Bioresource Technology 137 (2013) 409–413

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Short Communication

Biological treatment of mining wastewaters by fixed-bed bioreactors

at high organic loading
Svetlana Bratkova a,⇑, Bogdana Koumanova b, Venko Beschkov c
a
Department of Engineering Geoecology, University of Mining and Geology, ‘‘St. Ivan Rilski’’, Prof. B. Kamenov Str.,1700 Sofia, Bulgaria
b
Department of Chemical Engineering, University of Chemical Technology and Metallurgy, 8 Kliment Ohridsky Blvd., 1756 Sofia, Bulgaria
c
Institute of Chemical Engineering, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Blvd. 103, 1113 Sofia, Bulgaria

h i g h l i g h t s

 Water contaminated with Fe and Cu was remediated by microbial hydrogen sulfide.

 Sulfate-reducing bacteria were immobilized on saturated zeolite in fixed-bed bioreactor.
 Solution containing ethanol, lactate, citrate and glycerol was used as donor of electrons.
2
 Influences of SO4 volume loading rates, temperature and pH to the rates of the process were studied.
 Efficient removal of COD, H2S, N and P was achieved in a three-sectional bioreactor.

a r t i c l e i n f o a b s t r a c t

Article history: Acid wastewaters contaminated with Fe – 1000 mg L1 and Cu – 100 mg L1 were remediated by micro-
Received 21 January 2013 bial sulfate-reduction at high organic loading (theoretical TOC/SO2
4 ratio 1.1) in a laboratory installation.
Received in revised form 25 March 2013 The installation design includes a fixed-bed anaerobic bioreactor for sulfate-reduction, a chemical reac-
Accepted 27 March 2013
tor, a settler and a three-sectional bioreactor for residual organic compounds and hydrogen sulfide
Available online 6 April 2013
removal. Sulfate-reducing bacteria are immobilized on saturated zeolite in the fixed-bed bioreactor.
The source of carbon and energy for bacteria was concentrated solution, containing ethanol, glycerol, lactate
Keywords:
and citrate. Heavy metals removal was achieved by produced H2S at sulfate loading rate 88 mg L1 h1. The
Sulfate reduction
Fixed-bed anaerobic bioreactor
effluent of the anaerobic bioreactor was characterized with high concentrations of acetate and ethanol.
Heavy metals The design of the second bioreactor (presence of two aerobic and an anoxic zones) makes possible the
High organic loading occurrence of nitrification and denitrification as well as the efficiently removal of residual organic
compounds and H2S.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction
Acid mine drainage is a major environmental hazard that affects
the aquatic ecosystems around mines. Conventionally, hydroxide
precipitation is the most commonly applied method for the treat-
ment of metal containing waters. In recent years, the use of sulfate-
reducing bacteria (SRB) has been proposed as an alternative to
hydroxide precipitation (Kaksonen and Puhakka, 2007). Sulfate-
reducing bacteria oxidize simple organic compounds with sulfate
under anaerobic conditions. The main products from the process
are hydrogen sulfide and bicarbonate ions. The HCO 3 ions increase
the alkalinity of the water. The soluble sulfide reacts with the met-
als to form insoluble metal sulfides.
Numerous reactor designs for microbial sulfate reduction and a
variety of used donors of electrons have been reported (Kaksonen
⇑ Corresponding author. Tel.: +359 2 8060 498.
E-mail address: s_bratkova@yahoo.com (S. Bratkova).
et al., 2004; Bayrakdar et al., 2009; Nevatalo et al., 2010). The pre-
ferred configurations for anaerobic wastewater treatment are
mainly the upflow anaerobic sludge bed reactor, the fluidized
bed reactor and the anaerobic filter. Biological sulfate reducing
reactors used for metal precipitation can have either one or more
stages, i.e., the sulfate reduction and metal precipitation can occur
simultaneously, or in separate process units (Hao et al., 2000). The
separation of microbial sulfate reduction and metal precipitation
alleviates toxicity on SRB. Various parameters such as temperature,
pH, sulfide and metal concentrations in the acid mine drainage af-
fect the growth and activity of sulfate-reducing bacteria. Other
important parameter is the total organic carbon SO2 4 ratio. Velasco
et al. (2008) reported that the feed chemical oxygen demand
(COD)/SO2 4 ratio can be an useful parameter to control the hydro-
gen sulfide production in the metal precipitation process. Kakso-
nen et al. (2004) showed that the stoichiometric COD/SO2 4 ratio
of 0.67 was adequate to attain around 60% of sulfate reduction with
an initial sulfate concentration of 2000 mg L1.

