You are on page 1of 10

Chemosphere 153 (2016) 244e253

Contents lists available at ScienceDirect

journal homepage:

Biochemical passive reactors for treatment of acid mine drainage:

Effect of hydraulic retention time on changes in efciency,
composition of reactive mixture, and microbial activity
Yaneth Vasquez a, b, Maria C. Escobar b, Carmen M. Neculita c, Ziv Arbeli b, Fabio Roldan b, *
a , Colombia
Departamento de Ciencias Naturales, Universidad Central, Cra. 5 No. 21-38, Bogota
Unidad de Saneamiento y Biotecnologa Ambiental (USBA), Departamento de Biologia, Ponticia Universidad Javeriana, Cra. 7 No. 40-62, Bogota,
Canada Research Chair on Passive Treatment of Contaminated Mine Water, Research Institute on Mines and Environment (RIME), University of Quebec in
Abitibi-Temiscamingue (UQAT), 445 Boulevard de l'Universite, Rouyn-Noranda, QC J9X 5E4, Canada

h i g h l i g h t s

 Biochemical passive treatment represents a sustainable approach for treating acid mine drainage.
 Hydraulic retention times had a strong inuence in the sulfate-reducing bacteria and dehydrogenase activity.
 Long hydraulic retention time (4 d) generated higher alkalinity and excess residual suldes in the efuent.
 The hydraulic retention time strongly inuenced performance of biochemical passive reactors.

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

Article history: Biochemical passive treatment represents a promising option for the remediation of acid mine drainage.
Received 4 August 2015 This study determined the effect of three hydraulic retention times (1, 2, and 4 days) on changes in
Received in revised form system efciency, reactive mixture, and microbial activity in bioreactors under upward ow conditions.
11 March 2016
Bioreactors were sacriced in the weeks 8, 17 and 36, and the reactive mixture was sampled at the
Accepted 12 March 2016
Available online 25 March 2016
bottom, middle, and top layers. Physicochemical analyses were performed on reactive mixture post-
treatment and correlated with sulfate-reducing bacteria and cellulolytic and dehydrogenase activity.
rg E. Drewes
Handling Editor: Jo All hydraulic retention times were efcient at increasing pH and alkalinity and removing sulfate (>60%)
and metals (85e99% for Fe2 and 70e100% for Zn2), except for Mn2. The longest hydraulic retention
Keywords: time (4 days) increased residual suldes, deteriorated the quality of treated efuent and negatively
Acid mine drainage (AMD) impacted sulfate-reducing bacteria. Shortest hydraulic retention time (1 day) washed out biomass and
Sulfate-reducing bacteria (SRB) increased input of dissolved oxygen in the reactors, leading to higher redox potential and decreasing
Metal removal metal removal efciency. Concentrations of iron, zinc and metal suldes were high in the bottom layer,
especially with 2 day of hydraulic retention time. Sulfate-reducing bacteria, cellulolytic and dehydro-
Hydraulic retention time (HRT)
genase activity were higher in the middle layer at 4 days of hydraulic retention time. Hydraulic retention
Biochemical passive reactor (BPR)
time had a strong inuence on overall performance of passive reactors.
2016 Elsevier Ltd. All rights reserved.

1. Introduction from prolonged exposure of sulde ores to air and water

(Nordstrom et al., 2015). First, there is formation of sulfuric acid and
The major type of contaminated efuent generated by the progressive acidication of water, which then dissolves metals in
mining industry is acid mine drainage (AMD), which originates the rock, resulting in low pH and high metal and sulfate loading.
These characteristics make AMD responsible for physical, chemical,
and biological degradation of aquatic habitats and surrounding soil
* Corresponding author. quality (Adams et al., 2014).
E-mail address: (F. Roldan).
0045-6535/ 2016 Elsevier Ltd. All rights reserved.
Y. Vasquez et al. / Chemosphere 153 (2016) 244e253 245

A sustainable approach to remediate AMD-impacted water is 2. Materials and methods

