Biosci. Biotechnol. Biochem.

, 75 (11), 2264–2268, 2011

Communication
Use of Bottom Ash of Waste Coal as an Effective Microbial Carrier
Min Keun K IM,3 Shah Md. Asraful I SLAM,1 Myoung Geun Y UN,1 Jong Min K IM,1
Ji Joong C HO,1 Tae Ho K ANG,1 and Han Dae Y UN1;2; y
1
Division of Applied Life Science (BK21 Program), Gyeongsang National University,
Chinju 660-701, Republic of Korea
2
Research Institute of Agriculture and Life Sciences, Gyeongsang National University,
Chinju 660-701, Republic of Korea
3
Environment-Friendly Research Division, Gyeongsangnam-do Agricultural Research and Extension Service,
Chinju 660-360, Republic of Korea

Received June 30, 2011; Accepted September 17, 2011; Online Publication, November 7, 2011

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[doi:10.1271/bbb.110495]

An experiment was done to determine the efficacy of
waste bottom ash as an effective microbial carrier.
Bottom ash found to be a suitable microbial carrier. The
average of viable cells of Paenibacillus polymyxa GS01
(as a test biocontrol agent) in bottom ash samples was
about 108 cfu/10  2 mg. The surface of bottom ash
coated with 5% PVA w/v was most effective for
improvement of cell viability. TSB medium containing
50 mg/L of MnSO4 . H2 O was the best for spore
production of P. polymyxa GS01. Thus waste bottom
ash coating with 5% PVA is likely to be suitable for use
as a microbial carrier.
Key words: coal bottom ash; microbial carrier; Paeni-
bacillus polymyxa GS01; sporulation
There are three types of coal ash that emanate from
thermoelectric power plants: fly ash, cinder ash, and
bottom ash.1) Bottom ash, which consists of porous
particles, is a by-product of thermoelectric power
plants.2) Coal combustion by-product coal ash, known
as bottom ash and fly ash, account for 12% to 20% of the
original coal when coal is burned to produce steam for
electricity generation.3) Approximately 6 million tons of
coal ash is annually produced in Republic of Korea
(Korea Ministry of Environment, 2005). Bottom ash is a
relatively coarse, gritty material, in contrast to fly ash. It
has particle sizes generally within a range of 0.1 to
10 mm.4) Fly ash is effectively reused in cement
industry. Bottom ash is also slightly recycled as
aggregates on construction sites, but most remains as
waste material. Bottom ash can be used as a construction
material,2) as a source material for the production of
geopolymers,5) to remove organic pollutants in coking
wastewater and paperindustry wastewater,6) and in soil
improvement.7) Major constituents include calcium,
aluminum, iron, magnesium, potassium, silicone, so-
dium, and titanium. These typically constitute up to 95%
of the mass of the ash. Of these materials, Ca, Fe, Mg,
K, and Si are essential plant nutrients.4) Moreover,
bottom ash has a dimensionally stable macro-porous
structure for the attachment of microorganisms and
hence is effective microbial carrier. Soil microorganisms
influence above-ground ecosystems by contributing to
plant nutrition, plant health, soil structure, bioremedia-
tion, and soil fertility.8) Bacillus and Paenibacillus spp.
elicited plant growth promotion and suppression of plant
diseases.9,10) Among the microorganisms, Paenibacillus
polymyxa (formerly Bacillus polymyxa) has been iso-
lated from different soils, rhizospheres, and roots of
plants cultivated all over the world.11) P. polymyxa is
also known as a free-living nitrogen fixer, and it has the
nifH gene, an indicator of nitrogen fixation in many
plant species.10) Moreover, P. polymyxa GS01 had
shown potential activity as a biocontrol agent against
phytopathogenic fungi.12) However, a microbial carrier
should be low in cost. There are some carriers for
microbial inoculants. Use of waste material as microbial
carrier is one of the best means of eco-friendly waste
management. In view of this, this study was carried out
to observe the efficacy of bottom ash as an microbial
carrier. In addition, various coating materials for the
carrier were studied to increase the viability of P. poly-
myxa strain GS01 as a biocontrol agent.
In this study, to select suitable microbial carriers,
different waste materials, zeolite, rubble, bottom ash,
and fly ash, were collected from the Environment
Laboratory of Gyeongsang National University, and
Samcheonpo Thermal Power Plant, located at Gosung in
southern Korea. After drying of the material by sunlight,
these were refined by sieving with a 2.0-mm diameter
sieve and preserved for study. The surfaces of candidate
materials were observed by naked eye and FE-SEM (a
field emission scanning electron microscope, Philips
XL30S FEG, AE, Netherlands) at 2,000
and 4,000

