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Content may be subject to copyright.
Uapanese Journal ol llbter Tleatment Biolo&y Vo1.49 No.1 I l0 20131
Application of Lactobacillus plantarum
ATCC 8014 for Wastewater Treatment in
Fisheries lndustry Processing
MARINO S. MORIKAWA-SAKURA', MARTIN MIGUEL CASCO-ROBLES" MIJUNG KIM',
KAZUYA SH|Mlzu'?, YINGNAN YANG', NORIO SUGIURA', and ZHENYA ZHANGI*
rGraduate School of Life and Environmental Sciences, University of Tsukuba
/1-1-1, Tennodai, Tsukuba, 305-8572, Japan.
2Department of Life Science Toyo University/Ora-gun, Gunman 374-0193, Japan.
Abstract
In the present study, the bacterial inoculation and colonization of marine food
wastewater with inoc]u.lated Laclobacillus plantarum ATCC 8014 was analyzed. Firstly, an
inoculation standardization procedure of the bacteria in sterile wastewater, and examined
the effect of this inoculation testing for pII kinetics, acidity, soluble proteins and bacterial
grolrth were conducted. The results demonstrate that the inoculated strains have a
tendency to decrease pH and to increase acidity, whereas non-inoculated strains had a
tendency to keep or decrease acidity, Soluble proteins from the inoculated strains reached
a maximum peak before non-inoculated strains. Colonization growth from the non-
inoculated strains had a significant higher mortality compared to the inoculat€d bacteria,
The effect of the inoculated L. plantarum on the raw wastewater colonization, with
inoculum percentages of 17o and syo, both with and without molasses were investigatod,
The results suggested that for both pH and acidity, the most effective inoculums were rt
17o, whereas 57o inoculums proved to be effective in decreasing MPN of fecal coliforms
and in BOD removal.
Keywords: Lutoba.cillus plantarurn, marine wastewater treatment, MPN, BOD
INTRODUCTION
In the last few decades, in developed
countries a growing trend for the consumption
of marine products has been observed in
light of increasing cardiovascular diseases
and cancer associated with the consumption
of red meatt) and other problems, such as the
mad cow disease. On the other hand, strict
measures have been imposed to restrict the
import of suspicious sea products that pose
risks to consumers' health. One of the factors
accountable for the decrease in quality of
these products seems to be associated with
the deterioration of the coastal areas, which
are polluted by the industries in developing
countries 2).
Recently several fish processing plants
have a more diversified output, with more
commercial and added value, have been
established. However, these plants generate
Liquid Industrial Waste (LIW) with high
organic load, when discharged into coastal
waters significantly decreases the dissolved
oxygen, thus affecting and modifying the
biological communities3). Moreover, these
LIW, already carry a microbial load resulting
from the processing, which combined with
the high organic load, resu-Its in an extremely
high level of microbial pollutionli.
Lactobacilli plantarum are facultative
heterofermentative capable of developing and
growing at low temperatures and in presence
of oxygens). Some species of Lactobarillus
* Corresponding author
Phone:+81-29.853-4712 Fax:+81-29-853-4712
E-mail: tyou6688@sakura.cc.tsukuba.ac.jp (CC: zhatg.zhenya.fu@u.tsukuba.ac-jp)
Japanese J Wat. Treat. Biol. Vol.49 No.1
actively participate as part of the self-
purification microflora, for instance in
wetlands6'. In addition this microorganism
has a strong stability with respect to the
oxygen and its capacity to generate a series
of antimicrobial compounds that do not
compromise the bacteria as it continues to
grow even at pH as low as 4? e). L. plantarum
is ubiquitous in the environment, and it has
been found even in sewagess), which implies
an extraordinary adaptability with respect to
negative physical-chemical factors and a
response capacity that has not been assessed
suf6cientlyr0). Moreover, the safety of lactic
bacteria suggests that they can be used as
anti-microbial agentsrr'12).
The objective of this study was to examine
the BOD removal and decreasing percentage
of fecal coiiforms in defined L. plantarum
cultures for the treatment of wastewater
with high organic load. In addition, we
assessed inoculation of L. plantarum in
sterile wastewater, evaluation of growth
kinetics, and wastewater colonization.
