Indones. J. Chem.

, 2017, 17 (2), 203 - 210 203

Removal of Sulphate and Manganese on Synthetic Wastewater

in Sulphate Reducing Bioreactor Using Indonesian Natural Zeolite

Endah Retnaningrum1,* and Wahyu Wilopo2

1
Faculty of Biology, Universitas Gadjah Mada,
Jl. Teknika Selatan, Sekip Utara, Yogyakarta 55281, Indonesia
2
Geological Engineering Department, Faculty of Engineering, Universitas Gadjah Mada,
Jl. Grafika No. 2, Bulaksumur, Yogyakarta 55281, Indonesia

Received March 3, 2017; Accepted April 4, 2017

ABSTRACT

The present research was conducted to investigate sulphate and manganese removal from synthetic
wastewater. The continuous laboratory scale of down-flow fluidized-bed reactor (DFBR) using sulphate reducing
bacteria (SRB) consortium and Indonesian natural zeolite as a bacterial support material was designed. At 9 days
operation, maximum sulphate and manganese removal was observed to be 23% and 15.4%, respectively. The pH
values were also changed to neutral. The population of SRB increased which effect on the raising of their activity for
removing sulphate and manganese. Using the scanning electronic microscopy (SEM), it was observed that natural
zeolite possesses excellent physical characteristics as a bacterial support material in DFBR. The imaging SEM
result of SRB consortium on zeolite surface clearly showed the developed SRB biofilm on that particle. Analysis
result of EDX confirmed that manganese was precipitated as manganese–sulfides.

Keywords: DFBR; SRB consortium; natural zeolite; SEM-EDX; manganese-sulfides

ABSTRAK

Penelitian dilakukan untuk menyelidiki penghilangan sulfat dan mangan dalam limbah cair sintetik. Desain
penelitian berupa down-flow fluidized-bed reactor (DFBR) skala laboratorium dengan sistem kontinyu menggunakan
konsorsium sulphate reducing bacteria (SRB) dan zeolite alam dari Indonesia sebagai material penyangga bakteri.
Pada pengoperasian hari ke-9, penghilangan sulfat dan mangan secara maksimum teramati masing-masing
sebesar 15,4% dan 23%. Nilai pH juga berubah menjadi netral. Populasi SRB meningkat sebagai akibat kenaikan
aktivitas bakteri dalam penghilangan sulfat dan mangan. Pengamatan menggunakan scanning electronic
microscopy (SEM) terlihat bahwa zeolit alam mempunyai karakter fisik unggul sebagai material penyangga bakteri
dalam DFBR. Hasil pencitraan SEM terhadap konsorsium SRB pada permukaan zeolit secara jelas memperlihatkan
terjadi pembentukan SRB biofilm pada partikel tersebut. Hasil analisis EDX menegaskan bahwa senyawa mangan
mengalami presipitasi dalam bentuk mangan sulfida.

Kata Kunci: DFBR; konsorsium SRB; zeolite alam; SEM-EDX; mangan sulfida

INTRODUCTION sulphate remediation have been carried out all around

the world.
Due to rapid industrialization, particularly from Anaerobic sulphate reducing bioreactors are
mining industries have been generated acid mine becoming the biological alternative treatment of acidic
drainage (AMD). Those polluted waters occur when metal-containing wastewaters due to low cost and high
sulphide rock are exposed to oxygen and water in the efficiency. These reactors contain sulphate reducing
absence of sufficient neutralizing minerals. The AMD is bacteria (SRB) that have the ability to reduce sulphate
characterized by a low pH and high concentrations of to sulphide and then this sulphide reacts with certain
heavy metals and sulphate [1-2]. The presence of metals dissolved, such as manganese, copper, iron
elevated concentrations of manganese and other heavy and zinc, forming insoluble precipitates [1]. Numerous
metals in wastewater can exert detrimental effect on reactor designs for microbial sulphate reduction have
human health and environment due to their toxicities [3- been reported [4-8]. The applying of those reactor
5]. In order to remove those pollutants from industrial designs have also been reported to be used for the
effluents, many attempts related to both heavy metal and treatment of industrial effluents, particularly from acid

* Corresponding author. Tel : +62-274-580839 DOI: 10.22146/ijc.22710

Email address : [email protected]