0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2013.03.177
410 S. Bratkova et al. / Bioresource Technology 137 (2013) 409–413
This work reports the experimental results from the treatment
of synthetic acid mine water, containing Fe and Cu in a laboratory-
scale installation. Microbial sulfate-reduction was realized in a
fixed-bed bioreactor with saturated zeolite. The effect of SO2
4 vol-
ume loading rates, temperature and pH on rate of sulfate-reduction
at high organic loading were determined. Further, the removal of
residual concentrations of organic compounds, sulfide, N and P
were demonstrated by performance of the second bioreactor.
2. Methods
2.1. Reactors
2.1.1. Fixed-bed reactor for microbial sulfate-reduction
The geometric volume of the anaerobic fixed-bed reactor is
1.2 dm3. The carrier was produced by saturation of natural oc-
curred zeolite, clinoptilolite of 2.5–5.0 mm size fraction, with the
following composition, %: SiO2 – 67.96, Al2O3 – 11.23, Fe2O3 –
0.83, K2O – 2.85, Na2O – 0.74, CaO – 3.01, MgO – 0.06, TiO2 –
0.90. The whole 6 L solution containing NH4Cl – 10 g L1, K2HPO4
– 5 g L1, MgSO47H2O – 4 g L1 was used for saturation of
1.20 kg zeolite. The reactor is filled with 1.10 kg zeolite and
0.70 dm3 modified Postgate medium, containing lactate and theo-
retical TOC/SO2
4 ratio of 1.1. It is inoculated with 70 ml enriched
microbial culture of SRB belonging to genera Desulfotomacilum,
Desulfomicrobium, Desulfovibrio and Desulfobacterium. The adher-
ence of biofilm of SRB onto the saturated zeolite is carried out for
a period of 3 months through repeated periodic replacement of
50% of the liquid phase of the bioreactor with fresh medium.
2.1.2. Three-sectional bioreactor
The construction of the three-sectional bioreactor is inserted
into each other of the three cylinders with increasing diameters.
The total volume of bioreactor is 2.8 dm3. The outermost and
innermost zones of the reactor are aerated. The gravel with a frac-
tion size of 5–9 mm was used as biomass carrier in the aerobic
zones of the bioreactor. The water is fed at the bottom of innermost
cylinder. Water overflow enter in the middle digester anaerobic
zone. In the perforated bottom of the second cylinder, water enters
the outermost zone of the aerobic bioreactor in which the motion
is upstream. The bioreactor was inoculated with 80 ml activated
sludge taken from a wastewater treatment plant.
2.2. Influences of SO2
4 volume loading rates, temperature and pH on
the rates of the microbial sulfate-reduction in anaerobic fixed-bed
reactor
The continuous cultivation was started up after the formation of
active biofilm of SRB. The new medium is mixture of two solutions.
First of them is a highly concentrated solution, containing organic
compounds (TOC – 200 g L1). It contained per liter of distilled
water: ethanol, 96% – 333 ml, lactic acid, 80% – 36 ml, sodium cit-
rate – 20 g, glycerol – 23.4 g, yeast extract – 5.0 g, NH4Cl – 10.0 g
and K2HPO4 – 5.0 g, pH 7.2–7.5. The second solution contains Na2-
SO4 – 2 g L1, Mg2SO47H2O – 4 g L1 and conc. H2SO4 – 0.1 ml (pH
2.8–3.0). Adjustable flow peristaltic pumps support the mainte-
nance of the theoretical TOC/SO2 4 ratio of 1.1 and concentration
of sulfates 3.0 g L1. The influence of volume loading rate with sul-
fates on the rate of the process is examined through seven different
HRT: 13.8, 17.3, 23.0, 34.5, 46.1, 69.3 and 86.4 h for a period of
3 months. The effluents from anaerobic bioreactor were sampled
5–7 times after reaching a dynamic equilibrium for every mode.
The influence of temperature and pH on the rate of the process
were evaluated at SO2 4 volume loading rate 0.176 g L
1 1
h (HRT
17.3 h). The effect of temperature (21, 27 and 37 °C) was deter-
mined for a period of 10 days per each temperature as the samples