to use biochemical passive reactors (BPRs), which are columns
lled with mixtures of organic and inorganic substrates, referred 2.1. Passive reactors: set-up, operation, and sampling
to as reactive mixture. BPRs have potential advantages, such as
low costs, ability to work in remote areas, use of recycled or waste Seven 5 L laboratory reactors were constructed using acrylic
organic materials, and natural appearance (Adams et al., 2014). columns (73  10 cm) and acrylic caps at both ends. The bottom cap
Because diverse contaminants, specic environments, and locally contained a valve used as an inuent port for AMD, and the top cap
available substrates require appropriate design of BPRs, labora- contained a double valve: one for collecting treated AMD and
tory testing is still necessary (Gusek, 2008). This is usually per- another for instantaneous sampling of efuent. The columns were
formed through testing in columns to determine the effectiveness packed, using the procedure described by Neculita et al. (2008a)
of the treatment and select essential design parameters, and Song et al. (2012). Briey, each column was lled with the
including hydraulic retention time (HRT) and the composition of same reactive mixture, consisting of 15% cow manure, 10% mush-
reactive mixtures (Adams et al., 2014). The HRT has a direct in- room compost, 25% sajo sawdust, 15% gravel, 20% limestone, and
uence on the performance of BPRs (Younger et al., 2002; 15% wetland sediment as inoculum (dry weight in % w/w); this was
Neculita et al., 2008a). Flow velocity, which determines the slightly compacted in layers of 10 cm. This reactive mixture (com-
HRT, plays an important role in the overall activity of microor- ponents and proportions) was selected because was found highly
ganisms in the reactors. Very short HRT may not allow adequate efcient for treating synthetic AMD in batch testing (Vasquez et al.,
time for bacteria activity to neutralize acidity and precipitate 2016). The reactive mixture was sandwiched in the reactor between
metals or may result in biomass being washed out of the reactor two porous plates coated with a ne geotextile (25 mesh), with a
(Neculita et al., 2007). Very long HRT may deplete the substrate or 7 cm layer of gravel (0.6e1.0 cm) at the bottom inuent port and a
built up bacterial growth and mineral precipitates that eventually 3 cm layer of gravel at the top efuent port to prevent loss of
lead to low hydraulic conductivity and short-circuiting of the organic matter and clogging. Finally, the columns were sealed and
treatment system (Younger et al., 2002). saturated with synthetic AMD prepared according to the maximum
To date, the effect of the HRT on the efciency of BPRs has been concentrations for metals and sulfate found in real efuent quality
studied with a focus on metal-removing efciencies, mainly in data found at ve active mine sites in the Zipaquira  Mining District,
iron-rich AMD (Chang et al., 2000; Neculita et al., 2008a). Perfor- Colombia (Vasquez et al., 2016). The synthetic AMD, which was
mance of these systems in the treatment of AMD, characterized by prepared weekly (Zagury et al., 2006), had the following compo-
high sulfate concentrations with low metal loading, has not yet sition (in mg L1): 201 44 Fe2; 30 2 Mn2; 19 2 Zn2;
been addressed. 215 11 Ca2; 128 13 Mg2 and 2500 105 SO2 4 , at pH 3.0e3.7.
The key parameter in an efcient BPR is the activity of sulfate- After the packing procedure, the columns were wrapped in
reducing bacteria (SRB), because they reduce sulfates to suldes, aluminum foil to exclude light and prevent growth of phototrophic
which then react with dissolved heavy metals to form solid metal bacteria (Waybrant et al., 2002; Neculita et al., 2008a). The columns
suldes that are immobilized in the reactor. Although SRB meta- remained at room temperature in the dark for two weeks. During
bolism conducts the sulfate reduction reaction, these bacteria rely this acclimation period, 20 mL samples were collected twice
on activity of anaerobic cellulolytic and fermentative bacteria to weekly, from the top port and tested until favorable conditions for
break down complex organic materials, such as cellulose, to pro- SRB (pH 5e8, ORP < 100 mV) prevailed (Postgate, 1984).
vide them with available carbon and energy sources (Pruden et al., Following acclimation, continuous upward ow of synthetic AMD
2007). Therefore, the cellulose and dehydrogenase enzymes can be was started, with three columns at a ow rate of 0.74 mL min1 (for
used as indicators of general microbial activity in anaerobic systems 2-day HRT), and four columns at a ow rate of 0.37 mL min1 (for 4-
(Serra-Wittling et al., 1995). However, there are limited data day HRT). Continuous ow was maintained for 36 weeks, using
available related to the changes in microbial activity under different precision peristaltic pumps (Masterex L/S, Cole-Parmer, Vernon
HRTs in the long-term operation of BPR, under laboratory Hills, IL), which were calibrated prior to starting the tests. After 17
conditions. weeks, a strong increase of soluble sulde concentrations in treated
The composition of reactive mixture changes during the life of efuents from the columns with 4-day HRT was recorded. These
BPRs by precipitation of oxy-hydroxide, carbonate, and sulde results justied the decision to change one of the reactors from 4-
minerals (Neculita et al., 2007). Metals extracted during the acid- day HRT to 1-day HRT, at a ow rate of 1.48 mL min1. Columns
volatile sulde (AVS) process are known as simultaneously were sacriced throughout the study to monitor the changes in the
extracted metals (SEM) and the molar ratios of both (i.e. SEM/AVS) post-treatment reactive mixture and microbial activity. Four col-
indicate which are the predominant metal removal mechanisms. umns, two of 2-day HRT and two of 4-day HRT, were sacriced at
When this ratio is  1.0, it indicates that metals are mainly present weeks 8 and 17. The other three columns (one each of 1, 2, and 4-
as solids in the form of sulde minerals, whereas at ratio > 1.0, the day HRT) were analyzed at the end of the study (36 weeks). The
oxy-hydroxide and carbonate minerals are predominant (Di Toro reactive mixture from the sacriced columns was removed in three
et al., 2005). Although the SEM/AVS molar ratio can provide valu- cores (20  10 cm), one from the bottom (at 0e20 cm), one from the
able information on reactor efciency and metal removal mecha- middle (at 20e40 cm), and one from the top (at 40e60 cm). Each
nisms in AMD, limited data is available about the inuence of HRT sample was homogenized and refrigerated at 4  C until analyzed.
on the potential mobility of metals in the reactive mixture evalu-
ated after sacricing the reactor (post-treatment) (Neculita et al., 2.2. Effect of the hydraulic retention time on efciency of
2008b). biochemical passive reactors
The main objective of this study was to measure the effect
(temporal and spatial) of HRT (1, 2 and 4 days) on the efciency of To determine the effects of HRT on the efciency of reactors for
BPRs for the treatment of sulfate-rich AMD. In addition, the cor- increasing pH and alkalinity, as well as promoting reduction of
relation between microbial activity (cellulolytic and dehydroge- sulfates and metals during treatment of AMD, 50 mL efuent was
nase) and physicochemical characteristics of the reactive mixture collected with a syringe each week from the valve located at the top
after treatments was determined. cap of each column. The pH and redox potential (ORP) were
immediately measured with a multi-parameter probe (HI 9828,
246 Y. Vasquez et al. / Chemosphere 153 (2016) 244e253

