]. was applied to reflect the relative
importance o f v a rious factors involved in the
production of thes e bioactive compounds by the chos en
s train. For each v a ria ble a high (+) and low (-) levels
were tes ted. The examined variables in t h is e xperiment
a n d t h eir levels are s hown in Table 2. Eight different
trials were performed in duplicat e s . Rows in Table 3
repres ent the different trials (row no. 9 repres ents the
bas al contro l). The main effect of each variable was
determined with the following equation:
Exi = (M i+ – M i-) / N
W here Exi is the variable main effect, a n d M i+,
M i- are the radius of the clear zone arou n d each well
in the trials , where the independent variable w a s
pres ent in high and low concentrations , res pectively,
and N is the n umber of trials divided by 2. Statis tical
t-values for equal u n paired s amples were calculated
us ing M icros oft Excel to determine t h e variable
s ignificance.
Effect of Mutations on B i oac ti ve C ompounds
Production: To s tudy the effect of mutations on the
productivity of the s elected s train again s t the different
pathogens , phys ical mutat ion was performed us ing UV
lig h t (254 nm) at different time intervals in order t o
plot the s urvival c u rv e . Induction of mutation was
carrie d out at a dos e that yielded 90 % mortality as
determined from the previous s urvival curve following
the procedure of Kun g & Lee [3 3 ]. To s tudy the effect
of chemical mutation, ethidium b romide was us ed as
chemical mutagen according to [ 2 1 ]. Each mutant was
tes ted for its potent ability agains t the previous ly
mentioned pathogens us ing the well cut diffus ion
technique.
Effect of Immobilization on B i oactive Compounds
Production: Immobiliza t ion was performed us ing both
entrapment and ads orption techniques as was des cribed
by Eikme ie r & Rehm, [1 3 ] aiming to enhancing the
production of the bioactive compounds agains t different
pathogens .
micros cope. Cells grown in LB were harves t e d by
centrifugation, was hed with phos phate buffer and fixed
with 2 % glutaraldehyde followed by 1 % o s mium
tetroxide treatment. A fter complete fixation, s amples
were was hed in buffer s olution, and then dehydrated in
as cending order of ethanol concentrations . The s amples
were dried completely in a critical point dryer, and
finally coated with gold in JEOL-JFG1100 E ions putter-coater. The s pecimen s w ere viewed in JEOLJSM 5300 mic ros cope operated at 20 kV with a beam
s pecimen angle of 45o .
RES ULTS AND DIS CUS S ION
Res ults : The marine is olate S14 was the mos t
promis ing s train , it inhibited the growth of all
pathogens es pecially Staphyllo c o ccus aureus, the
inhibition zone was (9mm) on s ea water a g ar
medium.This s train was is olated from the s urface of the
alga (Ulva s p .) from Sidi-Bis hr. It is biochemically
identified as Enterococcu s sp. This was confirmed
us ing the molecular techniques .
Molecular Characterization of Enterococcus S pecies :
DNA of the promis ing Enterococcus s p. was extracted
a nd the extracted 16S rRNA gene was amplified us in g
the univers al primers 16F 27 and 16R1492. The
produced amplicons was analyzed u s ing agaros e gel
electrophores is as s hown in Figure 1. It wa s c le ar that
this s t rain s howed nucleotide s ize of 1490 bas e pair
c ompared with phage ë DNA Hind III cut molecula r
weight marker.
S equencing of PCR -DNA Products : The amplified
DNA was s equenced us ing A BI 310. T he s equencing
data obtained utilizing this s trategy was 500 bas e pair
which repres ents the partia l coding s equence of 16S
rRNA gene. The s equencing data was analyzed us ing
nucleotide BLA ST s earch c omputer bas ed program
where this s equ ence was compared with that of any
other rRNA (or rDNA ) genes that have been s equenced
s o far. The res ulting data indicated that the is olate
under s tudy was identified as Enteroc o c cus faecium
with identity percentage 92% w h ich confirms the
identification us ing the traditional biochemical tes ts .
The phylogenetic t ree was dis played us ing the
TREEVIEW program as s hown in Figure 2
Opti mi zation of Bioactive Compounds Production by
E. Faecium:
Effect of Medium Type on the Production of the
Bioactive Compounds : E. fa e c ium was examined for
the antagonis tic activity us ing three diffe rent media;
nu t rie n t agar (NA ), Luria Bertani (LB ) and ZoBell s ea
water agar . A s s h o w n in Table 1, the highes t activity
was obs erved us ing LB.
El ectron Micros copy: The adhes ion of the s elected
bacteria on or in the s upporting materials and formation
of the biofilm was carried out us in g s c a nning electron
1447
J. Appl. Sci. Res., 5(10): 1445-1454, 2009
Fig. 1:
16S a g a ro s e gel electrophores es of the extracted and amplified DNA . Lanes 1& 2= purified PCR product s
of the is olate, and lane 3 is phage ë DNA Hind III cut molecular weight marker.