MATERIALS AND METHODS
Culture conditions and bacterial strain L-
plantarum, ATCC 8014 was used and kept
routinely in vials with MRS agar (Vlerck,
Darmstadt, Germany) at 4t to 5T, for 24
hours, and then at 4t in 15% glycerol (voU
vol). In all cases, we started with a single
colony grown in plates with MRS agar at
30tl for 48 hours that was later sub-cultured
in MRS broth (Casein peptone 10 gll; Beef
extract 8 g/l; Yeast extract 4 g/l; D(+) glucose
20 g/l; Give-hidrogen potassium phosphate
KHzPOo 2 g/l; Tween 80 1g/l; Give-ammonium
citrato hidrogen CoHuO'(NH.)rH 2 g/l; Sodium
acetate CHsCOONa S Sll: Magnesium
su.lphate MgSOr.THzO 0.2 g/l; Manganese
sulphate MnSOo.HrO 0.04 gl I) at 30t for 24
hours.
Inoculation oI L. plantatun in sterile
wastewater Samples were taken from a
fishilg industrial company at Kanto Region
(Japan). The characteristics of the water
samples obtained contained primari.Iy marine
waste products.
MRS broth was mixed with wastewater in
test tubes, in triplicate, up to a 10 ml
volume, with the following proportions: tube
1 (9:1), tube 2 (8:2), tube 3 (6:4), tube 4 (2:8),
tube 5 (0:10) and tube 6 (0:10), and sterilized
at 121t for 15 minutes. Subsequently, 0.1
m/ (1oi,) from a bacterial suspension grown in
MRS broth is transferred to the tubes marked
as 1 and left to grow at 30"C for 24 hours;
then 0.1 ml is transferred from tube 1 to
tube 2 at 30tl for 24 hours, and so on until
reaching tube 6. The bacteria from the final
tube 6 are called inoculated. Non-inoculated
bacteria were obtained by direct transfer
from the MRS broth to tubes 1 through 6
and kept at 30b for 24 hours.
Growth comparison between inoculated and
non-inoculated cultur€s The growth was
measured before inoculation and non-
inoculation (STARD with MRS agar and
after inoculation and non-inoculation (END)
with TSB (Merck) + 2o/o agar Merck), the
exclusion method was used both at the start
and the end. The results were expressed as
mortality percentage.
Evaluation of pH kinetics and acidity In
glass jars with 400 ml of sterile wastewater,
inoculated bacteria and non-inoculated
bacteria were inoculated at lOo/o, in triplicate,
and incubated at 30t and room temperature
(18-2111, Iaboratory form F-41). The pH was
then measured'3) with a Hanna Instruments
8521 pH meter at different times (1, 2, 3, 5,
7,9, 7I, 13, 15, 17, 19, 21, 23 and 25 days)
as well as the total acidityl3) with 0.1 N
NaOH and 0.02 N NaOH using the
potentiometric method, expressed in % Iactic
acid. It was observed that the jars with non-
inoculated bacteria were caramel-colored as
MRS broth was added.
Evaluation of soluble protein kinetics In
the same jars used in the previous experiment,
soluble proteins were measured but at longer
intervals (0, 6, 12, 18,24 and 30 days).
Samples were taken in duplicate and
centrifuged at 10000 rpm for 5 minutes
(Eppendorf 5415c microcentrifuge), then I ml
of Bradford reagent (Sigma 86916) was
added, and read in the spectrophotometer at
595 nm (Spectrophemeter UV - 200s double
bean Shimadzu); then 0.2 ml of TCA at 10%
and 1 ml of Bradford reagent were added to
the other sample and read at 595 nm; in the
end, both quantities were subtracted.
Evaluation of growth kinetics In glass
:
:
,
Applicaliun of L plot4arum for Uaslor{ater Trpabrrcnt
jars, in triplicate, with 400 ml of sterile
wastewater inoculated with inoculated
bacteria at 10% and incubated at 30tl and
room temperature, the growth was measured
at di-fferent intervals (0, 1, 2, 3, 6,9, 12, 75,
18, 21 and 24 d.ays) using the TSB + 2% agar
exclusion method.
Wastewater colonization Inoculated .L.
plantarum at 1% and 5% with molasses and
without molasses, in duplicate, were
transferred to glass jars with 400 ml of non-
sterile wastewater, and kept at 25'C for 3
days. The pH, acidity, MPN of fecal coliforms
(MPN / 100m1) were then measured as per
APHA et aI., 1992 (13); BOD (mg O,/f as per
APHA et aI., 199213) using a Hanna HI9143
oxymeter. This was time 0. The pH, acidity,
fecal coli.forms I\{PN and BOD were measured
again at day 5, and then inoculated again at
lYo and, 5o/o. The same procedure was followed
on day 12, 19, 26 and 33.