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204 Indones. J. Chem., 2017, 17 (2), 203 - 210

mine drainage (AMD) [9-12]. However, there are two heavy metal of MnSO4.H2O was added separately to
problems for applying those anaerobic sulfate reducing the bottles reach to final concentration of 10 mg/L.
bioreactors: (1) lack of bacterial resistance to metals, The natural zeolite used as SRB biomass support
and (2). the loss of bacteria biomass in the systems with was obtained from a deposit located in the Province of
high hydraulic loading rates. In the literatures, zeolites Gunung Kidul, Yogyakarta, Indonesia. The chemical
have reported shown a great capacity for adsorption of composition (%, w/w) of the zeolite used was SiO2
heavy metal (Mn, Cu, Cd, Pb and Zn) [13-15], aflatoxins 86.3%; Al2O3 13.7%; Fe2O3 2.4%; CaO 2.3%; MgO
[16], and cationic dye [17]. In addition, previously 0.4%; Na2O, 1.7%; and K2O 1.8%. It has mineral
researchers reported that zeolites can be useful as a composition as clinoptilolite; mordenite and
microbial support in anaerobic processes of different montmorillonite. The surface area, pore volume and
2
wastewaters [18-20]. Therefore the bioreactor could be pore size of zeolite was 20.0 m /g, 12.4 cc/g, and
operated at higher heavy metal concentration by 23.8 Å, respectively. It was powdered and grouped into
immobilization using natural zeolites. This immobilized three groups based on its diameter size (R1, R2 and
SRB could lead to its biofilm formation. Moreover, SRB R3 Groups). Diameter size of group R1, R3 and R3
cells have better stability and higher metabolism [21]. were 0.4-0.6 cm, 0.6-0.8 cm, and 0.8-1.2 cm,
Hence, that formation may reduce metals toxicity and respectively. Before utilization it was washed twice and
the loss of that SRB biomass. This process may further soaked with deionized water for 24 h at 30 °C. After
stimulate their activities in the sulphate reducing this treatment, zeolite was dried for 24 h at 80 °C and
bioreactor. Due to highly abundance of natural zeolites used as a carrier for immobilization of mixed SRB
obtained in some area of Indonesia. It is important to cultures in both batch and continuous experiments.
evaluate possibilities for utilizing Indonesian natural
zeolite as a carrier for SRB immobilization in the Instrumentation
sulphate reducing bioreactor. The main objective of this
research was to operate the sulphate reducing Centrifuged (Eppendorf, 5810R) apparatus was
consortium in a controlled bioreactor system using used for separation between bacterial cells and
natural zeolite as supporting materials, to determine the medium. For analysis of sulphate and manganese
sulfate reduction rate, manganese removal and pH removals in DFRB were used a spectrophotometer
profile. The laboratory scale reactor was selected which (Shimadzu UV-1601) and Atomic Absorption
can be control all the influential environmental Spectroscopy (AAS) apparatus (Hitachi, Z-2000),
parameters in well-organized manner. The optimized respectively. Decreasing of pH values in the bioreactor
conditions for that reactor were obtained from batch was measured using pH meter (Metrohm). The
experiments conducted previously. characteristics of biofilm and the manganese interacted
on zeolite surface were observed using Scanning
EXPERIMENTAL SECTION Electron Microscope- Energy Dispersive- X-ray
Spectroscopy (SEM-EDX) (JEOL JSM-T300).
Materials
Procedure
The mixed culture of SRB used in the present
study was obtained from sheep manure samples. The Anaerobic batch experiments
culture was placed in a glass bottle with Postgate B The anaerobic batch tests were performed in
medium and incubated at 35 °C [22]. This medium sealed flasks (250 mL). The flask experiments were
contained (in g/L): K2HPO4 0.5, NH4Cl 1.0, CaSO4 1.0, seeded with 20 mL of the mixed SRB cultures into
FeSO4.7H2O 0.5, sodium lactate 8 mL, MgSO4.7H2O 180 mL synthetic wastewater under the anaerobic
2.0, yeast extract 1.0 and ascorbic acid 0.1. The medium condition. Three groups of zeolites in different diameter
was aerated with nitrogen gas to maintain anaerobic size (R1, R2 and R3) in the amount of 20 g were added
conditions before inoculating. Every week 20% of the to each batch SRB cultures. Therefore the inoculated
volume of the culture in the bottle was replaced by fresh flasks were incubated at 30 °C for 14 days. During the
medium. After three months, a culture containing high incubation, samples were collected at 0, 1, 3, 7 and 14
density of bacteria of different morphological types was days and were further analyzed.
achieved.
The synthetic wastewater (pH 5 and 2500 mg/L Anaerobic continuous experiments
sulphate) contained (in g/L): MgSO4.7H2O 2.563, The zeolite of R1 added in the batch experiment
Na2SO4 1.479; KH2PO4 0.056; NH4CI 0.11, ascorbic acid previously which demonstrate the highest removing
0.011, yeast extract 0.05 and sodium lactate 8 mL. The pollutants (sulphate and manganese metal), increasing
pH and population number of SRB obtained, was