magnification. The selected materials were sputter-
coated with gold (JFC-1100E ion sputtering device,
EG&G, CA) and their microstructures were analyzed
with an FE-SEM operated at 10 kV. Heavy metals from
bottom ash (Cd, As, Cu, Pb, Zn, Ni, Cr, and Hg) were
extracted with a ternary solution (HNO3 :H2 SO4 :HClO4 ,
10:1:4 volume/volume) and quantified using ICP-OES
(an inductively coupled plasma-optical emission spec-
trophotometer, Perkin Elmer Model OPTIMA 4300DV,
Shelton, CT). Various harmful materials, PCE (per-
chloroethylene), TCE (trichloroethylene), and OP

y
To whom correspondence should be addressed. Tel: +82-55-772-1962; Fax: +82-55-772-1969; E-mail: hdyun@nongae.gsnu.ac.kr
Min Keun Kim and Shah Md. Asraful Islam contributed equally in this study.
 Bottom Ash an Effective Microbial Carrier 2265

(organic phosphate) were analyzed in compliance with .

solid waste management law (Korea Ministry of
Environment, 2007).
Paenibacillus polymyxa GS01 was previously isolated
as a potential biocontrol agent against phytopatho-
gens.12) Bottom ash was autoclaved at 121  C for 15 min
for use as a microbial carrier. Paenibacillus polymyxa
GS01 was cultured at 30  C for 16 h in 200 mL of
Tryptic soy agar (TSB, Difco, Detroit, MI) medium. The
culture was pelleted for 10 min at 4  C at 2;455
g and
then resuspended in 5 mL TSB for use an inoculant. One
g of bottom ash (about 100 pieces) was mixed with 5 mL
of bacterial suspension and then left for 3 min for natural
absorption of the bacterial suspension by the bottom ash,
and transferred to a petri dish for drying at 37  C for 3 h.
The surface of the absorbed bottom ash was observed by
different concentrations of MnSO4 H2 O, and resuspend-
ed in 0.1 M MgSO4 . To remove vegetative cells from this
suspension, the suspension was heat-treated at 70  C for
30 min and cooled to room temperature over 30 min. The
suspension was serially diluted, spread on a TSA plate,
and then incubated at 30  C for 48 h to determine the
number of viable cells germinated from endospore.
P. polymyxa GS01 was incubated on a TSA plate at
30  C for 72 h. The endospore of strain GS01 was
stained by the Rakette and Ziehl-Neelsen staining
method and observed by compound microscope.14) On
the other hand, for spore formation, P. polymyxa GS01
was incubated at 30  C for 72 h in TSB medium
.
containing 50 mg/L, MnSO4 H2 O, and this was effec-
tive for spore formation. Then bottom ash was immo-
bilized with the bacterial culture and coated with