Hanna Instruments; Woonsocket, RI). Alkalinity was also deter- cooling to room temperature, the supernatant was diluted to 50 mL
mined (TitroLine alpha plus 20, Schott, Mainz, Germany) by using with de-ionized water and ltered (0.45 mm; Sartorius). Dissolved
method 2320B (APHA, 2005). The remaining sample was ltered metals (Fe2, Mn2, and Zn2) were then quantied by atomic
(0.45 mm, Sartorius, Goettingen, Germany) before sulfate analysis absorption spectrometry. Removal mechanisms for metals were
by UVeVIS spectrophotometry (Genesys 10, Thermo Scientic; assessed with the SEM/AVS procedure, as modied by Neculita et al.
Waltham, MA) using method 4500eSO4 (APHA, 2005) and sulde (2008b).
analysis using the methylene blue method (4500 D; APHA, 2005).
Dissolved metals (Fe2, Mn2 and Zn2) were quantied by atomic 2.4. Effect of hydraulic retention time on changes in microbial
absorption spectrometry (Varian 240 FS; Agilent Technologies; activity
Santa Clara, CA), using method 7000B (USEPA, 2001), with a
detection limit ranging from 0.03 to 1.0 mg L1. Once a month, Dehydrogenase and cellulolytic activity were measured, ac-
dissolved organic carbon (DOC) was analyzed, using high temper- cording to Serra-Wittling et al. (1995). Microbial activity was tested
ature combustion in method 5310B (APHA, 2005), in an external in triplicate with 1.0 g reactive mixture that was air-dried for 24 h
accredited laboratory. and sieved (mesh No. 50, 0.297 mm). For dehydrogenase analysis,
To identify the potential precipitation mechanisms responsible samples were incubated in darkness for 24 h with 4 mL 1% 2,3,5-
for changes in metal concentrations, saturation indices (SI) were triphenyl-tetrazolium chloride at 30  C, using this chemical as the
calculated, using the geochemical modeling software Visual MIN- electron acceptor and reduced to triphenyltetrazolium formazan
TEQ 3.0 (Gustafsson, 2011) on the treated efuent collected from (TPF; displayed as red color), which was extracted with methanol
some representative phases of treated water. This modeling soft- (10 mL) and quantied by UVeVIS spectrophotometry. For
ware considers solution chemistry and equilibrium distribution of measuring cellulolytic activity, the sample was incubated at 30  C
chemical species according to geochemical processes, such as for 24 h with 3.5 mL 0.2 M acetate buffer and 3.5 mL 1% carbox-
dissolution/precipitation, oxidation/reduction, complexation, and ymethyl cellulose as the substrate. The Antrone method was used
ion exchange. However, VMINTEQ does not consider SRB activity to quantify total soluble carbohydrates (Dische, 1962).
and precipitation of biogenic suldes (Waybrant et al., 2002;
Zagury et al., 2006). 2.5. Statistical analysis
SRB counts were determined once a month, using the most
probable number technique, method D4412e84 (ASTM, 2009). Statistical analysis were based on SPSS Statistics 19 (IBM,
Formation of black precipitates (after 25 of days inoculation) was Armonk, NY), using Pearson correlation between physicochemical
interpreted as generation of iron sulde with SRB activity. When variables and/or microbial activity.
the black color was not obvious, the presence of suldes was
conrmed by a blue color after adding a few drops of 0.005 M ferric 3. Results and discussion
chloride hexahydrate and 0.005 M N-N-dimethyl-p-phenylenedi-
amine dihydrochloride. 3.1. Effect of hydraulic retention time on changes in efciency