were collected every 2 days. The concentration of sulfates, organic
acids and alcohols and pH were analyzed. The effect of pH in fixed-
bed bioreactor on the rate of the process was determined in the
range 5.45–7.25 for period of 2 months. For this purpose, the pH
of the solution containing sulfates was additionally acidified by
adding HCl from pH 3.5–1.7. In this procedure, the following inter-
vals with decreasing values of pH are studied: 7.2–7.3, 6.7–6.9,
6.2–6.4, 5.8–5.9, 5.6–5.7, 5.5–5.6, 5.4–5.5. 3–4 Samples for every
mode were collected when bioreactor achieved a steady state.
2.3. Treatment of waters polluted with heavy metals in the laboratory
installation
2.3.1. Scheme of laboratory installation
The fixed-bed bioreactor (1) was fed with concentrated nutrient
medium through a peristaltic pump (5) with adjustable flow rate
(Fig. 1). Homogenization with upward flow in the reactor is per-
formed by a recirculating pump (7). The microbial produced H2S
contacts with the solution of heavy metals in a chemical reactor
(2). The solution of heavy metals was fed into the chemical reactor
through a peristaltic pump (6). The geometric volume of the chem-
ical reactor is 0.5 dm3.The insoluble sulfides precipitated in a ver-
tical-flow settler (3) with a volume of 0.85 dm3. The greatest part
of the effluents of settler was pumped into anaerobic fixed-bed
bioreactor by a recirculating pump (8). The other part of effluents
was entered into three-sectional bioreactor (4).
2.3.2. Treatment of waters polluted with heavy metals
The acid solution of heavy metals contains heavy metals Cu and
Fe, added as FeSO47H2O – 4.98 g L1 and CuSO45H2O –
0.393 g L1. The concentration of sulfates was adjusted to
3.0 g L1 by addition of Na2SO4 and MgSO47H2O. This solution
was treated in fixed bed anaerobic bioreactor at two different
SO2
4 volume loading rates – 0.176 and 0.088 g L1 h1 at 21 °C
and TOC/SO2 4 ratio 1.1 for a period of 2 months. The efficiency of
removal of heavy metals was found by sampling two times per
week. Each sample was analyzed for pH, Eh, sulfate, sulfide, COD
and soluble Fe and Cu. The effluents from anaerobic bioreactor at
mode 0.088 g L1 h1 were treated in the second bioreactor. The
concentrations of ammonium, nitrate and phosphate were also
analyzed. The change in exchangeable cations and the amount of
organic carbon of natural zeolite after saturation and after forma-
tion of biofilm of sulfate-reducing bacteria were also determined.
2.4. Analytical methods
The sulfate concentration was determined using spectrophoto-
metric method by BaCl2. The total sulfide concentration was mea-
sured immediately after sampling using Nanocolor test 1-88/05.09.
The phosphate concentration was determined by the molybde-
num-blue ascorbic acid method. The ammonium concentration
was measured by the Nessler method. The nitrate concentration
was measured by sodium salicylate method. Organic substrate uti-
lization was estimated by measuring the chemical oxygen demand
(COD). The dissolved metal concentrations were determined by ICP
spectrophotometry. Organic acids and alcohols were analyzed by
high-performance liquid-chromatography. An Aminex HPX-87H
column from Bio-Rad coupled to a RI detector (LC-25RI) was used
for HPLC; sulfuric acid (0.01 N) was used as an eluent. Exchange-
able cations (Na, K, Ca and Mg) were extracted from zeolite using
an extracting solution (ammonium acetate) at pH 7.0. Na and K
were determined by Flame photometer and Ca and Mg by EDTA
complexometric titration. Standard statistical parameters includ-
ing mean and standard deviation (for more than duplicate data
points) were used. The experimental data were also analyzed by
one-way ANOVA (95% confidence interval) using STATGRAPHICS
Centurion XV software.
 S. Bratkova et al. / Bioresource Technology 137 (2013) 409–413 411