RESULTS AND DISCUSSIONS
Evaluation of the inoculation oI Lactobacillas
plantarum in steril€ wastewater Growth
comparison between inoculated and non-
inoculated cultures For the count of viable
colonies the TSB agar I 2o/o agry was used
(optimum medium, non-reported data). A full
random design (DCA) was applied for the
statistical evaluation, where the experimental
unit is -L. plantorum and the treatments are
the inoculation and non-inoculation.
The respective ANVA table showed
significant differences at 5% between the
survival caused by inoculated cultures and
non-inoculated cultures (data not shown); the
mortality of inoculated cultures was 43.60%,
compared to 57 .59% mottality of non'
inoculated cultures. We therefore conclude
that the inoculation is a method that causes
a significant mortality degree compared to
the non-inoculation. Conducting the inoc-
ulation can therefore be justifed although it
takes more time than the non-inoculated as
the loss of bacteria is sensibly lower. It
would be convenient, since the basic principie
of the wastewater treatment is to convert the
majority of dissolved substances into a mass
of organisms so that they can be extracted as
settieable matte as suggested by Lavee,
2010'. It is possible that enzymes in the
inocuiated cultures are being induced,
whereas those in the non-inoculated are not
or are i.nduced slowly. Moreover, several
characteristics of the Lactobacillus are caused
by physiological induction, such as the
acetate generation under aerobic conditions"'
or the induction of amino acid-degrading
enzymestu'.
Evaluation of the pH kinetics, acidity and
soluble proteins of inoculated and non-
inoculated Lactobacillus plantarum in sterile
wastewater at two different temperatures
The pH kinetics is shown in Figure 1. The
pH decreased in inoculated cultures and
increased in non-inoculated cultures irre-
spective of the incubation temperature. This
behavior with regards to the pH downward
and upward trend depending on how the
inoculums was obtained (inoculated or non-
inoculated) suggest that in inoculated
cultures there are induced physiological
changes that in non-inoculated cultures do
Fig. 1 pH kinetic curve of inoculated L. plantarum and non-inoculated L. plantarum at two temperatures:
30t incubation lemperature and room temperature. Closed square shows non-inoculated at 30t,
open square shows inoculated at 30C, closed triangle shows non-inoculated at Room Temperature,
and op€n triangle shows inoculated at Room Temperature.
Japanese J Wat. Treat. Biol. Vol.49 No.1
not occur (Fig. 1).
The induction of gene expression may
include: response to osmotic stress by the
synthesis of compatible solute transporters
induced by a change of the medium osmolarity
to keep the high turgor pressule characteristic
of Lactobacillus and positive gram in
generalt: response to energy generation in
shortage of sugar as a proton-motive
metabolic cycle, where ATP is created from
the aminoacid decarboxylation in a single
metabolic sequence in Lactobacillus and aci.d
lactic bacteria in general, has been proposedr'':
response to the carbonate source for growth,
as the existence of proteinases associated
with the cellular wall and intracellular
peptidases that would use milk proteins and
help sausage ripening wrlh L. plantarum, L.
sahe and. L. curuatust" '"'. Hence, the trend to
decrease the pH in inoculated cultures would
be an indirect way to measure the gene
expression, as the mai.n fermentative flow of
these bacteria is oriented to acid generation,
such as lactic and acetic acid (in the aerobic
metabolism), i,e. a bacteria that is using
wastewater for growth should produce acids
and therefore reduce the pH, as confumed in
Figure 2, for acidity data, as inoculated
cultures that reduce pH are those that
increase acidity (Fig. 2).
We observed that non-inoculated cultures
increased pH and maintained acidity with no
increase or with a 1ow one, and not having
induced specific proteins to help them grow
in wastewater led them to have a greater
mortality when compared to the inoculated
cultures.
Next we measured the values for soluble
protei.ns, shown in Figure 3. The dye selected
for the method used in this research is
Coomassie Brilliant Blue and it bonds to the
soluble proteins as described by Bradford,
1976'!". Indirect growth of bacteria can be
monitored based on their proteins (proteinases
bonded to their ce1l wall), which causes the
breaking of wastewater proteins and the
Fig. 2 Acidity kinetics curve of inoculated L. plantarum and non-inoculated L. plantarum al lv,to temperatures:
30t incubation temperature and room temperature. Closed squares show nonjnoculated at 3O'C,
open squares show inoculated at 30'C, closed triangles show non-inoculated at room temperature,
open triangles show inoculated at room temperature.
