Co esponding au ho : A i ah Si ak Ap ilia
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Isola ion and Cha ac e iza ion o Bac e ia om Fly Ash Samples as Bio emedia ion
Agen s
A i ah Si ak Ap ilia 1, *, Kusuma Handayani 1, Fika Ro iek Mu akhi 2 and Tugiyono 1
1 Depa men o Biology, Facul y o Ma hema ics and Na u al Sciences, Uni e si y o Lampung, Banda Lampung,
Indonesia.
2 Na ional Resea ch and Inno a ion Agency, Sou h o Lampung, Indonesia.
GSC Biological and Pha maceu ical Sciences, 2025, 33(01), 223-229
Publica ion his o y: Recei ed on 11 Sep embe 2025; e ised on 19 Oc obe 2025; accep ed on 22 Oc obe 2025
A icle DOI: h ps://doi.o g/10.30574/gscbps.2025.33.1.0403
Abs ac
Fly ash, a esidue gene a ed om coal combus ion a coal- i ed powe plan s (CFPPs), p esen s bo h economic po en ial
due o i s a e ea h elemen (REE) con en and en i onmen al isks as a haza dous pollu an . The e o e, an app op ia e
emedia ion s a egy is equi ed o mi iga e i s nega i e impac s while u ilizing i s aluable componen s. This s udy
aimed o isola e and cha ac e ize bac e ia om ly ash samples collec ed a ound he Ta ahan CFPP, Sou h Lampung,
wi h po en ial as bio emedia ion agen s o a e ea h elemen s, pa icula ly Y ium (Y). The esea ch employed an
explo a o y expe imen al app oach, including sampling, bac e ial isola ion and cul i a ion, mac oscopic and
mic oscopic cha ac e iza ion, and op imiza ion es ing o bac e ial ole ance o Y ium. The esul s e ealed wo
bac e ial isola es (FA1.2 and FA2.2), iden i ied as G am-nega i e cocci ha do no o m spo es bu a e capable o
adap ing o pollu ed en i onmen s. Op ical Densi y (OD) analysis showed op imal g ow h a 50 ppm and esis ance up
o 400 ppm. These indings indica e ha bo h isola es possess s ong po en ial as bio emedia ion and bio-ex ac ion
agen s o a e ea h elemen s in ly ash om CFPPs.
Keywo ds: Coal Fly Ash; Bac e ia; Bio emedia ion; Ra e Ea h Elemen s (REE); Coal-Fi ed Powe Plan s (CFPP)
1. In oduc ion
Compos ing Coal- i ed powe plan s (CFPPs) ha use coal as hei p ima y uel p oduce solid esidues in he o m o ly
ash and bo om ash. Fly ash is a pollu an composed o e y ine pa icles ha can easily dispe se in he ai and se le in
su ounding en i onmen s, leading o ai , soil, and wa e pollu ion i no p ope ly managed [1]. In addi ion o i s small
pa icle size (0.5–200 µm) and ligh weigh na u e ha allows i o emain suspended and sp ead h ough wind [2], ly
ash also con ains haza dous compounds such as hea y me als and silica es, which may nega i ely a ec bo h he
en i onmen and human heal h when no p ope ly mi iga ed [3].
Ne e heless, ly ash is known o con ain a e ea h elemen s (REEs) such as y ium, neodymium, and eu opium, which
ha e high indus ial alue, especially in mode n echnology. Indonesia, howe e , s ill elies hea ily on impo ed REEs
[4]. The limi a ions o ex ac ion echnology and he en i onmen al isks o con en ional mining, which ul ima ely
equi e emedia ion, ha e es ic ed he u iliza ion and managemen o ly ash esidues [5]. The e o e, explo ing
al e na i e, eco- iendly app oaches o u ilizing ly ash is highly necessa y.
One po en ial app oach is bio emedia ion, which u ilizes mic oo ganisms such as bac e ia o educe oxici y o con e
hea y me als in o less ha m ul compounds [6]. Ce ain bac e ia a e e en capable o ex ac ing me als h ough a
bioleaching p ocess, which no only educes con amina ion bu also enables he eco e y o REEs om solid was e such
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as ly ash [7]. Bac e ia ha can su i e in con amina ed en i onmen s o en exhibi high esis ance o oxic subs ances,
making hem p omising candida es o his p ocess [8].
Hence, he isola ion and cha ac e iza ion o bac e ia om he su ounding en i onmen o he Ta ahan CFPP in Sou h
Lampung, pa icula ly om ly ash dump a eas, we e conduc ed o iden i y mic oo ganisms ha a e no only esis an
o ex eme condi ions bu also capable o biologically ex ac ing REEs, pa icula ly y ium (Y). This s udy is expec ed
o con ibu e o he de elopmen o sus ainable solu ions o ly ash was e managemen and he u iliza ion o a e ea h
elemen s (REEs) con ained wi hin hese esidues.