2.3. Effect of the hydraulic retention time on changes in During the initial acclimation period, pH increased from
composition of the reactive mixture 3.0 0.8 to 7.0 0.5 in efuent from all reactors, regardless of the
HRT. This increase was associated with increased alkalinity,
Samples of reactive mixtures (n 21) were collected from the 1500 54 mg CaCO3 L1 for 2-day HRTs and 2400 32 mg
several layers in the bioreactors sacriced at weeks 8, 17, and 36. CaCO3 L1 at 4-day HRTs (Fig. 1). Increased alkalinity mostly
Physicochemical analyses were carried out in triplicate. Easily resulted from the dissolution of limestone in the reactive mixture
available substances (EAS), organic nitrogen and total organic car- and occurred within the rst several hours. After the beginning of
bon (TOC) were tested on air-dried and sieved (mesh No 70, continuous-ow operations, the pH remained relatively constant,
0.210 mm) samples. The pH was measured (Lab 870, Schott; Mainz, in the range of 6.5e7.0, and alkalinity decreased to 800 28 mg
Germany) using method 4972e01 (ASTM, 1995a) and a ratio of CaCO3 L1 for 2-day HRT and 1200 60 mg CaCO3 L1 for 4-day
solid to de-ionized water of 1:1. Moisture was determined by dif- HRT. Thereafter, alkalinity was relatively constant at both HRTs
ference in weight between wet samples and samples dried at 105  C until there was a strong increase in the 4-day HRT starting at 21
for 20 h (Method D 2216e98; ASTM, 1995b). EAS were analyzed by week until the end of the study (Fig. 1). In previous studies, higher
forage ber analysis, according to Prasad et al. (1999). The organic alkalinity in the reactor was associated with formation of carbonate
nitrogen was measured by Method 4500eNorg (APHA, 2005), and species through dissolution of limestone from reactive mixtures or
cellulose content was determined, according to Harper and Lynch oxidation of readily available organic substrates by SRB, as well as
(1981). The organic carbon was analyzed by the WalkleyeBlack from dissolution of surface-bound hydroxyl ions in the substrate
method (Schumacher, 2002). To ensure complete digestion of materials under acidic conditions (Neculita et al., 2008a; Robinson-
extraction mixtures, 5 mL 1 N K2Cr2O7, 10 mL H2SO4 (Analytical- Lora and Brennan, 2011; Song et al., 2012).
grade), and 0.2 g reactive mixture were gently boiled at 140  C for The reactors developed reducing conditions during the accli-
30 min, cooled overnight, ltered (0.45 mm; Sartorius). Cr3 was mation period (Fig. 1). The ORP initially decreased to 260 mV and
quantied by UVeVIS spectrophotometry (Genesys 10, Thermo declined to 360 mV by the fourth week. Afterwards, the ORP
Scientic; Waltham, MA). remained relatively constant, without signicant difference be-
Total metal concentrations immobilized in the reactive mixtures tween the 2- and 4-day HRTs, until the end of the study. These
were determined by the digestion method described by Neculita results indicate that the acclimation period was necessary and
et al. (2008b), where 0.5 g samples were weighed in 50 mL poly- important for establishing an appropriate geochemical environ-
propylene centrifuge tubes, 10 mL concentrated HCl was added to ment for anaerobic bacteria, including the SRB. Then, during the
react for 24 h at room temperature. Then, 15 mL HNO3 was added continuous ow, higher pH and alkalinity together with lower ORP
and the tubes were heated in a water bath at 75 3  C for 50 min, led to a more rapid development of SRB, favoring the consumption
followed by adding 10 mL HClO4 and 1 mL HF (analytical-grade). of readily available organic substrates, especially at longer HRT.
The samples were heated in a water bath at 75 3  C for 2.5 h. After Sulfate concentration in synthetic AMD, used to saturate the
Y. Vasquez et al. / Chemosphere 153 (2016) 244e253 247

columns during the set-up, was 2500 170 mg L1; however two concentration of suldes was usually higher in the reactors under
weeks later, the sulfate concentration in efuents increased to as 4-day HRT because longer residence time allowed greater oxidation
much as 2800 32 mg L1 for 2-day and 3382 31 mg L1 for 4- of available organic carbon and reduction of sulfate by SRB in the
day HRT (Fig. 2). This was caused by leaching from the organic efuents. Sulde concentrations signicantly increased at the 2-
substrate (cow manure and mushroom compost) in the reactive day HRT (1952 52 mg H2S L1) and 4-day HRT (2826 185 mg
mixture, as observed during batch testing (Vasquez et al., 2016). H2S L1) after week 16. The increase in concentrations of suldes is
Release of sulfate (27.5 g kg1) from organic substrates, especially a response to the excessive sulfate added in AMD in the week 15
manures and compost, was previously reported (Song et al., 2012). and the growth of SRB that removed the sulfate leached from the
After the beginning of continuous-ow, sulfate declined to organic substrates. This eventually created a sharp decline in SRB
1300 32 mg/L for 2-day and 1000 50 mg/L for 4-day HRT, and counts between weeks 12 and 20 (Fig. 2). Sa ez-Navarrete et al.
remained relatively constant in both treatments until week 16. In (2009) found that soluble suldes (H2S or HSand S2) reduce
following weeks, the sulfate increased to 1700 98 mg/L, probably cell size and growth, when present in concentrations > 108 mg
due to the reduction of SRB counts caused by increased soluble H2S L1. Okabe et al. (2003) found that cell production fell slightly
suldes until 1952 52 mg H2S L1 for 2-day and 2826 185 mg at a concentration of 100 mg H2S L1, whereas at 180 mg H2S L1,
H2S L1 for 4-day HRT. Between weeks 23 and 36, sulfate removal the rate of growth declined sharply. Concentrations of sulde
increased, higher in the 4-day HRT columns. There was no differ- decreased in 2-day HRT reactors (357 12 mg H2S L1) and in 4-day
ence between 1-day and 2-day HRT. HRT reactors (1822 67 mg H2S L1) after week 24. These changes
The concentration of DOC at the beginning of continuous-ow favored growth of the SRB in 2-day HRT reactors. Suldes in re-
operations was 740 mg L1, but decreased to ~10 mg L1 by week actors with 4-day HRT remained high (1066 19 mg H2S L1),
8 and remained nearly constant until the end of the study. This whereas in reactors with 2-day HRT were reduced to 218 37 mg
pattern was similar for the three HRTs tested (Fig. 2). The reactive H2S L1, at the end of the study. With these results, after week 17,
substrate released DOC at a nearly constant rate, which was avail- one of the 4-day HRT reactors was changed to a 1-day HRT reactor.
able for the microorganisms in the reactors to reduce sulfates. As expected, after the change, alkalinity (Fig. 1) decreased from 767
These results are consistent with Song et al. (2012) who observed to 510 mg CaCO3 L1 and remained relatively constant until week
that substrates with high initial DOC is likely to leach a considerable 36. This change did not affect pH. However, there was an effect on
amount of DOC to the efuent, especially at low pH condition, the ORP, which increased rapidly from 375 to 108 mV from the
which is not available for microbial growth. dissolved oxygen (DO) in the AMD. The new ow released a large
Suldes were detected by week 4, with signicant differences amount of sulfate (3115 mg L1) (Fig. 2), probably retained in the
between the 2-day and 4-day HRTs (Fig. 2). As expected, the reactive mixture as gypsum (CaSO4$2H2O). Another source of