Fig. 3 Soluble proteins kinetic curve of inoculated L. plantarum and non-inoculated L. plantarum at lvto
incubation temperature, 30oC and room temperature, respectively. Closed squares show non-
inoculated at 30t, open squares show inoculated al 30C, closed triangles show non-inoculated at
room temperature, open triangles show inoculaled at room temperature.
Applicatior of L. pknttdrunt for Wastewater Tleatnrent
)
[
bacterial proteins synthesis from hydrolyzed
proteins (turnover). A description of soluble
proteins kinetics is carried out because of the
bacterial activity. Figure 3 shows that in the
starting values (day 1) there is an important
difference between the inocuiated and the
non-inoculated cultures, and this difference
is repeated in the corresponding control
points (non-reported data), which could be
due to the culture medium that transfers at
the time of inoculation of non-inoculated
ones, which could contain non-degraded
peptones and other proteins. Furthermore,
bacteria already grown in the MRS broth
that, compared to wastewater with a lower
number of inoculated bacteria, have generated
a difference in the amount of soluble proteins
detected. This turnover of soluble proteins is
consistent with Gulahmadov et al., 2009'3)
(Fie.3).
L. plantarum inoculated culture incubated
at 30tl reached the maximum peak before
the non-inoculated culture, and at room
temperature both reached this maximum
turnover at the same time. which is
corroborated by the data obtai.ned from the
comparison of the growth produced by
inoculated and non-inoculated cultures with
pH kinetics and acidity (see Figures 1, and
2), since it is likely that inoculated cultures
are capable of using wastewater nutrients
faster than the non-inoculated ones and,
therefore, reach the maximum protein
turnover first; however, they also fell faster
since the experiment was carried out as a
closed culture.
Based on the results obtained for the
1o'
tot
rou
inoculation standardization. it was decided to
work with inoculated cultures according to
the results previously described.
Evaluation of the growth kinetics of
Lactobacillus plantatum inoculated to two
different temperatures Figure 4 shows the
growth curve of L- plantarum inoculated and
incubated at 30'C and at room temperature,
where the specific growth rate F is expressed
in days' (d I) was evaluated. It was observed
that the highest p was reached on the second
day of growth, which agrees with the values
obtained in Figure 4, where the curve reaches
its peak on the third day and then it
remained the same. When comparing the u
values in L. plantarum at both test
temperatures, it can be seen that they are
similar on the second day of incubation: 0.24
d-t at 30"C while 0.18 d ' at room temperature.
It should be noted that g is a value that
mainly depends on composition and
concentration of the culture medium.
inhibitors, temperature and pH'?or. In this
sense, sterilized wastewater contains
disinfectant residues (A.T.V., 1993"' and
communication with HACCP employees at a
canning plant), and a high pH (non-reported
control point values), which makes the
growth of these microorganisms difficult in
wastewater. On the other hand, a good
growth has been obtained, which is favorable
during wastewater colonization stage. It is
interesting to compare these results in their
ideal MRS medium, since in this experiment
on the second day they reach their maximum
growthr') instead of on the third day, and this
may be due to the acidity and pH above
012 3 6 912 15 18 ?1 74
Iim. (daF)
Fig. 4 Growth kinetic curve ol L. plantarum inoculated at two incubation
temperatures. Closed squares show at 30'C and open squares show
at room temperalure.
」apanese J VVat Treat Blol vOi49 No l
mentioned (Fig. 4).
Evaluation of wastewater colonization with
inoculated L plantarum Det€rmination of the
ideal inoculum age Based on the results
obtained for the growth kinetic in Figure 4,
colonization with L. plantarzrn was carri.ed
out and it was incubated in sterile wastewater
at room temperature for 3 days. Then.
colonization was carried out in non-sterilized.
wastewater.
Determination of the ideal inoculum and the
colonization time in wastewater The
experiment was carried out in non-sterilized
wastewater. Molasses was added in the
colonization stage to give some advantage to
L. plantarum, since it is aciduric, aside from
the fact that bacteria had already been
inoculated. Table 1 shows the results of pH
and acidity (Table 1).