Fig. 1. Schematic diagram of the laboratory installation for active treatment of wastewaters polluted with heavy metals: 1 – anaerobic fixed-bed biofilm reactor; 2 – chemical
reactor; 3 – settler; 4 – three-sectional bioreactor; 5 and 6 – peristaltic (roller) pump; 7 and 8 – recirculating pump; 9 – sludge; I, II and III – sampling points.

3. Results and discussion There is also a statistically significant linear relationship be-
tween the rate of the process and pH in the range from 5.9 to
3.1. The influences of SO2
4 volume loading rates, temperature and pH
7.25 (R2 = 99.84%):
on the rates of microbial sulfate-reduction
1
Rate of sulfate-reduction ðmg L1 h Þ
The influence of volume loading rate of sulfates on the rate of sul-
¼ 86:1711 þ 30:3677  pH
fate-reduction for seven different modes showed that with the
increasing of sulfate volume loading rate from 0.035 to 0.130 SO2 4 Maximal rate of the process – 134 ± 3 mg SO2 4 L1 h1 was
g L1 h1 leads to an increase of the rate of the process in a linear achieved at pH 7.25. The rate of sulfate-reduction was notably re-
relation. At the same time, the efficiency of the process decreases duced at pH below 5.7, as at pH 5.45 the rate of the process was
from 97.8 ± 0.3 to 84.5 ± 0.9%. During the study, it was established only 25.3 ± mg SO2 4 L1 h1. This lower rate of sulfate-reduction
a maximum rate of sulfate-reduction – 130.3 ± 1.6 mg SO2 4 L
1 1
h could be related to the increased toxicity of total dissolved sulfide
1 1
while volume loading rate was 0.176 g L h (HRT 17.3 h). At to SRB. At pH below 7, the fully protonated form of hydrogen sul-
HRT 13.8 h, the rate of the process decreases to 128.3 ± 3.4 mg fide (H2S) dominates, and this form is believed to be responsible for
SO2
4 L
1 1
h , and the concentration of sulfates in the effluent solu- inhibition of bacterial respiration (Moosa and Harrison, 2006).
tion were high – 1.23 ± 0.047 g L1. The statistical analysis showed The effluent from sulfidogenic bioreactor was characterized with
that there was no meaningful difference between the measurements high values of COD (8970 ± 500 mg L1). Data from HPLC analyses
of the rate of the process for each HRT (P = 0.99 > 0.05). showed that these results are related to the incomplete oxidation
It was found that at volume loading rate 0.176 g SO2 4 L
1 1
h in of organic compounds mainly to acetate and residual ethanol. It
2
the range 21–37 °C, the process rate increases linearly (R = 97.4371%): was found ethanol – 2.57 g L1, and acetate – 2.35 g L1 in the efflu-
ent at sulfate volume loading rate 0.176 g L1 h1and at temperature
1
Rate of sulfate-reduction ðmg L1 h Þ 21 °C. The concentrations of the abovementioned organic com-
pounds showed 37.7% removal of TOC under these conditions. At
¼ 89:5434 þ 2:04847  Temperature ð CÞ
temperature 35 °C and at the same sulfate volume loading rate in
Q10 temperature coefficient under these conditions of cultiva- the effluent were measured ethanol – 1.64 g L1 and acetate –
tion is 1.14. It was determined maximal rate of sulfate-reduction 2.79 g L1, corresponding to 43.4% removal of TOC. There are not
– 165 ± 2 mg L1 h1at 37 °C. established residual concentrations lactate, glicerol and citrate, nei-