Fig. 2:
Phylogenetic relations hips among repres entative experime n t a l s t rain and the mos t clos ely related
Enterococcus s pecies The dendogram was generated us ing Tree View Program.
Effect o f t h e growth media on the production of the
bioactive compounds by E. faecium.
Inhibition zone (mm) produced by the selected
strain using different media
LB
NA
ZoBell
------------------------------------------------------------------------------------10
9
9
Table 1:
Optimization of th e Fermentation Factors : The
Placke t t-Burman des ign was applied to reflect the
relative importance of various factors in v o lved in the
production of thes e agents by E. fae c i u m Ep1 .The
main effect of each variable on t h e production of the
bioactive compounds as well as t-values were es timated
for each independent variable as s hown in Table 4 and
graphically pres ented in Figure 3. Res ults in this Figure
indic a t e d that the main effect of all variables were
pos itive on the production by E. faeciu m except for
yeas t extract where high concentra t ion of yeas t extract
in the medium caus es decreas e in the production.
Statis tical analys es of the res ults (t-tes t) s h owed that
varia t ions in yeas t extract and the incubation period in
1448
the tes ted ranges had the mos t cons iderable effects on
the production of bio a ctive compounds by E. faecium.
The interacting effect o f y eas t extract with the
incubation period is des cribed in three-dimentional
repres entation (Figure 4). A s illus terated, the inhibit ory
effect of high levels of yeas t extract on the antagonis tic
activity of E. fa e c i um can be partially overcome by
preparing cultures with decreas ing level of yeas t extract
and extending the incubation period.
A ccording to the obtained res ult s , the predicted
medium for cultivation of E . faecium to enhance
maximum production of the bioactive c o mpounds was
formulated as follows : (g / l): peptone, 15; yeas t extract,
2.5; concentrated s e a w a t e r ( >100%) , adjus ted to pH
8 and inoculum s ize (1.5 ml for e a ch 50ml medium)
all of which are incubated for 48 h at 35o C.
In order to evaluate the accuracy of the a pplied
Pla c ke t t -Burman s tatis tical des ign, a verification
experiment was ap p lie d to compare between the
predicted near optimum levels of independent variables
J. Appl. Sci. Res., 5(10): 1445-1454, 2009
and the bas al condit io n s ettings . Res ults in Table 5,
confirmed that,the p ro d u c t io n of the bioactive
compounds increas ed and the inhibition zone increas ed
by 1.6 fold increas e.
Table 2: Indepen d en t variables affecting production of the bioactive
compounds and their l evels in the Plackett- Burman design
Factor
Symbol
Level
---------- - -- - -- - -- - -- - -- - -- ---------1
0
1
Peptone (g/l)
P
5
10
15
Yeast extract (g/l)
Y
2.5
5
7.5
Inoculum's size (ml)
IS
0.5
1
1.5
Sea water concentration (%)
C
50%
100%
>100%*
pH
pH
6
7
8
T emperature (o C)
T
25
30
35
Incubation period (h)
IP
12
24
48
*150 ml of sea water was concentrated to100 ml by evaporation
Table 3: Experimental results of the Plackett- Burman design
T rials
Factors symbols
(Response)
Diameter of
inhibition zone
P
Y
IS
C
pH
T
IP
(mm)
1
-1
-1
-1
1
1
1
-1
11
2
1
-1
-1
-1
-1
1
1
16
3
-1
1
-1
-1
1
-1
1
0
4
1
1
-1
1
-1
-1
-1
0
5
-1
-1
1
1
-1
-1
1
14
6
1
-1
1
-1
1
-1
-1
12
7
-1
1
1
-1
-1
1
-1
0
8
1
1
1
1
1
1
1
15
9
0
0
0
0
0
0
0
10
Table 4: Statistical analysis of the Plackett-Burm an ex p erimental
design
Variable
Main Effect
t-value
Peptone
4.75
0.8
Yeast extract
-9.75
-2.4
Inoculum size
1.75
0.36
Sea water conc.(%)
3.25
0.6
pH
2.75
0.55
T emperature
4.25
0.76
Incubation period
5.75
1.09
t-value significant at the 1% level = 3.70
t-value significant at the 5% level = 2.45
t-value significant at the 10% level = 1.94
t-value significant at the 20% level = 1.37
Standard t -va lues are obtained from Statistical Methods (Cochran and
Snedecor, 1989).
Fig. 3: Elucidation of fermentation conditions affecting
the production of t h e antagonis tic agents of
E.faecium.