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FE-SEM. On the other hand, to count absorbed cells 5% w/v PVA. To determine, the number of viable cells
shortly after drying, a piece of bottom ash containing in bottom ash was counted by spread-plate method by
cells was stirred at 4  C for 4 h in 3 mL of TSB medium. serial dilution during 200 d of immobilization.
The stirred TSB medium was serially diluted, spread on The surfaces of candidate materials were observed by
a TSB agar plate, and then incubated at 30  C for 48 h to naked eye, and by FE-SEM. By observation of FE-SEM,
determine the number of colony forming units. The macro- and micro-porous structures were more distinct
washed bottom ash was observed by FE-SEM. Data on the surface of bottom ash than other materials zeolite,
were collected with three replications. rubble, and fly ash (Fig. 1). In addition, FE-SEM
To improve the viability of immobilized cells in analysis showed that these pore openings were present
bottom ash, CMC (carboxymethyl cellulose, Sigma, St. throughout the entire surface of bottom ash, and were
Louis, MO) and PVA (polyvinyl alcohol, Sigma, St.
Louis, MO) were used as coating materials to study the
a b
effectiveness of the two materials. Both coating materi-
als were prepared by autoclaving at 121  C for 15 min
after they were dissolved in distilled water. For CMC,
2%, 3%, and 5% concentrations w/v were evaluated,
because CMC is sticker than PVA. On the other hand,
for PVA, 2%, 5%, and 10% concentrations w/v were
evaluated because PVA is less sticky than CMC. In case
of CMC after bacterial cell immobilization (previously
described), 1 g of immobilized bottom ash was mixed
with CMC, and PVA solutions were prepared according
to the concentration. Then bottom ash was transferred to
a Petri dish and dried to the point of hardening at 37  C
for 1 h. Some of the coated bottom ash granules were
measured by FE-SEM for surface observation, and
others were stored at room temperature for 10 d. After
10 d, the viable cells in the coated bottom ash were
counted by spread-plate method by serial dilution. We
maintained control without using any coating materials.
To evaluate the effects of the media depending on
.
concentration of MnSO4 H2 O for endospore production,
sporulation of P. polymyxa GS01 was carried out using
1/10 TSB, a nutrient broth (NB) containing 10, 30, and
.
50 mg/L, MnSO4 H2 O, and TSB medium containing
.
10, 50, and 100 mg/L of MnSO4 H2 O.13) To calculate
Fig. 1. Surface Observation of Candidate Microbial Carrier Materials.
Observation was conducted by naked eye (a) and FE-SEM (field
the number of endospores, P. polymyxa GS01 was emission scanning electron microscopy (2,000
)) (b). A, zeolite; B,
grown for 90 h in three media (100 mL each) containing rubble; C, bottom ash; D, fly ash.

Table 1. Soil Contamination Standards and Amounts of Harmful Materials Detected in Bottom Ash

Test items (unit: mg/kg)

Sample
Cd As Cu Pb Zn Ni Cr Hg PCE TCE OP
Soil contamination standards 4 25 150 200 300 10 5 4 4 8 10
Bottom ash <LLDa <LLD 0.35 0.021 <LLD <LLD 0.075 <LLD <LLD <LLD <LLD
a
<LLD: Below lower limit of detection
Lower limits of detection: 0.0006 mg/kg for Cd, 0.0135 mg/kg for As, 0.0005 mg/kg for Zn, 0.0022 mg/kg for Ni, and 0.004 mg/kg for Hg
2266 M. K. KIM et al.

readily accessible to microorganisms through openings

in pores. Similarly, single bottom ash particles were
investigated by optical microscopy, scanning electron
microscopy with quantitative energy-dispersive X-ray
microanalysis (SEM/EDX), and electron probe micro
analysis (EPMA). The fresh bottom ash consisted of
amorphous (>30 wt.%) and major crystalline phases
(>1 wt.%), including silicates, oxides, and carbonates.15)
Moreover, bottom ash was a relatively coarse, gritty
material, in contrast to fly ash, which consisted of very
fine particles. Harmful materials in bottom ash (copper,
lead, and chromium) were detected at very low concen-
trations, and other what were below the limit of
detection,16) as shown in Table 1.
The immobilized bottom ash was observed by FE-
SEM (Fig. 2). Before immobilization of bacteria, there

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were good numbers of macro- and micro-porous
structures (Fig. 2A and B). The cells of strain GS01
on entire surface of bottom ash and, many cells were
observed in grooves and pore openings (Fig. 2C and E).
The average number of viable cells in bottom ash
samples was about 108 cfu/10  2 mg. The washed Fig. 2. Field Emission Scanning Electron Microscope (FE-SEM)
bottom ash, which was stirred for 4 h due to measure- Analysis.
ment of the viable cells, was observed by FE-SEM. Most A, bottom ash surface (2,000
). B, bottom ash surface (4,000
).
C, immobilized bacteria in bottom ash (2,000
). D, immobilized
of the cells were removed from the surface of bottom bacteria in bottom ash (4,000
). E, washed bottom ash (2,000
).
ash after stirring for 4 h (Fig. 2D and F). Bottom ash can F, washed bottom ash (4,000
). Observation was done 3 d after
be used as a source of oligoelements by bacteria, and the immobilization.
extent of microbial colonization of the bottom ash and
the intensity of microbial processes can impact the rate
of leaching of potentially toxic elements.17)

A B

C D

Fig. 3. Field Emission Scanning Electron Microscope (FE-SEM) Analysis.