Table 1
The operational data of the operated reactors.

Parameter Solution of heavy metals Effluent from fixed-bed anaerobic bioreactor at sulfate volume loading rate Effluent from three-sectional bioreactor
0.176 g L1 h1 0.088 g L1 h1
pH 2.88 ± 0.08 6.14 ± 0.11 7.27 ± 0.03 8.24 ± 0.35
Eh (mV) +363 ± 6 181 ± 50 248 ± 21 +197 ± 18
SO2 1 3.0 ± 0.1 1.45 ± 0.1 0.23 ± 0.035 0.14 ± 0.01
4 (g L )
H2S (mg L1) – 0 35.5 ± 2.5 0
Cu (mg L1) 94.1 ± 3 0.135 ± 0.015 <0.004 <0.004
Fe (mg L1) 959.4 ± 10 144.1 ± 12.34 5.15 ± 2.45 1.2 ± 0.6
NHþ 1
4 (mg L )
0 50.1 ± 4.3 18.35 ± 1.55 1.1 ± 0.8
NO 1
3 (mg L ) 0 – – 6.55 ± 2.06
PO3 1 0 28.7 ± 5.8 24.1 ± 2.6 1.47 ± 0.12
4 (mg L )
COD (mg L1) – 9080 ± 360 8825 ± 415 29.5 ± 5.5
412 S. Bratkova et al. / Bioresource Technology 137 (2013) 409–413
Table 2
Cation exchange capacity, exchangeable cations and organic carbon in zeolite.
Type of the Ca2+, meq Mg2+, meq K+, meq Na+, meq
clinoptilolite 100 g1 100 g1 100 g1 100 g1
Natural 43.20 1.60 23.02 15.22
Saturated 15.70 4.40 28.77 3.81
After treatment 11.40 7.0 21.74 50.02
ther propionate, 1,3-propanediol and 3-hydroxypropionic acid,
which are the other possible products from the microbial metabo-
lism under these conditions.
Dar et al. (2008) reported that at the electron donor limitation,
lactate oxidation was mainly carried out by incompletely oxidizing
SRB, which formed 80–85% of the total bacterial population. In the
near absence of sulfate acetogens and methanogenic archaea were
the dominant microbial communities. Choi and Rim (1991) also re-
ported that at COD/SO2 4 ratios exceeding 2.7 methanogenic ar-
chaea can compete with SRB for hydrogen and acetate. In present
study experimental COD/SO2 4 ratio was in the range 3.4–3.5 but
the gas production was not observed in the system.
3.2. Treatment of waters polluted with heavy metals at TOC/SO2
4 ratio
of 1.1
Initially, the acid solution containing Fe and Cu was treated by
the laboratory installation at sulfate volume loading rate
0.176 g L1 h1. Although that highest rate of sulfate-reduction
was achieved at these conditions in previous studies, it was found
a high concentration of ferrous ions (144.1 ± 12.34 mg L1) in the
effluent (Table 1). Moreover, the effluents pH was 6.14 ± 0.11 –
the values close to those in which the process collapses. The heavy
metals ions in the solution also are toxic for microorganisms
including SRB, because of their ability to deactivate enzymes and
to denature proteins.
Effective removal of Fe and Cu was achieved at sulfate volume
loading rate 0.088 g L1 h1 (Table 1). In this mode (HRT 34.5 h),
the efficiency of the sulfate-reduction is 91 ± 0.7%. Due to the buf-
fering effect of the bicarbonate ions, the pH of the solution is
7.3 ± 0.05. Under these conditions, sulfide speciation equilibrium
tends toward HS- ions and therefore, the precipitation of ferrous
and cupric ions as metal sulfides is enhanced.
The efficiency of removal of COD in high organic loading fixed-
bed bioreactor was in the range 12–19% because of the incomplete
oxidation of organic compounds. The values of COD, hydrogen sul-
fide, ammonium and phosphates decreased below the permeable
level for water intended for use in the agriculture and/or industry
by adding of second bioreactor in technological scheme (Table 1).