2. Ma e ial and me hods
2.1. Time and Loca ion
This esea ch was conduc ed om Janua y o June 2025. Sample collec ion o ly ash was ca ied ou in he Ta ahan
Coal-Fi ed Powe Plan (CFPP) a ea, Sou h Lampung Regency, Lampung P o ince, Indonesia. Labo a o y analyses we e
pe o med a he In eg a ed Mine al Labo a o y, Na ional Resea ch and Inno a ion Agency (BRIN), KS Iskanda
Zulka nain, Tanjung Bin ang, Lampung.
2.2. Equipmen and Ma e ials
The equipmen used included s e ile sampling spoons, d oppe s, s e ile sample ubes, analy ical balance, 50–500 mL
E lenmeye lasks, Pe i dishes, es ubes, es ube acks, shake , spa ula, inocula ing loop, Bunsen bu ne , o en, bulb,
incuba o , lamina ai low, olume ic pipe e, 250 mL measu ing cylinde , mic opipe e, o ex, D igalski spa ula,
mic oscope slides, wooden clamps, mic oscope, and UV-Vis spec opho ome e .
The ma e ials used we e ly ash samples, dis illed wa e , s anda d y ium (Y) s ock solu ion, Nu ien Aga (NA),
Nu ien B o h (NB), physiological saline (0.8% NaCl), 70% e hanol, co on and gauze s oppe s, spi i bu ne uel, labels,
plas ic w ap, aluminum oil, c ys al iole , iodine solu ion, 95% e hanol, sa anin, and malachi e g een.
2.3. Sample Collec ion
Samples we e collec ed om wo di e en loca ions a ound he ly ash dump si e o he Ta ahan CFPP, Sou h Lampung.
Sampling poin s we e de e mined based on ield obse a ions and isible ly ash accumula ion. A each loca ion,
samples we e aken om h ee poin s wi h wo sub-samples pe poin ( eplica es) wi hin a adius o 1 m and a dep h
o 20–30 cm using s e ilized ools. Sub-samples we e composi ed in s e ile con aine s o ob ain a ep esen a i e sample
om each poin . Each sample was labeled acco ding o loca ion and sampling ime, hen s o ed in a cool box o main ain
mic obial s abili y du ing anspo o he labo a o y.
2.4. Bac e ial Isola ion
Fly ash samples we e cul u ed by adding 5 g o ly ash in o 100 mL o Nu ien B o h (NB) and incuba ing in a shake
o 24 hou s o s imula e mic obial g ow h. The cul u e was hen se ially dilu ed wi h s e ile 0.8% physiological saline
o ob ain dilu ions o 10⁻³ and 10⁻⁴. Each dilu ion was inocula ed on o Nu ien Aga (NA) medium supplemen ed wi h
ly ash and 1 ppm y ium using he sp ead pla e me hod wi h a s e ile D igalski spa ula. The pla es we e incuba ed a
37°C o 24 hou s. G own colonies we e obse ed and sub-cul u ed o ob ain pu e isola es.
2.5. Bac e ial Cha ac e iza ion
Pu e bac e ial colonies we e cha ac e ized based on mac oscopic and mic oscopic ea u es.
Mac oscopic cha ac e iza ion included obse a ions o colony shape, colo , edge, and ele a ion on solid media. G am
s aining was pe o med o iden i y cell wall ypes (G am-posi i e o G am-nega i e) and cell shapes (coccus o bacillus).
The G am s aining p ocess consis ed o p ima y s aining (c ys al iole ), mo dan (iodine), decolo iza ion (e hanol),
and coun e s aining (sa anin), ollowed by mic oscopic obse a ion.
Spo e s aining was also ca ied ou o de ec spo ula ion abili y using malachi e g een ollowed by sa anin as a
coun e s ain.
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2.6. Bac e ial Pe o mance Op imiza ion Tes
The pu i ied bac e ial isola es we e es ed o ole ance limi s by obse ing hei g ow h in media con aining y ium
(Y) a a ying concen a ions. Nu ien B o h media we e p epa ed wi h y ium concen a ions o 50, 100, 200, 300,
and 400 ppm, each inocula ed wi h one loop o bac e ial isola e. Posi i e and nega i e con ols we e also p epa ed.
Incuba ion was ca ied ou a 37°C o 72 hou s. E e y 24-hou in e al, abso bance was measu ed using a UV-Vis
spec opho ome e a 600 nm o de e mine g ow h a es. The abso bance da a (Op ical Densi y, OD) we e hen used o
e alua e bac e ial ole ance le els and e iciency in handling inc easing concen a ions o REEs.
