Studies on bioflocculant production by Methylobacterium sp. Obi isolated from a freshwater environment in South Africa

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African Journal of Microbiology Research Vol. 5(26), pp. 4533-4540, 16 November, 2011
Available online at http://www.academicjournals.org/AJMR
ISSN 1996-0808 ©2011 Academic Journals
DOI: 10.5897/AJMR11.376
Full Length Research Paper
Studies on bioflocculant production by
Methylobacterium sp. Obi isolated from a freshwater
environment in South Africa
Luvuyo Ntsaluba, Oladele Agundiade, Leonard Mabinya1* and Anthony Okoh
Applied and Environmental Microbiology Research Group (AEMREG), Department of Biochemistry and Microbiology,
University of Fort Hare, Private Bag X1314, Alice 5700, South Africa.
Accepted 15 June, 2011
A bioflocculant-producing bacterium isolated from river water was identified as Methylobacterium sp.
Obi by 16S ribosomal ribonucleic acid (rRNA) gene sequencing. The nucleotide sequence was
deposited in the Genbank with accession number HQ537130. The bioflocculant secreted by the isolate
showed a peak flocculating activity of 72% in 1 g/L kaolin suspension supplemented with CaCl2. The
bioflocculant yield was maximal when glucose and peptone were used as carbon and nitrogen sources
respectively, at an optimum pH of 7. Calcium was the most effective cation in stimulating
bioflocculating activity. Chemical analyses of the purified bioflocculant revealed it to be a
polysaccharide.
Key words: Freshwater, Methylobacterium sp. Obi, bioflocculant, flocculating activity, polysaccharides.
INTRODUCTION
Bioflocculants are compounds produced by
microorganisms, which promote flocculation by causing
colloids and other suspended particles in liquid to aggre-
gate or form a floc. They are nontoxic, biodegradable and
harmless to both humans and the environment (ABD-EL-
Haleem et al., 2008). Louis Pasteur was the first person
to discover the presence of flocculants in a micro-
organism system (Gong et al., 2008). Further studies
established the existence of a correlation between the
accumulation of extracellular bioflocculants and cell
aggregation (Jie et al., 2006).
The use of bioflocculants is encouraged due to the fact
that most chemical flocculants that are in use showed
side effects to human health and also contribute to
environmental pollution. For instance, although
polyacrylamide is a frequently used chemical flocculant,
*Corresponding author. E-mail: LMabinya@ufh.ac.za. Tel: +27
40 602 2444. Fax: +27 86 628 2554.
Abbreviations: EBFs, Extracellular biopolymeric flocculants;
PCR, polymerase chain reaction; rRNA, ribosomal ribonucleic
acid.
there is evidence that the acrylamide monomer is not
only neurotoxic but also non-degradable (Zheng et al.,
2008).
Bioflocculants have a lot of characteristics that make
them a perfect substitute for chemical flocculants, such
as safety, biodegradability and the absence of secondary
pollution (Li et al., 2008), and as such, can be safely
applied in drinking water, wastewater purification and
downstream processes in fermentation industries.
Recently, bioflocculants have attracted research interest;
hence more focus is being directed to the application of
microbial bioflocculants in various fields (Gong et al.,
2008).
Even though bioflocculants are potentially suitable for
application in various industries, there is a need to
improve productivity by screening for new micro-
organisms which can produce flocculants with high
flocculating activity (Feng and Xu, 2008). Most of these
bioflocculants are mainly composed of polysaccharides,
proteins, nucleic acid and some other macromolecular
compounds and are mainly used in wastewater to
remove the dyes, inorganic solid suspensions such as
bentonite, activated carbon, kaolin, calcium hydroxide,
aluminium oxide, humic acid and other suspensions

4534 Afr. J. Microbiol. Res.
(Deng et al., 2005).