Treatments that included molasses resulted
in greater decrease in pH and a greater
increase in acidity, and this is due to the fact
that molasses contain up to 40% of sucrose, a
disaccharide composed of glucose and
fructose. Both through the EMp or
phosphogluconate pathway produce lactic
acid and acetic acid in L. plantarum and.
other bacteria, causing a greater total acidity
and a lower pH. In the treatments with
molasses, it can be seen that compared to
their control point, if values in Table 1 are
followed, with 1% inoculum a better response
(in pH and acidity) is obtained in the same
time that with 5olo inoculum and the control
point. This may be because a 1% inoculum
does not inhibit completely the native
bacteria in wastewater, and these, by having
a p (specific growth rate) greater than tr.
plantarum, do not generate a greater aciditv
bul a more accelerated one, which results in
an apparent superiority of the control point
both in acidity and pH compared to the b%
inoculum. Thus, the obtained final values
(day 33) for 1% inoculum were 3,66% of lactic
acid in pH and 1,38% in acidity, which can
be compared to the values obtained in silages
pH < 4,,, and in lactic acid ferrnentation in
gherkins between 0,9% and 1% of lactic acid
and a pH of 3,6'!3). If the acidity and pH of
the 5% inoculum were left for a longer period
of time, it is likely that they will be higher
than those of the 1% inoculums (Table 2).
Tabie 2 shows the data for fecal coliforms
over time. Due to the closed culture. fecal
Table I Variation of pH and acidity during colonizalion in non_sterilized wastewater
With molasses Without molasses
Acidity
Я
鶏賜hOCmum 5% Inoculum Control point 1% Inoculum b% Inoculum Oontrol
0
12
19
26
33
374
373
392
404
398
004
006
051
08
086
112
121
012
02
023
033
041
546
53
53
528
525
517
497
489
494
492
381 062
368 092
366 097
38 121
365 138
063 375
077 865
084 379
101 354
12 378
012 529
01 553 008
011 528 008
017 542 007
023 525 008
pH Acidity pH
0 728 004 728 004 728 0o4 pH Al・idity
7.28 0.04 7.28 0.04 7 .28
Table 2 Variation ot l\,lPN and BOD during colonization in non_sterilized wastewater
With molasses Without molasses
Я
tt h∝dum当
MPN BOD Inoculum 5% Control point Inoculum 1% Inoculum 5% Control point
MPN BOD MPN BoD BOD MPN BOD
MPN MPN BOD
0
5
12
19
26
33
2100
1374
2× 10° 2100 2100 2X10°
4x10 | 1398 20 2x103 1608 5x10{
1.4x10r 546 20 438
2× 10° 2X10° 2100 2x105 2100
1572 1.6x101 1584
531 1.1x102 645
2xtos 2100
1.3x101 1740
4x103 1536
20
く
20
く
20
159
90
75
く
20
く
20
く
20
2X102
20
く
20
く
20
く
20
く
20
186
184
185
20
く
20
く
20
80
20
く
20
874
534
442
555 17× 102
320 20
:
Application of L plorLtotum for Wasterrater fYeatment
coliforms naturally decreased in the control
points. However, the speed of this process
should be observed. In fact, inoculation
treatments both at 1% and 5% decrease
faster than the control points with fecal
coliforms. However, one exception is the
control point with molasses, where the
decrease in fecal coliforms is also considerable.
This may be due to the fact that water has
not been sterilized and contains a great
number of initial bacteria, that when growing
are inhibited by their own metabolites
derived from the added rnolasses, in addition
to having less substrate avaiiable as time
goes by'o "i. Among the treatments without
molasses, the decrease response in fecal
coMorms is better than in the 5% inoculum.