Fig. 1. Changes in pH, oxide reduction potential (ORP), and alkalinity during treatment of acid mine drainage (AMD) in biochemical passive bioreactors (BPR) over 36 weeks. In week
17, one column of 4 days hydraulic retention time (HRT) treatment was changed to 1 day HRT treatment.
248 Y. Vasquez et al. / Chemosphere 153 (2016) 244e253

Fig. 2. Changes in dissolved organic carbon (DOC), sulfate, suldes, and sulfate-reducing bacteria (SRB) during the treatment of acid mine drainage (AMD) in biochemical passive
bioreactors (BPR) over 36 weeks. In week 17, one column of 4 days hydraulic retention time (HRT) treatment was changed to 1 day HRT.

residual sulfate may have come from re-oxidation of suldes that 23. The major contributor to ORP is DO and other redox active
were previously precipitated in the reactor (Dvorak et al., 1992). species, such as sulfate and Fe2. The reactions that remove metals
From weeks 26 through 33, the reactor stabilized and ORP in reactors are highly sensitive to redox conditions and are
decreased from 189 mV to 270 mV. In the last three weeks of the reversible (Cheong et al., 2010).
experiment, the ORP increased; this affected the efciency of sul- During the acclimation period, Mn2 could be removed by
fate reduction. Excessive production of suldes declined at week 20 sorption on fresh organic substrate and precipitation as carbonate
and decreased to 23 4 mg H2S L1 at week 36 (Fig. 2). The increase (MnCO3 e rhodochrosite). Lewis (2010) found that adsorption on
in ow and the consequent increased loading rate of oxygen organic substrates is the predominant mechanism that provides
affected anaerobic conditions and subsequent capacity of SRB to some Mn2 removal at this phase. After the start of continuous-
produce soluble suldes. Increased ow also washed out the SRB in ow operations, Mn2 in efuent was 77 4 mg L1 for 4-day
the reactive mixture (Fig. 2), which led to clogging the efuent HRT reactors and 60 2 mg L1 for 2-day HRT reactors, which
tubing with leached biomass and metal sulde precipitates. Once exceeded levels in the synthetic AMD (31 mg L1). By week 21,
the tubing was replaced, outow was slowly re-established. Mn2 concentrations decreased again (efciencies up to 60% were
Removal of Fe2 and Zn2 in the AMD was observed during the measured), without signicant differences between the three
36 weeks of operation of the reactors, without signicant differ- retention times. Removal of Mn2 is tied to increasing alkalinity, a
ences between 2- and 4-day HRTs (Fig. 3). During the start-up and condition present in all reactors. Relatively high alkalinity is usually
acclimation, metals could be removed by precipitation in the form necessary to ensure appropriate abiotic oxidation rates of Mn at
of hydroxides and carbonates, as well as by sorption on fresh pH < 8 (Mariner et al., 2008). Low removal of Mn by bioreactors
organic substrate, with respect to the pH of pore water. Then, results from high solubility of MnS relative to other metal suldes
during continuous-ow operation, at steady-state condition, metal (Cheong et al., 2010), and the inhibition of its precipitation as hy-
removal might be partially explained by the formation of sulde droxides if the concentrations of Fe2 or Zn2 are high (Yoo et al.,
minerals (Zhang and Wang, 2014). Removing metal is generally 2004). Mn2 has been acknowledged as the most challenging
closely related to the ORP. In the reactor changed to 1-day HRT, metal to remove in bioreactors under reducing conditions (Chang
efciency of removing Fe2 and Zn2 decreased (75% and 67%, et al., 2000; Neculita et al., 2007; Robinson-Lora and Brennan,
respectively) when ORP increased from 360 to 108 mV at week 2011). In the present study, after removal of other metals in AMD,
Y. Vasquez et al. / Chemosphere 153 (2016) 244e253 249

Fig. 3. Metals removal (Fe2, Mn2, and Zn2) during the treatment of acid mine drainage (AMD) in biochemical passive bioreactors (BPR) over 36 weeks. In week 17, one column of
4 days hydraulic retention time (HRT) treatment was changed to 1 day HRT.