Over the past decades, a number of microorganisms,
including algae, bacteria, actinomycetes and fungi, have
been reported to produce bioflocculants (Takagi and
Kadowaki, 1985; Zhang et al., 1999; Huang et al., 2005),
but majority of microbial bioflocculants have come from
Bacillus species (Salehizadeh and Shojaosadati, 2001;
Deng et al., 2003; Kwon et al., 1996; Suh et al., 1997).
He et al. (2004) investigated the production of a
polygalacturonic acid bioflocculant REA-11 from a newly
isolated Corynebacterium glutamicum CCTCC M201005
strain while Kurane et al. (1986) also reported that
Nocardia restricta, Nocardia calcarea and Nocardia
rhodnii could produce biopolymer flocculants. In this
paper, we assess the bioflocculant production potential of
Methylobacterium sp. Obi, a freshwater bacterium
isolated from the Tyume River, in South Africa.
MATERIALS AND METHODS
Source of bacteria and culture media
Over 200 freshwater bacteria isolated from the Tyume River were
screened for bioflocculant production using a cultivation medium
described by Zhang et al. (2008), and composed of the following:
10 g glucose, 1 g peptone, 0.3 g MgSO4.7H2O, 5 g K2HPO4, 2 g
KH2PO4 in a litre of distilled water. The initial pH was adjusted to 7.0
with either NaOH (0.1 M) or HCl (0.1 M).
Screening for flocculant producing microorganism
Each bacterial isolate was inoculated into 5 ml of sterile growth
medium contained in a McCartney bottle and incubated at 25°C
with shaking (120 rpm) for 7 days. The fermentation broth was
centrifuged at 4000 × g for 30 min at 4°C to sediment the cells. The
cell free culture supernatant was used assayed for flocculating
activity.
Determination of flocculating activity
Using a suspension of kaolin clay as test material, flocculating
activity was measured according to the method described by
Kurane et al. (1994) and Zhang et al. (2008). Three millilitres of 1%
(w/v) CaCl2 and 2.0 ml of bioflocculant were added into 100 ml of
kaolin suspension (4.0 g/l) in a 250 ml conical flask, the mixture was
vigorously stirred, poured into 100 ml of measuring cylinder and
allowed to stand for 5 minutes at room temperature. The optical
density (OD) of the clarifying solution was measured with a
spectrophotometer at 550 nm. A control experiment was prepared
in the same way but the bioflocculant was replaced with distilled
water. The flocculating rate (FR) was determined by using the
formula:
FR = {(A – B)/A}* 100
Where, A and B are optical densities of the control and samples
respectively at 550 nm.
Factors affecting bioflocculant production and flocculating
activity
The assessment of the effects of different carbon and nitrogen
sources on bioflocculant production by the test bacterium was done
according to the method described by Lachhwani (2005). Carbon
source candidates included glucose, sucrose, fructose, lactose and
starch, while the nitrogen source candidates were peptone,
ammonium sulphate, ammonium chloride (inorganic nitrogen
sources) and urea (organic nitrogen sources). The effect of salt ions
on flocculating activity was investigated by using the following
electrolyte solutions as sources of cations: calcium chloride, ferric
chloride, magnesium chloride, ferrous sulphate, potassium chloride
and the flocculating activity measured as previously described. The
effect of pH on flocculating activity was assessed by adjusting the
pH of kaolin suspension using HCl or NaOH prior to measuring
flocculating activity. The pH values ranged between 3, 4, 5, 6, 7, 8
and 9.
Time course assay for bioflocculant production
The composition of the medium for bioflocculant production was
prepared according to the method described by Zhang et al. (2007)
by dissolving the following in a liter of deionized water: 10 g of
glucose, 1.0 g of peptone, 0.3 g of MgSO4 .7 H2O, 5 g of K2HPO4
and 2 g of KH2PO4. The pH was adjusted to 6.5 with either NaOH or
HCl. The selected strain was pre-cultured in 50 ml medium
contained in 250 ml flasks on the rotary shaker (120 rpm) at 25oC
for inoculation preparation. After 16 h of cultivation, 1% (v/v) of
culture broth was used as seed culture to inoculate 400 ml of
medium in 1000 ml flasks. Batch fermentation was carried out
under the same cultivation conditions as those for pre-cultivation.