A, carboxymethyl cellulose (CMC) coated bottom ash; the surface of bottom ash was coated with 2% CMC, 3% CMC, and 5% CMC (arrow
head indicates crack formation on bottom ash surface). B, polyvinyl alcohol (PVA) coated bottom ash; the surface of bottom ash was coated with
2% PVA, 5% PVA, and 10% PVA, and microscopic observation was conducted at 100
and 4,000
magnification. The prevalence of viable
cells after immobilization with Paenibacillus polymyxa GS01. C, coating with CMC. D, coating with PVA.
 Bottom Ash an Effective Microbial Carrier 2267

Table 2. Effects of Various Culture Conditions on Cell Growth and Spore Production during 72 H of Fermentation at 30 C

Maximum number Maximum number

Sporulation
Culture media (100 mL) of viable cells of spore cells
(%)
(CFU/mL) (CFU/mL)
1/10 TSB agar þ 10 mg MnSO4 H2 O . 4:72
105 3:10
105 66.7
1/10 TSB agar þ 20 mg MnSO4 H2 O . 3:21
105 2:78
105 86.6
1/10 TSB agar þ 30 mg MnSO4 H2 O . 5:12
105 3:67
105 71.6
NU þ 10 mg MnSO4 H2 O . 1:18
105 3:77
104 31.9
NU þ 20 mg MnSO4 H2 O . 1:08
105 5:34
104 49.4
NU þ 30 mg MnSO4 H2 O . 1:06
105 5:14
104 48.5
TSB agar þ 30 mg MnSO4 H2 O . 1:58
108 5:31
107 33.6
TSB agar þ 50 mg MnSO4 H2 O . 2:11
108 8:74
107 41.4
TSB agar þ 100 mg MnSO4 H2 O . 9:43
107 2:31
107 24.5
TSB agar 3:88
108 3:25
107 8.4

Sporulation (%) ¼ Spore cells/Viable cells
100

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The surface of bottom ash coated with CMC and with
PVA after cell immobilization was observed by FE-
SEM. In case of bottom ash coated with CMC, the
surface was cracked due to the higher coating concen-
tration, but a lower concentration was used for CMC,
because it is stickier than PVA (Fig. 3A). The surface
of bottom ash coated with 2%, 5%, and 10% w/v PVA
was not cracked, and cells were uniformly covered
with PVA, but the surface of bottom ash coated with 10%
w/v PVA was thickly covered with PVA, and cells rarely
showed on the surface (Fig. 3B). Cell viability in bottom
ash was measured at 10 d after coating. The viable cells Fig. 4. Changes in Cell Viability on Bottom Ash against Storage
in bottom ash coated with PVA (Fig. 3D) were more Time.
Both of the samples were coated with 5% PVA.
numerous than those of bottom ash coated with CMC
(Fig. 3C), and 5% w/v PVA was most effective for
improvement of cell viability (Fig. 3D). Bio-pesticides Acknowledgments
and bio-fertilizers require delivery to the field of viable,
active microorganisms in high numbers, which requires This research was supported by Basic Science
high-quality inoculants.18) For this reason, the surface of Research Program through the National Research
bottom ash coated with PVA 5% w/v might be the most Foundation of Korea (NRF), funded by the Ministry of
effective in improving of cell viability. Education, Science, and Technology of Korea (03-2010-
After 90 h of incubation, the maximum number of 0250). T. H. Kang is supported by a scholarship from the
endospores of strain GS01 was calculated (Table 2). On BK21 Program, Ministry of Education, Science and
the media containing 20 mg/L, MnSO4 H2 O, the strain . Technology, Republic of Korea.
GS01 sporulation rate was highest on 1/10 TSB
medium, at 86.6% sporulation. Among all media, TSB References
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