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 Indones. J. Chem., 2017, 17 (2), 203 - 210 205

selected for biomass carrier of continuous experiments. samples were fixed with 2.5% glutaraldehyde, ethanol
The continuous laboratory scale of down-flow fluidized- (dehydrated) and coated with gold under vacuum in an
bed reactor (DFBR) inoculated with mixed SRB was argon atmosphere [25]. The surface morphology of the
used in this study [23]. The DFBR was fed with synthetic gold coated samples was visualized by a SEM (JEOL
wastewater that prepared freshly every day. The volume JSM-T300). The surfaces elemental analyses of a Mn
of the DFBR reactor is 1.2 L and it is filled with 1.13 kg interacted in zeolite were carried out by Energy
natural zeolite and 0.67 L synthetic medium with the dispersive X-ray.
result that the surface of zeolite in the reactor is
completely covered. The reactor is then inoculated with Statistical analysis
40 mL of SRB culture from batch experiments. The The experiments were done in triplicates. All
DFBR was operated in a temperature controlled at 30 °C values are expressed as means ± standard deviation.
for 9 days. During this operation, samples of influent and Mean values of treatments were compared by the
effluent were collected at 0, 3, 5, 7 and 9 days and were analysis of variance (one-way ANOVA) followed by
further analyzed. Duncan Multiple Range Test (DMRT) method for mean
differences testing. Differences were considered
Chemical analysis significant at p < 0.05.
At certain intervals, 10 mL samples of both batch
experiment and continuous experiment were collected RESULT AND DISCUSSION
using a syringe, and centrifuged at 5000 rpm to prepare
the cell free supernatant at 4 °C. This supernatant was Effect of Different Diameter Size of Natural Zeolite
used for analysis of concentrations of sulphate and on Mixed SRB Activity
manganese metal. Sulphate concentration was
measured according to the turbidimetric method using a Effects of different diameter size of natural zeolite
Shimadzu UV-1601 spectrophotometer, and the (R1, R2 and R3) on the mixed SRB activities were
absorbance of the sample was measured at wavelength investigated for 14 days in anaerobic batch
of 420 nm [24]. For determination of the dissolved experiments. During those incubations, all mixed SRB
manganese the supernatant samples were acidified with cultures revealed the black precipitates. Those
nitric acid and analyzed by Atomic Absorption precipitates were indicated on mixed SRB activity that
2
Spectroscopy (AAS) using a Hitachi, Z-2000 model can utilize sulphate (SO4 -) as an electron acceptor,
spectrometer. The pH samples were measured therefore generating hydrogen sulfide (H2S). Synthetic
immediately without centrifugation using pH meter acid mine drainage containing lactate was used by
(Metrohm). The number of SRB was enumerated by the SRB as organic substrate for generating bicarbonate
three-tube Most Probable Number (MPN) assay with ions and hydroxides [1]. Furthermore, those sulfides,
serial dilutions [22]. Performance of down-flow fluidized- carbonates and hydroxides can react with manganese
bed reactor (DFBR) on the pollutants removal (sulphate metal and increase pH value in the AMD. However
and manganese concentration) was evaluated at the their activity rates were influenced significantly by
inlet (initial concentration) as well as the outlet (final different size of natural zeolite addition (P < 0.05) and
concentration). The pH value during that bioreactor the highest result was analyzed on the zeolite R1,
operation was also measured. followed by zeolite R2, R3 and control (Fig. 1). Since
zeolite R1 had the smallest diameter compared with
Characterization of developed SRB biofilms zeolite R2 and R3, therefore increasing the surface
population area of zeolite for attachment and colonization of SRB.
The developed SRB biofilms populations were These enhancements influenced microbial and
taken at 5 and 9 days DFRB operation. Samples on the enzymatic transformation of a variety of substances for
zeolites surface of DFBR were enumerated by the three- sulphate reducing bacterial during their attachment and
tube Most Probable Number (MPN) assay with serial colonization in the zeolite surface [26-28]. In addition
2
dilutions. One cm biofilm samples on zeolite surface of zeolites have been reported to absorb manganese and
DFBR were scraped from the substrates using a sterile other heavy metals [29-30]. As the result, number SRB
scalpel and dispersed in 10 mL of sterile water. Samples cells of zeolite R1 was highest compared with zeolite
were then diluted serially and inoculated in Postgate B R2 and R3. Hence, that SRB metabolism for biological
medium [22]. The cultures furthermore were incubated at sulfate reduction process was also highest. Their
30 °C for 5 days. The characteristics of biofilm sulphate reducing process induced reducing sulphate
attachment on zeolite surface were observed using and manganese concentrations that lead to higher pH
Scanning Electron Microscope- Energy Dispersive- values.
X-ray Spectroscopy (SEM-EDX). For SEM the biofilms