Medium samples were withdrawn at appropriate time intervals and
monitored for pH, cell growth and flocculating activity. Five milliliters
of culture broth was centrifuged at 8000 x g for 15 min, and the cell
free supernatant was used as the test bioflocculant to determine the
flocculating activity.
Chemical analysis of the bioflocculant
Total carbohydrate content was determined by the Phenol
Sulphuric acid method with glucose used as a standard solution
(Chaplin and Kennedy, 1994). The protein content was measured
with Folin-Lowry method as described by Lachhwani (2005) with
bovine serum albumin (BSA) as the standard.
Identification of the bioflocculant-producing bacterium
The bacterium was identified by 16S rRNA gene sequencing. For
this purpose, DNA was first extracted using boiling method whereby
2-3 pure colonies of the bacterium were suspended in 70 µl of
sterile double distilled water in a 1.5 ml eppendorf and boiled in a
heating block at 100ºC for 10 min, allowed to cool for 5 min and
thereafter centrifuged at 3000 rpm for 5 min to pellet cell debris.
The supernatant was transferred to a clean tube and stored at 4ºC.
This serves as the template in the PCR assay.
PCR was carried out in 50 µl reaction volume containing 2 mM
MgCl2, 2 U Supertherm Taq polymerase, 150 mM of each dNTP,
0.5 mM of each primer (F1: 59-AGAGTTTGATCITGGCTCAG-39; I
= inosine and primer R5: 59-ACGGITACCTTGTTACGACTT-39)
and 2 ml template DNA. Primer F1 and R5 binds to base positions
7-26 and 1496-1476 of the 16S rRNA gene of Streptomyces
ambofaciens ATCC 23877, respectively (Cook and Meyers, 2003).
The primers in this study were used to amplify nearly full-length 16S
rDNA sequences. The PCR programme used was an initial
denaturation (96ºC for 2 min), 30 cycles of denaturation (96ºC for
45 s), annealing (56ºC for 30 s) and extension (72ºC for 2 min), and
a final extension (72°C for 5 min). Gel electrophoresis of PCR
products were conducted on 1% agarose gels to confirm that a

Ntsaluba et al. 4535
Figure 1. PCR product agarose gel (1%) electrophoresis. Lane 1:
DNA markers and Lane 2: PCR product.
fragment of the correct size had been amplified.
Statistical analysis
The results obtained on factors affecting bioflocculant production,
flocculating activity and time course for bioflocculant production
were analyzed with Statistical Package for Social Scientists (SPSS
version 17). A significance level of p≤0.05 was used. The mean
values obtained were subsequently used to chart graphs using
Microsoft Office Excel 2007.
RESULTS AND DISCUSSION
Screening and identification of bioflocculant-
producing strain
Over 200 freshwater bacteria isolated from the Tyume
River in the Eastern Cape Province of South Africa were
screened for bioflocculant-producing capabilities. Among
these was our tested bacterium which showed a
flocculating activity of 72%. PCR amplification of the 16S
rRNA gene of the bacterium produced a PCR product of
approximately 1.5 kb size (Figure 1). BLAST (Basic Local
Alignment Search Tool) analyses of the nucleotide
sequence of the amplified product showed a 98%
similarity to Methylobacterium sp. 440 and the sequence
was submitted in GenBank as Methylobacterium sp. Obi
with accession number of HQ537130. The morphological
characteristics of the bacterium revealed the colony to be
reddish in colour, round, flat, smooth, and glistening with
a colony size of approximately 2 mm in diameter.
Methylobacterium genus is strictly aerobic, Gram-
negative and rod-shaped facultative methylotrophs, which
use methanol and other reduced mono-carbon
compounds via the serine pathway (Green, 1992).