Mn removal as carbonates was probably favored by the presence of 3.2. Effect of hydraulic retention time on changes in composition of
high alkalinity; this coincides with the results from Visual MINTEQ the reactive mixture
modeling which did not indicate the formation of MnS.
The SI of the potential minerals in the reactors was calculated by The analysis of the reactive mixture at weeks 8 and 17 showed
VMINTEQ modeling, using some representative phases of water pH > 8.0, with similar trends in all layers (Table 1). At week 36, pH
quality. Sphalerite [(Zn, Fe)S] super saturation (SI > 1) over time decreased, especially in the bottom layers (<6.3), probably from
was the highest in the column with 1-day HRT. The results also depletion of neutralizing agents caused by constant AMD input and
indicated that Zn concentrations could be controlled by the solu- upward ow in the reactors. The decline in pH in the reactors did
bility of a zinc sulde phase. The SI of calcite (CaCO3) indicated that not affect activity of the SRB, which requires a pH 5.0e8.0 range for
this mineral could be higher at the beginning of continuous ow, optimal growth (Postgate, 1984).
when alkalinity increased. Mackinawite (FeS) and siderite (FeCO3) The biodegradable portion of the substrate, composed of soluble
were probably present in the efuents of the reactors with 1- and sugars, amino acids, starch, certain proteins, and hemicellulose
4-day HRTs. At the end of the study, rhodochrosite supersaturation (Zagury et al., 2006) rapidly decreased in the rst 8 weeks (Table 1)
probably occurred in reactors with 1-day HRT. because it is the soluble fraction that is readily used by microor-
HRT strongly affected changes in the efciency of reactors ganisms. The EAS increased at weeks 17 and 36, and showed a
treating sulfate-rich AMD. 4-day HRT, with low up ow velocity, negative correlation with cellulose content (0.672) and organic
favored growth and activity of SRB. As a result, high alkalinity and nitrogen (0.754;p < 0.01). The nitrogen was more greatly reduced in
excess sulde concentrations could leave the reactor, leading to reactors under 4-day HRT, which may be related to the higher count
toxicity of the efuent. However, excessive sulde content from of SRB in the reactive mixture after treatment at week 36. The
biological sulfate reduction can result in physical and biological concentrations of organic nitrogen were positively correlated with
inhibition of SRB and eventually contributes to failure of the total organic carbon (0.764; p < 0.01), increasing the C:N ratio.
reactor. 1-day HRT more rapidly washes out SRB and introduces Available carbon and nitrogen are among the very important fac-
more DO in the reactor, leading to increased ORP. Anaerobic con- tors affecting the treatment of AMD with SRB (Waybrant et al.,
ditions are disrupted, which adversely affects SRB activity. As a 2002; Zagury et al., 2006).
result, increased ORP reduced removal of metal by up to 30%. Metal (Fe and Zn) in the reactive mixtures were concentrated in
the bottom layer of all reactors, especially in reactors with 2-day
250 Y. Vasquez et al. / Chemosphere 153 (2016) 244e253

Table 1
Physicochemical and microbiological characterization of the reactive mixture post-treatment in biochemical passive reactors.

Week HRT (d) Sample location pH % w/w (dw) SRB  103 (cells/100 mL)

EAS TKN COT Cellulose

Reactive mixture 6.3 0.1 51.8 0.6 7.1 0.1 60.8 1.3 60.9 0.25 7.00
2 Top 8.1 0.6 27.0 0.5 6.0 0.2 34.5 2.1 55.8 2.5 6.0
Middle 8.3 0.1 30.0 0.1 6.6 0.1 33.8 1.6 52.0 0.8 20.0
8 Bottom 8.1 0.4 23.1 0.1 6.0 0.1 32.7 0.8 50.7 1.4 89.1
4 Top 8.5 0.2 30.8 1.2 5.3 0.5 35.5 1.0 56.0 0.2 8.3
Middle 8.5 0.1 26.3 0.5 5.6 0.2 37.1 1.1 51.1 0.4 14.5
Bottom 8.3 0.1 28.3 0.6 5.4 0.2 35.4 1.6 59.4 0.6 122.0
2 Top 7.5 0.5 62.6 5.6 5.6 0.8 31.3 1.2 43.5 0.3 2.0
Middle 7.3 0.9 64.2 0.4 5.3 0.2 29.9 2.1 45.8 1.6 3.7
Bottom 7.5 0.2 63.7 4.0 5.1 0.2 30.2 1.0 42.0 1.8 1.9
4 Top 7.5 0.2 58.6 2.1 4.3 0.1 22.8 1.7 41.4 3.6 2.2
Middle 8.0 0.1 55.1 2.0 4.6 0.2 25.7 0.4 43.2 0.5 3.5
Bottom 8.0 0.1 60.0 0.6 4.3 0.1 23.3 1.9 40.2 1.5 2.5
2 Top 7.2 0.7 70.3 3.8 3.1 1.0 27.7 2.7 33.2 3.4 13.4
Middle 7.1 0.3 76.0 1.8 3.5 0.2 22.2 1.0 34.8 2.6 17.5
Bottom 6.3 0.1 70.4 4.2 3.9 0.2 22.4 4.1 29.4 2.1 13.7
36 4 Top 7.4 0.3 65.0 1.2 2.7 0.1 15.3 2.1 36.4 2.4 182.2
Middle 7.4 0.2 61.5 1.0 2.6 0.2 17.3 0.5 31.1 3.4 350.0
Bottom 6.5 0.1 64.5 1.9 2.1 0.1 16.3 0.6 26.5 1.9 99.2
1 Top 7.1 0.3 61.1 1.2 3.4 0.1 22.6 2.5 31.6 0.4 15.3
Middle 6.5 0.9 60.2 1.5 3.4 0.1 25.0 1.6 34.8 0.9 93
Bottom 5.2 0.3 62.6 1.3 3.5 0.3 18.9 3.9 19.2 1.4 109.0

HRT: hydraulic retention time; TKN: Total organic nitrogen; SRB: sulfate-reducing bacteria; EAS: easily available substances; TOC: total organic carbon.