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206 Indones. J. Chem., 2017, 17 (2), 203 - 210

Fig 1. Variation sulphate concentrations (a), manganese concentrations (b), pH values (c) and the number of SRB
cells (d) during batch experiments of bioreactor using synthetic wastewater which treated by zeolite R1: diameter
size 0.4-0.6 cm, R2: 0.6-0.8 cm, R3: 0.8-1.2 cm and C: without treatment

The average of both sulphate and manganese Sulphate Reductions, Mn Removals and Ph Values
removals in these batch experiments of zeolite R1 were of Effluent on Anaerobic Continuous Experiments
50.3% and 56.4%, respectively. As can be seen, sulphate
concentration droplet rapidly to 148 mg/L on the first day Based on the results from the batch experiments,
of incubation and then decreased to 28 mg/L on the end zeolite R1 was applied in the continuous laboratory
day of incubation (14 days). The reduction of sulfate scale of down-flow fluidized-bed reactor (DFBR) in
concentration during that incubation was followed by a 1.2 L volume. The performance of DFBR in terms of
decrease in the concentration of manganese. sulfate reductions, Mn removals and pH values were
Consequently the pH values were also changed to presented in Fig. 2. The reactor started with 5.5%
neutral. The reduction manganese concentration during sulfate reduction, 1.9% Mn removal and pH increased
this process was influenced by the occurrence of in 3 days operation without any lag period due the
precipitation reactions between manganese metal and inoculation with active SRB biomass from an optimum
some compounds which produced during SRB activities batch experiment. The active SRB may have increased
(biological sulfate reduction process), such as sulfides, over time and the bacteria may have been adapted to
carbonates and hydroxides. Decreasing manganese the operating conditions [1-2, 31]. The performance of
concentration in the anaerobic batch culture caused a sulfate removal increased maximum to 23% in 9 days
reduction manganese toxicity in SRB cell. Furthermore operation. Therefore, metals were precipitated with the
the numbers of SRB cells as biofilms in the zeolite sulfide produced by sulfate reduction process. This
surface were significantly increased during 14 days performance was followed by a manganese removal
incubation. increased maximum to 15.4%. The sulfate reduction
rate and manganese removal rate of the DFBR
−1
operations were 3.2 and 6.4% day , respectively.