Methylobacteria are classified as α-Proteobacteria and
appear as pink to red in colour due to the synthesis of
carotenoids (Holland and Palacco, 1994). These rod-
shaped facultative methylotrophs are widely distributed in
nature but are mostly well known for being associated
with plants (Araujo et al., 2002; Corpe, 1985; Holland and
Palacco, 1994; Lidstrom and Christoserdova, 2002;
Lodexyckx et al., 2002a). Methylobacterium strains have
been shown to colonize the intercellular spaces and/or
vascular tissues of plants as endophytes (Idris et al.,
2004; Araujo et al., 2002; Lodexyckx et al., 2002b) and
have also been found as endosymbionts within the cells
of the buds of Scotch pine (Pirttila et al., 2000).
Bacteria of the genus Methylobacterium have been
reported to be non-pathogenic to their plant hosts
(Rughia et al., 2006). The host plants provide methanol
or methylated pectin as nutrients for their host bacteria,
while the bacteria can benefit the plants in different ways;
such as stimulating seed germination and plant develop-
ment, probably due to hormone or vitamin production
(Basile et al., 1985; Hiraish et al., 1995). In addition,
some strains showed 1-aminocyclopropane-1-
carboxylate (ACC) deaminase activity potential resulting
in improved stress tolerance by plants (Idris et al., 2004).
Methylobacteria can also contribute to a better iron
nutrition of plants by producing the siderophores (Bar-
Ness et al., 1992; Idris et al., 2004). In addition, evidence
exists that Methylobacteria contribute to the flavour
development of strawberries (Zabetakis, 1997).
Time course of bioflocculant production
The correlation between cell growth, pH and flocculating
activity was investigated over a growth period of 10 days.
The flocculating activity of the bioflocculant increased
with cell growth and an optimum activity of more than

4536 Afr. J. Microbiol. Res.
Figure 2. Time course assay of Methylobacterium sp. Obi showing correlation between cell growth
(OD660nm), pH and flocculating activity.
60% was obtained after 6 days of cultivation (Figure 2).
Similar results were reported for Bacillus sp. by Feng and
Xu (2008) and Xiong et al. (2010). A decrease in
flocculating activity with a corresponding decrease in pH
levels from 6.8 to 6.5 was observed in the first 6 days of
growth (Figure 2). The decrease in pH is thought to be
due to the production of organic acids either from glucose
metabolism or the presence of organic acid components
in the bioflocculant polymer being produced (Dermilim et
al., 1999). An increase in flocculating activity was
observed after a 5 day cultivation period and reached a
maximum of 62% at an optimum pH of 6.6 at 7 days of
cultivation (Figure 2). After seven days of cultivation, the
pH stabilized at 6.7 with a corresponding sharp decrease
in flocculating activity (Figure 2). The initial pH of the
culture medium determines the electric charge of the
cells and the oxidation-reduction potential which can
affect nutrient absorption and enzymatic reaction (Nakata
and Kurane, 1999; Salehizadeh and Shojaosadati, 2001).
According to Tago and Aida (1977), this observed sharp
decrease in activity may be attributed to the action of a
bioflocculant-degrading enzyme being produced by the
microorganism.
Several studies add been carried out on different
bacterial strains, some of which corroborate the findings
of this present investigation while some do not. Studies
have carried out to investigate the effect of pH on
flocculating activity of extracellular biopolymeric
flocculants (EBFs). The flocculating activity of Bacillus sp.
PY-90 is high in acidic pH (pH 3 -5) (Yokoi et al., 1995),
whereas the maximum activity of the flocculant produced
by Enterobacter sp. BY-29 is observed at pH 3 and the
activity decreases with increasing pH (Yokoi et al., 1996).