HRT because this rate of ow treated more AMD in less time 3.3. Effect of hydraulic retention time on changes in microbial
(Table 2). Formation of black precipitate and accumulation of iron activity
oxy-hydroxide minerals with yellow-brown color were present in
the bottom layers. Similar results were found in reactive mixtures SRB counts, based on the layer in the reactors, indicated that the
after the operation period and dismantling of reactors (Zaluski middle layer of reactors at 4-day HRTs had as much as twice the
et al., 2003). High concentrations of Fe and Zn in reactive mix- level of 2-day HRT reactors. The maximum number of cultivable
tures after treatment indicated that the reactors were efcient in SRB after treatment in the reactive mixtures was 350  103 cells
removing these metals from the inuent AMD. 100 mL1 in the middle layer in the 4-day HRTs at 36 weeks
The concentration of Mn was similar in all layers. As already (Table 1). Appropriate conditions (ORP, sulfate concentrations,
discussed, this metal was removed only at the beginning of the organic carbon and pH > 5.0) in the 4-day HRT reactors favored
treatment, when it was probably adsorbed on the reactive mixture growth of bacteria, whereas metal suldes accumulated in the 2-
or precipitated as rhodochrosite. After exhaustion of the adsorption day HRT reactors adversely impacted SRB growth (Table 1). Metal
capacity of the reactive mixture, no additional removal of Mn was suldes concentrated in the vicinity of the bacterial cells and hinder
recorded, whereas Fe and Zn were still being removed, probably further metabolism by preventing contact between the reactants
partially as biogenic metal suldes. Sulfate concentrations (sulfate, organic matter) and necessary enzymes (Utgikar et al.,
increased in all reactive mixtures, with higher concentrations in the
bottom layers. Positive correlations with Fe (0.421), Zn (0.750), and
Mn (0.381) were determined (p < 0.01) and negative correlation Table 2
Total metal in reactive mixture post-treatment in biochemical passive reactors.
with pH (0.773; p < 0.01) is explained by AMD and upward ow in
the reactors. Week HRT (d) Sample location Total metal (mg/kg dw)
The interpretation of results from SEM/AVS molar ratios > 1.0 Fe Mn Zn
showed that precipitation as oxy-hydroxide and carbonate min-
2 Top 7.3 0.9 0.5 0.0 0.3 0.0
erals represents the predominant mechanism of removing metals Middle 12.0 2.8 0.4 0.1 0.3 0.0
at weeks 8 and 17 (Table 3). The SEM/AVS molar ratio was higher at 8 Bottom 29.0 2.5 0.1 0.0 0.7 0.1
week 8, when the SRB had not been completely established. Over 4 Top 7.4 0.4 0.6 0.0 0.2 0.0
the next several weeks, the molar ratio decreased, probably as Middle 15.7 1.5 0.5 0.0 0.2 0.0
Bottom 26.0 3.1 0.5 0.0 0.4 0.0
removal of metals as suldes started. Results from the SEM/AVS 2 Top 25.4 1.2 0.9 0.1 7.6 0.0
procedure also showed higher mobility of metals under acidic Middle 33.5 0.4 0.6 0.0 9.0 0.1
conditions. In the bottom layer of the reactor under 1-day HRT Bottom 85.7 2.0 0.6 0.1 22.5 0.0
treatment, the pH decreased to 5.2, causing movement of metals 4 Top 11.2 1.7 0.8 0.0 7.4 0.0
Middle 15.5 0.7 0.2 0.1 8.0 0.3
toward the middle layer and reduction of the SEM/AVS molar ratio.
Bottom 51.5 0.1 0.7 0.0 16.2 0.3
Statistical analysis showed a strong positive correlation between 2 Top 31.1 6.2 0.7 0.0 26.1 0.3
the SEM/AVS molar ratio and sulfates, Fe, and Zn (0.556, 0.682, and Middle 43.1 2.8 0.5 0.0 34.3 1.7
0.631, respectively; p < 0.01) over 36 weeks, suggesting that sul- Bottom 87.0 3.0 0.2 0.1 64.6 0.2
des produced from sulfates by reduction following SRB activity is 36 4 Top 32.5 0.0 0.6 0.0 20.1 0.6
Middle 38.0 1.0 0.9 0.1 19.7 2.1
an important mechanism for removing metals. The TOC was Bottom 61.1 2.8 0.3 0.1 33.3 1.7
negatively correlated with the SEM/AVS molar ratio (0.792; 1 Top 36.2 5.5 0.4 0.1 30.1 1.2
p < 0.01) as a major factor controlling adsorption of metal (metal- Middle 61.7 5.2 0.4 0.0 27.0 0.1
binding phases; Di Toro et al., 2005). Bottom 40.3 1.0 0.6 0.0 21.1 2.5
Y. Vasquez et al. / Chemosphere 153 (2016) 244e253 251

Table 3
The acid-volatile sulde (AVS), simultaneously extracted metals (SEM), and SEM/AVS molar ratios in reactive mixture post-treatment in biochemical passive reactors.