3. Resul s and discussion
3.1. Mac oscopic Cha ac e iza ion o Bac e ial Isola es
Isola ion o bac e ial samples om ly ash and ai was ca ied ou using he sp ead me hod on media con aining NA +
ly ash (1 ppm) and NA + Y ium (1 ppm). Acco ding o [9], isola ion is he p ocess o ob aining bac e ia om hei
na u al en i onmen in o a sui able a i icial medium. In he ini ial s age o isola ion, bac e ia appea in he cul u e
mix u e, so a pu i ica ion p ocess using he s eak me hod is necessa y o ob ain pu e colonies.
Table 1 Mac oscopic Cha ac e is ics o Bac e ial Isola es om Fly Ash Samples
Isola e Codes
Colony Mo phology Cha ac e is ic
Fo m
Colo
Ma gin
Ele a ion
FA1.2
I egula
T anslucen Whi e
Undula e
Raised
FA2.2
I egula
T anslucen Whi e
Undula e
Fla
Two bac e ial isola es we e success ully ob ained om ly ash samples collec ed a ound he ly ash dump a ea o he
Ta ahan Coal-Fi ed Powe Plan , Sou h Lampung. Acco ding o [10], he oxici y o en i onmen al con aminan s
s ongly in luences mic obial di e si y, while [11] epo ed ha ly ash con ains a ious hea y me als ha inhibi he
g ow h o ce ain mic oo ganisms. Fac o s such as pH, chemical composi ion, and medium ex u e also a ec bac e ial
g ow h and ac i i y [12].
Mac oscopic mo phological iden i ica ion was ca ied ou o bo h isola es. The colony o isola e FA1.2 appea ed
i egula in shape, wi h aised ele a ion, undula e ma gins, and a anslucen whi e colo . Simila ly, isola e FA2.2
exhibi ed an i egula o m, la ele a ion, undula e edges, and a anspa en whi e colo . Acco ding o [13], a ia ions
in bac e ial colony mo phology a e closely associa ed wi h physiological esponses o en i onmen al s ess, nu ien
a ailabili y, and seconda y me aboli e ac i i y, including ex acellula polyme ic subs ance (EPS) sec e ion. The
p oduc ion o EPS enhances bac e ial adhesion, p o ides p o ec ion om desicca ion, and ac s as a physical ba ie
agains oxic compounds, allowing bac e ia o su i e in ha sh en i onmen s [14].
3.2. Mic oscopic Cha ac e iza ion o Bac e ial Isola es
Mic oscopic cha ac e iza ion was ca ied ou using g am s aining and hen obse ed unde a mic oscope a 200X and
500X magni ica ion.
Table 2 Mic oscopic Cha ac e is ics o Bac e ial Isola es om Fly Ash Samples.
Isola e Codes
Tes Type
G am S aining
Spo e S aining
G am Type
Fo m
A angemen
FA1.2
-
Coccus
Monococcus
-
FA2.2
-
Coccus
Monococcus
-
No es: G am s aining: – (G am-nega i e), + (G am-posi i e) Spo e s aining: – (non-spo e- o ming), + (spo e- o ming)
Mic oscopic obse a ion was conduc ed using G am s aining and spo e s aining o u he iden i y he physiological
cha ac e is ics o he isola es. Based on he G am s aining esul s, bo h isola es showed simila cha ac e is ics, iden i ied
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as G am-nega i e cocci wi h a monococcal a angemen . Mo phologically, he coccus o m wi h a monococcal pa e n
helps minimize di ec exposu e o oxic subs ances and educes wa e loss, pa icula ly in d y en i onmen s o unde
high osmo ic p essu e [15].
Meanwhile, he esul s o spo e s aining indica ed ha nei he isola e p oduced spo es. Al hough hese isola es did no
o m spo es like ce ain G am-posi i e bac e ia, G am-nega i e isola es possess dis inc physiological and
mo phological mechanisms ha enable hem o adap o ex eme en i onmen s. Physiologically, he cell wall o G am-
nega i e bac e ia consis s o a hin pep idoglycan laye and a complex ou e memb ane ich in lipopolysaccha ides
(LPS), which p o ide p o ec ion agains en i onmen al s esso s such as hea y me als, chemical agen s, and an ibio ics
[16].
Fu he mo e, acco ding o [14], bac e ia a e capable o p oducing ex acellula polyme ic subs ances (EPS), a
polysaccha ide ma ix sec e ed by cells o o m bio ilms. EPS ac s as a di usion ba ie agains hea y me als and
enhances wa e e en ion a ound he cell, he eby s eng hening bac e ial esis ance o oxida i e s ess and
en i onmen al luc ua ions [17]. Thus, e en wi hou o ming spo es, G am-nega i e bac e ia main ain e icien
physiological and mo phological adap a ion sys ems, allowing hem o su i e in oxic en i onmen s such as ly ash.