Shimofuruya et al. (1996) reported a flocculant produced
by Streptomyces griseus that is active in acidic conditions
ranging from pH 2 to 6 and with the maximum activity
observed at pH 4. Rhodococcus erythropolis produced an
EBF that is active at neutral pH (Kurane et al., 1994),
whilst the flocculant produced by Paceilomyces sp. show
maximum activity at the pH range of 4.0 - 7.5 (Takagi and
Kadowaki, 1985). The flocculating activity of Aspergillus
sojae increased when the pH exceeded 7 (Nakamura et
al., 1976b) and the maximum flocculating activity of
Aspergillus sp. JS-42 is reported in the pH range of 3 - 8
(Nam et al., 1996).
Effects of carbon and nitrogen sources on
bioflocculant production
Carbon and nitrogen sources have been reported to have
an effective role in enhancing the production of
bioflocculant by microorganisms (Nakamura, 1976d). The
composition of the production medium was supplemented
with different carbon and nitrogen sources to optimize
bioflocculant production. The effects of glucose, lactose,
sucrose, fructose and starch were investigated. From
results obtained, it was evident that glucose supported
the highest bioflocculant production with an optimum
flocculating activity of 72% at p≤0.05 compared to that of
lactose (42%), sucrose (31%) and starch (11%). Fructose
was the least preferred carbon source by

Ntsaluba et al. 4537
Figure 3. Effect of carbon source on bioflocculant production.
Figure 4. Effect of nitrogen source on bioflocculant production.
Methylobacterium sp. Obi and resulted in a low
flocculating activity of less than 10% (Figure 3). Overall,
the results reveal that glucose was the best carbon
source for bioflocculant production by Methylobacterim
sp. Obi, and the flocculating activity (in a kaolin suspen-
sion) was 72% after 7 days. Glucose has been reported
as a preferred carbon source in previous studies on
bioflocculant production by various microorganisms. Patil
et al. (2009) reported that the bioflocculant produced by
Bacillus subtilis is enhanced by glucose and sucrose as
carbon sources. In the case of Rhodococcus erythropolis,
glucose and fructose enhance elongation of the cells and
the production of the bioflocculant (Kurane et al., 1991).
The production medium was further optimized for
production of bioflocculant by supplementing it with
different nitrogen sources. The effects of the following
nitrogen sources on bioflocculant production were
investigated: peptone, ammonium chloride, ammonium
sulphate and urea. Although, they are all of natural
nitrogen sources, only peptone and ammonium chloride
showed significant support for bioflocculant production.
Peptone resulted in a flocculating activity of 72% which
was marginally better than 66 and 21% (p≤0.05) obtained
with ammonium chloride and ammonium sulphate res-
pectively (Figure 4). According Nakamura et al. (1976b)
in the presence of certain inorganic nitrogen compounds
(for example, ammonium chloride, ammonium nitrate,
and ammonium sulfate), the mycelial growth is poor and

4538 Afr. J. Microbiol. Res.
Figure 5. Effect of cations on flocculating activity.
and no flocculating activity could be detected.
Urea, with a flocculating activity of 8% was the least
preferred nitrogen source (Figure 4). Therefore peptone
remains the only suitable source of nitrogen for biofloc-
culant production by Methylobacterium sp. Obi. On the
contrary, Nakumura et al. (1976b) reported that the
production of the bioflocculant by Aspergillus sojae is
enhanced when casein, polypeptone and glutamic acid
were added to the medium.
Effect of cations on flocculating activity
Cations can neutralize negative charges of both
polysaccharide and suspended particles and increase the
adsorption of polysaccharide onto suspended particles
(Wu and Ye, 2007). These cations stimulate flocculation
by destabilizing the negative charge of the kaolin particle
(Gong et al., 2008). Divalent cations (Ca2+, Fe2+) were
found to be more effective in stimulating flocculating
activity than monovalent (K+) and trivalent (Fe3+) cations
(Figure 5). Flocculating activities of 72 and 63% (p≤0.05)
were obtained with Ca2+ and Fe2+, respectively, whilst an
insignificant activity of 4% was obtained with Mg2+, Fe3+
and K+ showed no stimulation of flocculating activity
(Figure 5).