1449
Effe c t of Mutation on Bioac ti ve C ompou n ds
Pr odu c tion: E. faecium was s ubjected to two types of
mutations (phys ic a l a nd chemical) to inves tigate the
productivity of bioac t ive compounds . It was s ubjected
to irra d iation with UV lamp at 254 nm as a phys ical
mut a g e n a n d ethidium bromide as a chemical
mutagen.Variants obtained from both types of mutation
were tes ted for the production of bioactive compounds
to s elect the mos t potent one.
Concerning the phys ical mutation by UV
irradiation, a s urvival curve was plo t t ed us ing s urvival
%of the treated s trains agains t the expos ure time (h) as
s hown in F ig u re 5 . Expos ure time at which about 90%
lethality of the bacterial population was 1 min.
Capacity of bioactive compounds production was
determined for each mutant.Comparis on between the
wild type and its variants is pre s e n ted in Table 6.
Res ults indicated that UV-mutan t of E. faecium
exhibited lo w e r activity (13 mm inhibition zone)
compared to its wild type (16 mm) while ethidium
bromide-mutant was better than UV-mutant (15 mm)
but s till lower than the activity of the wild type.
Effect of Immobilization on Bioactive Compounds
Production by E. Faecium: Living c ells of E. faecium
Ep1 were s ubjected to immobiliza t io n us ing ads orption
and entrapment techniques . A ds orption was carried out
u s ing both luffa pulp and pumice as s upport in g
materials . Figure 6 s howed the ads orption of the cells
on luffa pulp and pumice while entrapment w a s done
us ing s odium alginate as a gel matrix. The aim of this
experiment is t o c o mp a re t h e p ro duction of
antimicrobial agents by both t h e free and immobilized
cells of E. F a e c i um. The optimized medium containing
the ads orbed cells of E. faecium was us ed. Res ults in
Table 7 revealed that E. faecium biofilm on pumice
s howed relatively h igher activity in the production of
the bioactive compounds (1.2 fold increas e) in the
diameter of the inhibition zone.
Us ing luffa pulp as the s upporting material
decreas e d the production to about 0.81 fold in the
in h ibition zone and about 0.66 fold in the activity u n it
compared to the free non-fixed cells . By us ing
entrapped cells of the wild E . faecium. Res ults
indicat e d a d ecreas e in the inhibition zone diameter to
about 0.75 fold and about 0.56 in the activity unit
compared to the free cells .
Dis c u s s ion: M arine organis ms are a rich s ource of
s tructurally novel and biologically active metabolit e s
[7 ,18 ,]
. Secondary or primary metab olites produced by
thes e organis ms may be potential bioactive compounds
of interes t in the pharmac eutical indus try [2 0 ]. To date,
many chemically unique compou n ds of marine origin
with various biological activities have been is o la ted,
J. Appl. Sci. Res., 5(10): 1445-1454, 2009
Fig. 4: The interaction effect of yeas t e xt ra c t c o n c e ntrations (g/l) with incubation periods (days ) Levels , with
res pect to inhibition zone (mm) bas ed on Plackett-Burman res ults .
Table 5: A verification experimen t s h owing antagonistic activity of
E. faecium grown on basal against optimized media
Inhibition zone (mm)
Basal medium
optimized medium
--------------------------------------------------10
16
Activity unit (AU/ml)
4
10.24
Fig. 6: Scanning electron micrographs s ho w in g (A )
control luffa pulp; (B) the wild E. fa e c i um
ads orbed on luffa p u lp; (C) control pumice and
(D) the wild E. faecium
Fig. 5:
Survival curves of E. faeci u m treated with UV irradiation.
Table 6: C omparison among the wild type and their mutan t s fo r
production of the bioactive compounds
E.faecium
Inhibition zone (mm)
Activity unit (AU/ml)
W ild type
16.0
10.24
Mutant (UV)
13.0
6.76
Mutant (ET )
15.0
9.0
1450
and s ome of them are u n d e r inves tigation and are
being us ed to develop new pharmaceuticals [2 0 ,3 5 ] . The
ability of marine bacteria to pro duce s econdary
metabolites of potential interes t has been extens ively
documented.
The mos t promis ing is olate S14 (Enterococcus s p.)
identified us ing mo lecular techniques . Recently, 16S
rRN A s equence comparis on has been us ed as a
powerful tool for es tablis hing p h ylogenetic and
evolutionary relations hips among organis ms [3 7 ]. A n
approach pres ently employed in many laboratories us es
J. Appl. Sci. Res., 5(10): 1445-1454, 2009
Table 7: Effect of immobilization on production of the bioactive compounds by the E. faecium cells
Bacterial strains
Antagonistic activity using different support materials
- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - ------------------------------------------------------------------- - -- - -- - -- - -- - -- - -- Control
Luffa pulp
Pumice
Ca-alginate beads
W ild E. faecium EP1
IZ(mm)
AU/ml
IZ(mm)
AU/ml
IZ(mm)
AU/ml
IZ(mm)
AU/ml
--------------------- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - -- - ------------------------------------------------
16
10.24
IZ = Inhibition Zone (mm) - AU= Activity Unit/ml
13
6.76
t he polymeras e chain reaction [2 5 ,5 1 ] to obtain 16S
rRNA -s pecific genes for s equence analys is . Sequencing
of PCR-p roduct and comparis on of 16S rRNA (rDNA )
s equences has be e n reported among type s trains of
Enterococcus [2 4 ,6 ,5 1 ].