3.3. Op imiza ion Tes o Bac e ial G ow h (Op ical Densi y)
The wo isola es ha ha e been cha ac e ized as po en ial a e ea h me al bioex ac ion agen s we e hen es ed o
hei esis ance by obse ing hei g ow h on selec i e media NA + Ra e Ea h Me al Y ium (Y) wi h g aded
concen a ions o 50 ppm, 100 ppm, 200 ppm, 300 ppm, and 400 ppm.
Figu e 1 FA1.2 Isola e Op imiza ion Tes Cu e
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Figu e 2 FA1.2 Isola e Op imiza ion Tes Cu e
Bac e ial esis ance es ing agains a e ea h elemen s (REE), speci ically Y ium (Y), was conduc ed on bo h isola es
using media u bidi y obse a ion and Op ical Densi y (OD) measu emen wi h a spec opho ome e a a wa eleng h o
600 nm. Based on he u bidi y es esul s, media u bidi y began o appea ac oss all es ed concen a ions a e 72
hou s o incuba ion, indica ing a bac e ial adap a ion phase du ing he i s and second days, pa icula ly a
concen a ions o 300–400 ppm. Acco ding o [10], high u bidi y indica es op imal mic obial g ow h, sugges ing ha
he isola es we e able o wi hs and en i onmen al s ess caused by hea y me al con amina ion.
OD measu emen s o isola es FA1.2 and FA2.2 showed ha signi ican bac e ial g ow h occu ed a 50 ppm, wi h
abso bance alues con inuously inc easing up o 72 hou s. G ow h a concen a ions o 100–400 ppm showed a
empo a y decline du ing he 24–48-hou incuba ion pe iod, bu abso bance cu es ose again a e 48 hou s and
con inued o inc ease un il he hi d day, hough less p ominen ly a 400 ppm. This indica es ha he bac e ial isola es-
main ained esis ance e en a high Y ium concen a ions, demons a ing s ong po en ial as bioex ac ion agen s.
These esul s a e consis en wi h [18], who no ed ha OD alues e lec cell densi y and quan i a i ely ep esen
mic obial g ow h.
The e o e, bo h isola es exhibi a conside able abili y o ole a e Y ium con amina ion up o 400 ppm, highligh ing
hei p omising po en ial o bio emedia ion and bioleaching applica ions on a b oade scale. As s a ed by [19], mos
mic oo ganisms begin o expe ience g ow h inhibi ion a hea y me al concen a ions be ween 100–200 ppm; hus, he
abili y o su i e up o 400 ppm se es as an indica o o s ong physiological esilience. Fu he mo e, [20]
demons a ed ha success ul bioleaching o a e ea h elemen s such as neodymium and p aseodymium depends
g ea ly on isola es wi h high esis ance le els. Hence, he wo isola es ob ained in his s udy should be u he explo ed
and es ed o po en ial applica ion in pollu an bio emedia ion.
4. Conclusion
This s udy success ully isola ed and cha ac e ized wo bac e ial isola es om ly ash, a esidue om coal combus ion a
he Ta ahan S eam Powe Plan (PLTU), Sou h Lampung. Based on mac oscopic and mic oscopic cha ac e iza ion, he
isola es exhibi ed di e se colony mo phologies, indica ing high po en ial o adap a ion o ex eme en i onmen s. The
wo G am-nega i e, coccus-shaped bac e ial isola es, al hough non-spo e- o ming, demons a ed good physiological
adap a ion h ough an ou e memb ane s uc u e and possible EPS p oduc ion, which suppo s esis ance o oxic
condi ions.
Resis ance es ing o he a e ea h me al y ium (Y), showed ha bo h isola es con inued o g ow up o a concen a ion
o 400 ppm, wi h op imal g ow h a 50 ppm and a signi ican inc ease up o 72 hou s, which is conside ed high in he
GSC Biological and Pha maceu ical Sciences, 2025, 33(01), 223-229
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con ex o hea y me al con amina ion. The Op ical Densi y (OD) alues, which ini ially dec eased bu hen inc eased
again a e 48 o 72 hou s, indica ed ha he bac e ia we e able o unde go he adap a ion phase and esume ac i e
g ow h, and exhibi ed conside able physiological esis ance. The e o e, hese wo isola es a e wo hy o u he
de elopmen in ad anced esea ch and bio emedia ion and biome allu gy applica ions, pa icula ly in he bioleaching
p ocess o a e ea h me als.
Compliance wi h e hical s anda ds
Acknowledgmen s
The au ho s would like o exp ess since e g a i ude o he Na ional Resea ch and Inno a ion Agency, Sou h o Lampung,
Indonesia, o unding and p o iding he acili ies o conduc his esea ch.
Disclosu e o con lic o in e es
The e is no con lic o in e es .
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