As reported in previous studies, the Ca2+ ion enhances
both the cell growth and the flocculating activity of
Paecilomyces sp. (Takagi and Kadowaki, 1985). Good
floc growth has been observed for Kluyvera cryocrescens
in a medium containing a low concentration of Ca2+, but
flocs was not formed in the absence of Ca2+ (Kakii et al.,
1990). These results reveal that Ca2+ and the cell surface
are involved in bioflocculation (Endo et al., 1976). Salt
ions, such as, Ca2+, Co2+, Sr2+, Mg2+, Mn2+, and Al3+
promote flocculation of Kluyveromyces marxianus cells,
but not as efficiently as Fe2+ and Sn2+ (Sousa et al.,
1992).
Effect of pH on flocculating activity
The pH is said to have a major influence on flocculating
activity (Yokoi et al., 1996). In the case of
Corynobacterium xerosis, the flocculant is produced at
relatively low pH (Esser and Kues, 1983). The mycelial
growth of Aspergillus sojae is enhanced when the pH of
the culture is controlled at 6 and no flocculating activity is
observed in the cultures grown at pH 8 (Nakamura et al.,
1976d). The flocculant production by Rhodococcus
erythropolis is higher at alkaline pH values of 8.0 - 9.5
than at other pH values (Kurane and Matsuyama, 1994).
The effect of pH on flocculating activity of the
bioflocculant produced by Methylobacterium sp. Obi was
measured at pH values ranging from 3, 4, 5, 6, 7, 8, and
9, with the maximum activity peak of 72% obtained at an
optimum pH of 7 after which a gradual decrease in
activity was observed (Figure 6). Li et al. (2008) reported
that at low pH, both bioflocculant and kaolin particles are
likely to absorb hydrogen ions (H+), which weakened the
forming of complexes between bioflocculant molecules
and kaolin particles mediated by Ca2+. Similarly,
hydroxide ions (OH-) interfered with the combination of
the flocculant molecules and kaolin particles at high pH
values, resulting in lower flocculating activity. The
mediating effect of Ca2+ appeared to be the strongest at

Ntsaluba et al. 4539
Figure 6. Effect of initial pH on bioflocculantion suspension by Methylobacterium sp. Obi.
neutral pH values (Li et al., 2008).
Composition analysis of the bioflocculant
Studies carried out have shown that the chemical nature
of bioflocculants produced by different microorganisms
differs (ABD-EL-Haleen et al., 2008). Several types of
bioflocculants have been reported including
polysaccharides, proteins, lipids, glycolipids, and
glycoproteins (Zhang et al., 2002; Li et al., 2003). Phenol
sulphuric acid determination of total carbohydrates
indicated the presence of a polysaccharide as a major
component of the bioflocculant produced by
Methylobacterium sp. Obi with no protein peak detected
by the Lowry-Folin assay. Similar results were obtained
with Serratia ficaria (Gong et al., 2008), Vagococcus sp.
W31 (Jie et al., 2006), Klebsiella mobilis (Wang et al.,
2007). The total carbohydrate concentration of
bioflocculant produced by Methylobacterium sp. Obi was
measured at 23 mg/ml.
Conclusion
The strain Methylobacterium sp. Obi has proven to be an
important producer of bioflocculant composed mainly of
polysaccharide. Bioflocculant production by the bacterium
was optimal at neutral pH and in the presence of glucose,
peptone and Ca2+ as sole sources of carbon, nitrogen and
cations respectively. A detailed characterization of the
bioflocculant as well as establishment of process
conditions for pilot scale production of the bioflocculant is
necessary and is a subject of on-going investigation in
our group.
ACKNOWLEDGEMENT
The authors would like to acknowledge the National
Research Foundation of South Africa for funding this
research.
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