The research of Hosseini et al. (2013) also demonstrated high
capability of the developed process for removal of COD (more than
92%) using integrated anaerobic/aerobic fixed-bed sequencing batch
biofilm reactor. The high HRT (over 5 days) allows effectively to carry
out the processes nitrification and denitrification. Probably nitrifica-
tion occurs in both (the inner and in the outer) parts of the three-sec-
tional reactor. Gao et al. (2012) constructed the possible nitrogen
removal model of anammox-denitrifying system. Their results indi-
cate that the coexistence of anammox and denitrifying bacteria can
improve the environmental adaptability of anammox process. It is
possible to carried out of anammox reaction combined with hetero-
trophic denitrification in anoxic zone of the three-sectional reactor.
3.3. Changes in exchangeable cations and amount of organic carbon of
zeolite used as carrier in the anaerobic bioreactor
The mineral zeolite was chosen as carrier because of its impor-
tant properties (an excellent attachment surface for microorgan-
R cations, meq By ammonium ions sorbed, meq Organic carbon, mg
100 g1 100 g1 100 g1
83.04 70.55 199.91
52.68 104.20 198.31
90.14 106.9 255.42
isms and the high cation-exchange capacity (CEC)). Results from
our previous study (data is not published) shown that formation
of microbial biofilm on natural zeolite is delayed with a month be-
cause of the high capacity of zeolite to adsorb NHþ4 from the nutri-
ent medium. The saturation of the natural zeolite with solution
containing NH4Cl, K2HPO4 and MgSO47H2O has as a result increas-
2+
ing of NHþ 4 , Mg and K+ content at the expense of Ca2+ and Na+
(Table 2).
These changes of exchangeable cations in the zeolite are favor-
able for microbial growth because of the presence of available bio-
genic elements in solution. A significant change (increasing of the
sodium) was found in exchangeable cations in zeolite after perfor-
mance of 335 days as a result of chemical composition of polluted
water. The increase of the organic carbon concentrations from
198.31 to 255.42 mg 100 g1 is an evidence for the formation of
microbial biofilm.
There are some studies for different applications of zeolites in
wastewater treatment. Ion exchange properties of zeolite have
been found to provide more favorable environment for nitrifying
microorganisms (He et al., 2007). Stable microbial communities
also were established in a multimedia aerated biofilter containing
clinoptilolite media in treatment of a synthetic glucose-containing
wastewater (Ji et al., 2010).
4. Conclusions
An efficient removal of heavy metals Fe and Cu was achieved by
fixed-bed bioreactor at high organic loading (TOC/SO2 4 ratio of
+ +
1.1). The saturated zeolite with NHþ4 , K and Mg2 is suitable solid
substrate for immobilization of SRB. The maximal rate of sulfate-
reduction was found at 37 °C and pH 7.25. The rate the process
of was greatly reduced at pH below 5.7.
The design of the second bioreactor allows efficient removal of
residual organic compounds, H2S, N and P from wastewater. It can
be proposed for an efficient strategy for treatment of effluents from
sulfidogenic bioreactors.
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