This could be due to the fact that since they
are found in a greater number, they inhibit
wastewater flora more effectively than the
1% inoculum, which is found in a lesser
amount. These results agree with the values
obtained in pH and acidity, where the 5%
inoculum acidifies less and has a higher pH
because it is found in greater amount and
inhibits the surrounding flora that acidifies
faster. Among the treatment with molasses,
the superiority of 5% inoculum is more
significant and total hhibition is obtained on
the fifth day. This may be possible when
using molasses together with the higher
inoculum. Both factors are fatal for coliform
bacteria-
It is important to note, that if MPN and
acidity results are examined together, it can
be observed that there is an inverse
relationship as shown in Figure 5. This graph
shows a key point to be discussed; the 7o/o
inoculum was more elfective in acidity while
the 5% inoculum was more effective in fecal
coliforms. Thus, it can be inferred that
acidity was not the only direct responsible
for the death of coliforms, but that there are
other factors involved: These factors can be
explained by the food biopreservation theory
with Z. plantarum, which argues that tr.
plantarum has an antagonist mechanism
that provides a greater competitiveness
compared to the remaining microflora. The
main microbial antagonist mechanisms
compete for nutrients with the substrate and
the organic acids formation with the
subsequent decrease in pH'5). Other anti-
microbial substances produced are ethanol,
CO,, diacetyl, acetaldehyde, hydrogen
peroxide, and other oxygen metabolites and
bacteriocins'o '?6r. The foregoing may have
been mechanisms used by L. plantarum to
kill and displace fecal coliforms. In any case,
acidity is a useful parameter that partially
explains the decrease of fecal coliforms in a
Iesser time than the control point (Fig.5).
The data of BOD behavior in Table 2
shows that the 5% inoculum was the most
effective, which agrees with the results
obtained for fecal coliforms, since at this
concentration fecal coliforms were inhibited
with higher intensity, and this bacterium
Fig. 5 Relationship between acidity and MPN during colonization in non-sterilized wastewater by L. p/antarum
with molasses ad without molasses. Closed squares show acidity 1olo without molasses, open squares
show acidity l% with molasses, closed triangles show acidity control point without molasses, open
triangles show acidity control point with molasses, closed circles show MPN 1% without molasses,
open circles show MPN 1% with molasses, closed inverled triangles show MPN control point without
molasses, open inverted triangles show MPN control point with molasses, closed diamonds show
acidity 5% without molasses, open diamonds show acidity syo with molasses, closed double circles
show MPN 5% without molasses, open double circles show MPN 5% with molasses.
Japanese J. Wat. Treat. 8iol. Vol.49 No.1
could have used this condition to degrade
wastewater nutrients since it was inoculated,
and therefore, had induced many enzymes
that it would use to compete and degrade
advantageously wastewater proteins.
Likewise, this bacterium grows with a low
pH, which means that, once the pH has
decreased and with a high percentage of
acidity, it continues degrading organic
matter. This can be confirmed with the fecal
coliforms, since between days 12 and 19
there is a strong decrease in BOD curves,
which agrees with the inhibition of fecal
coliforms in treatments with 5% molasses,
5% without molasses and lo/o without
molasses. Our data suggests there is a
protection effect of pH and acidity in tr.
plantarum. that allows it to degrade organic
matter even after other microorganisms have
stopped metabolizing. Likewise, molasses
have no impact on the effectiveness of BOD
reduction.
The efficiency of the different treatments
with BOD are: The treatment with greater
BOD removal was the 5% inoculum without
molasses. where a 97.62% removal was
obtained; then, the 5% inoculum with
molasses with a 96.67% removal; and finally,
the L% inoculum with molasses with a
96.43% removal. These parameters show a
relative efficiency of the method used, since
it cannot be compared to other existing
methods.
In this case the biological method using
Lactobatillus. for wastewater treatment
would be recommended to use starting in the
tertiary treatment, or to a process of self-
purification. coNcLUsroNs
The present study examined (1) the
efficiency of different treatments with BOD.
The effective BOD removal was the 5%
inoculum without nolasses. which had
97 .62o/o removal; the 5% inoculum with
molasses had a 96.67% removal; and finally,
the 1% inoculum with molasses had a 96.43%
removal. These parameters show a relative
efficient method, since it cannot be compared
to other existing methods. (2) An inverse
relationship between MPN with 5% molasses
and acidity, which is consistent with the food
bio preservation theory with L. plantarum,
was observed. Then a practical use of .L.
plantarum (ATCC 8014 a GRAS speci.e) for
water treatment was provided. (3) Since the
value of l% and 5% inoculum with molasses
had similar rernoval percentage, as a cost-
effective method, 1% inoculum could be
suggested.
ACKNOWLEDGMENTS
This study was supported by Doctoral
Program in International Bioindustriai
Sciences-Graduate School of Life and
Environmental Sciences, University of
Tsukuba, The Ministry of Education, Culture,
Sports, Science and Technology (MEXT)
Japan and Forest &River Consultants in
Peru.
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