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 Indones. J. Chem., 2017, 17 (2), 203 - 210 207

Fig 2. Removal of sulphate, manganese concentrations

and pH values of effluent during anaerobic continuous Fig 3. The Number of SRB Biofilms attached on zeolite
bioreactor operation surface in anaerobic continuous bioreactor operation

Fig 4. Developed biofilm of SRB on the zeolite surface in the continuous reactor after a 5 days (a) and 9 days (b)
operation, 1: coccoid form bacterium cell; 2: bacillary form bacterium cell; 3: exopolysaccharide (EPS). Photographs
were taken at 10,000× amplification

Previously reported sulfate and manganese reduction Characteristics of Developed SRB Biofilms
rate in an ethanol-fed DFBR using zeolite obtained from Population
Turkey in 9 days operation were 0.4 and 0.3%,
respectively. It was also clearly demonstrated that the The number of SRB cells attached to zeolite in
bioreactor that feed by synthetic AMD with pH value of 5 the bioreactor that operated in 5 and 9 days were
.0 could change pH values of effluent were tend to presented in Fig. 3. During bioreactor operation, the
neutral due to alkalinity production during sulfidogenic population density of SRB increased approximately 5
electron donor oxidation [32-33]. times from 5 days to 9 days operation. This raising
This reactor was only setup for 9 days operation at altered of cross-feeding, co-metabolism, interspecies
optimum conditions 30 °C with sodium lactate as carbon hydrogen and proton transfer between SRB cells [36-
source, whereas the application this bioreactor in the 37]. Furthermore, its condition may further stimulate the
field scale reported in several literatures was operated in growth of micro colonies which effect on the increasing
the longer operation than 9 days. Longer operation of of their activity for removing sulphate and manganese.
the bioreactor could remove sulphate and manganese Those developed SRB biofilms on the zeolite
concentration more efficient and effective, thus can be surface were also observed in the bioreactor using
applied in the wastewater. Previously researchers SEM (Fig. 4). A large microorganism accumulation in
reported that sulfate reduction and metal precipitation in both the interior of the ruggedness and in the
the bioreactor operation of 250 days were 86% and superficial zones of natural zeolite was observed during
97%, respectively. [34]. Similar results also have been bioreactor operation. The attachment and colonization
reported by Yamashita et al. that denitrification reactor of SRB cells on the zeolite surface caused they were
packed with wood as a carbon source in the operation of more protected from friction [38]. It was also observed
1500 days could remove of sulphate and manganese that the support surface formed by a compact mass of
almost of 90% and 99%, respectively [35]. microorganisms, principally with coccoid and bacillary

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208 Indones. J. Chem., 2017, 17 (2), 203 - 210

Fig 5. EDX spectra of SRB biofilm attached on zeolite particle after interacting with manganese in the continuous
reactor after 5 days operation (a) and 9 days operation (b)

forms. These cells were directly attached to the zeolite CONCLUSION

surface and cells were immersed and covered with an
exopolysaccharide (EPS) which keeps them together. Indonesian natural zeolite was effective as a
EPS is produced by that bacterium which forms a dense sulphate reducing bacterial support material which
and sticky glycocalyx on the surface of bacterial cells designed in continuous laboratory scale of down-flow
[39-40]. fluidized-bed reactor (DFBR) for removing sulphate and
The composition of the metal precipitates which manganese. It has excellent physical characteristics
were accumulated from the surface of zeolite in the and the adequate environment for developing of SRB
bioreactor was determined by SEM-EDX. Those analysis biofilms on its surface particle material. The highest
of the zeolite surface indicated that the precipitated efficiency of sulphate reduction and manganese in this
compounds were most probably MnS (Fig. 5), this bioreactor were found to be 23% and 15.4%,
suggests that the removal of metal is mainly via sulfide respectively at 9 days operation. The imaging SEM of
precipitation. It is possible to determine that sulfide SRB consortium on zeolite surface clearly showed the
precipitation is the dominant metal removal mechanism developed SRB biofilm on that particle. Analysis of
in bioreactor operations. Therefore, two main sub- EDX confirmed that manganese was precipitated as
processes in bioreactors process can be referred to as manganese-sulfides.
diffusion and anaerobic reduction of sulfate, and the
reaction of biogenic sulfide with metallic ions and ACKNOWLEDGEMENT
precipitation of metal sulfide. Detection via EDX
confirmed the mechanism of the precipitation of metal This research was supported by KEMENRISTEK
sulfides occurred due to the certain sulfide-rich layer DIKTI Indonesia with the Research Implementation
around the zeolite surface. Therefore, metals ions might Letter No: 663/UN1-P.III/LT/DIT-LIT/2016.
not be inhibit SRB activities even in high concentrations.
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Endah Retnaningrum and Wahyu Wilopo