T h e phylogenetic relations hips among the new
experimental is olate S14 a n d the clos ely related
Ente rococcus s pecies have been des cribed in the
pres ent work and revealed that , s t rain S14 was
taxonomically pos itioned within the E. fa e c ium group
repres enting 92% identity. The data obtained by 16S
rRNA coincide with thos e found by t raditional,
morphological, phys iologic al and biochemical methods .
This s train was identified as E. faecium.
Different type cultures were tes ted for their
s ens itivity to the bioactive compounds produced by E.
faecium to achieve the highes t antagonis tic activity.
E. faecium was grown in Nutrient agar, ZoBell and
LB media and was tes ted for the antagonis tic e ffect
agains t S.aureus. LB was found to be the mos t s uitable
medium to achieve the highes t antagonis t ic activity as
was reported by [1].
In the pres ent s tudy, Plackett-Burman des ign was
employed which was s ucces s fully emplo y ed in enzyme
production and other optimiza tion experiments [2 5 ,5 1 ,2 ].
Res ults revealed that concentration of yeas t is the main
factor affecting the activity of the bioactive compounds
produced b y E. faecium where it was s ignificant at 1%
level. Decreas ing the levels of this factor yielde d t h e
highes t antagonis tic effect agains t the tes ted pathogen.
It was noticed that t h e highes t production of the
antimicrobial agent by E. faecium was obtained at pH
8. [1 7 ] reported that pH 8 was the opt imu m pH for the
maximum production of bioact ive compounds from
Nocardia brasiliansis.
Leal– Sánchez, [3 4 ]. reported that temperature was
found to have pos it ive s ignificant effects (p#5) on the
production of the bioactive compounds which is in
agreement with o u r s tudy. In the pres ent s tudy, s ea
water concent ra tion exhibited pos itive effect on the
p ro d u ction by E. faecium as s hown by [3 6 ]. Inoculu m
s ize had als o pos itive effect on the production [2].
A nother s tudy carried out by [2 8 ]. s tat e d that
fermentation time is very important optimizing factor.
1451
17
11.56
12
5.76
In this s tudy, the maxima l production of the bio-active
compounds was obtained by E. faecium after 48 h after
which, the activity decreas ed s ignificantly in the culture
medium. Similar res ults were als o reported by [1 0 ].
It was s hown that increas ing the incubation period,
peptone, inoculum s ize, t e mperature, pH and s ea water
concentration have pos itive effects on the production of
bio a c tive compounds by E. faecium. Therefore, res ults
concluded that to achieve the highes t antagonis tic effect
by E. faecium, the medium compos ition s hould be: (g/l)
peptone , 15; yeas t extract, 2.5; s ea water concentration
( <100%), pH 8 with inoculum s ize 1.5 ml (for e a ch
50 ml medium) for 48 hrs at 35o C. Under s uch
conditions , t h e activity unit produced by E. faecium
s howed 10.24 A U/ml (2.56 fold increas e) than that
obtained us ing the bas al growth medium.
The effect of mutation s on the production by
different bacterial s trains have been reporte d (Hos oya
et al., 1998) but in our s tudy, the mutation s d e creas ed
the production of bioactive compounds .
Immobilization on pumic e s howed (1.13) fold
increas e in the productivity compared t o t h e free cells
while immobilization on luffa and ins id e t h e beads of
s odium algin a te reduces the productivity and thes e
res ults may be due to one or more of the following
reas ons : the support may pos s es s es poor mech a n ical
s tability, as reported by Kloos ter and Lilly , [3 2 ].
Diffus ion limitation is a s e c o n d important factor where
s ubs trate limitation/product inhibition in entrapped
immobilization s ys tems may als o affect t h is proces s [5].
A third factor is the individual characteris tics of the
bacte riu m [3 1 ]. where Ivanova et al., 1998 s tated that
the antibiotic production of 12 s trains of epibiotic
bacteria was enhanced after immobilization.
W e can conclude that ma rine bacteria E. faecium
have the po t entiality to produce higly effective
bioactive compou n des which mus t be applied in
aquaculture or in the production of pharmacutical
agents .
ACKNOWLEDGEMENT
The authors would like to thank Dr. Nermeen A .
El-Sers y for her help in s tatis tical des ign.
J. Appl. Sci. Res., 5(10): 1445-1454, 2009
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