Week HRT (d) Sample location SEM (mg/kg, dw) AVS SEM/AVS (molar)

Fe Mn Zn (mg/kg, dw)

2 Top 0.1 0.0 0.1 0.0 0.2 0.0 0.2 0.0 4.8 0.1
Middle 0.1 0.0 0.1 0.0 0.1 0.0 0.2 0.0 4.8 0.1
Bottom 26.1 4.0 0.2 0.0 0.4 0.2 1.3 0.1 12 1.8
4 Top 0.7 0.1 0.2 0.0 0.1 0.0 0.2 0.0 4.8 0.1
Middle 0.8 0.1 0.2 0.0 0.1 0.0 0.2 0.0 4.8 0.1
Bottom 23.5 3.0 0.1 0.0 0.2 0.0 4.1 0.5 3.4 0.1
2 Top 6.7 1.3 0.1 0.0 6.6 0.0 3.7 0.1 2.8 0.3
Middle 14.1 0.7 0.6 0.0 7.0 0.1 3.9 0.1 3.1 0.2
17 Bottom 38.6 10.3 0.7 0.4 12.5 0.0 6.9 0.1 8.6 0.2
4 Top 5.0 0.8 0.9 0.0 7.4 0.0 1.9 0.3 3.0 0.0
Middle 5.7 2.2 0.6 0.0 7.0 0.3 3.7 0.3 2.4 0.6
Bottom 42.4 2.8 0.9 0.0 12.7 0.3 9.3 0.6 4.5 1.0
2 Top 6.3 2.0 0.4 0.0 27.7 0.2 5.4 0.1 1.3 0.2
Middle 35.2 0.4 0.5 0.0 31.8 0.4 5.7 0.3 3.3 0.9
Bottom 51.9 1.7 0.1 0.1 34.4 0.0 15.0 0.9 6.2 1.2
36 4 Top 1.6 0.7 0.6 0.0 20.0 0.0 5.8 0.3 0.7 0.1
Middle 5.9 0.1 0.5 0.1 20.1 0.3 5.0 0.3 2.2 0.0
Bottom 16.4 3.7 0.2 0.0 25.0 3.4 17.1 0.2 3.2 0.1
1 Top 13.9 1.9 0.3 0.0 31.3 0.2 3.2 0.5 3.4 0.1
Middle 32.4 1.0 0.3 0.0 25.2 0.0 3.5 0.0 6.3 0.0
Bottom 20.6 0.5 0.7 0.0 20.1 0.5 5.2 1.1 3.3 1.6

2002). Despite lower counts of SRB, microbial communities in the within the optimal range for hydrolytic fermenters, acetogenic
waste organic materials are diverse and this is advantageous in syntrophic bacteria, and SRB (5.0e8.0). Lower pH can cause
terms of resilience to stress (Hiibel et al., 2011). cessation of metabolic activity and result in an accumulation of
At weeks 8 and 36, SRB counts were consistent with dehydro- organic acids and potential process failure (Neculita et al., 2007).
genase activity. The 4-day HRT reactors had higher activity Interpretation of dehydrogenase activity is difcult because mi-
compared to 2-day HRT reactors (Fig 4). Dehydrogenase activity crobes are affected by many factors, including pH and chemical
was signicantly higher (p < 0.05) in the bottom layer of the 4-day composition of the reactive mixture (Serra-Wittling et al., 1995;
HRT reactors at week 8. At week 17, the second sacrice, there were Chu et al., 2007).
no signicant differences at any ow rate. At week 36, dehydro- Cellulolytic bacteria are very important for the overall perfor-
genase activity was greater in the middle layer of the 1-day HRT mance of anaerobic passive treatments because they provide labile
reactor. The changes in dehydrogenase activity depended of pH and organic carbon to the SRB (McCauley et al., 2009). Cellulolytic ac-
concentration of metal suldes in the reactive mixture determined tivity was not signicantly different (p < 0.05) among the three
after treatment. Concentration of Zn and dehydrogenase activity retention times, but signicant differences occurred at weeks 8, 17,
were negatively correlated in the bottom layer of all reactors at and 36. During the acclimation phase, cellulolytic activity was low
week 36 (0.332; p < 0.05). This metal inhibits enzymes by che- because the microorganisms consumed the starch, hemicellulose,
lation with sulfhydryl groups, which are primarily involved at the amino acids, some proteins, and soluble sugars in the matrix that
catalytically active sites of the enzymes (Mikanova, 2006). Enzyme are readily available (Zagury et al., 2006). At 17 and 36 weeks, EAS
activity may decrease because of physiological adaptations of bac- sources are scarce and the use of cellulose sources should be
teria under stress from heavy metals (Hu et al., 2014). A positive enhanced, thus promoting functional cellulolytic ability. No sig-
correlation (0.30; p < 0.05) occurred between dehydrogenase ac- nicant correlation was found between cellulolytic activity and pH,
tivity and pH in the bottom layer of the 1-day HRT reactor at the end although it has been reported that cellulose degradation can occur
of the treatments (Fig. 4). Passive reactors must maintain a pH after cessation of bacterial growth at a pH as low as 4.5 (optimal pH

Fig. 4. Dehydrogenase and cellulolytic activity. Whiskers indicate standard deviation. Asterisks indicate the presence of signicant differences (p < 0.05).
252 Y. Vasquez et al. / Chemosphere 153 (2016) 244e253

is 4.75; Lynd et al., 2002). reactors. Biotechnol. Bioeng. 40, 609e616.

Gusek, J.J., 2008. Passive treatment 101: an overview of the technologies. In: U.S.
EPA/National Groundwater Association's Remediation of Abandoned Mine,
4. Conclusions Lands, pp. 1e13. Denver, CO.
Gustafsson, J.P., 2011. Visual MINTEQ, v.3.0. Royal Institute of Technology, Depart-
ment of Land and Water Resources Engineering, Stockholm. http://www2.lwr.
Hydraulic retention time had a strong inuence in reactive
mixture composition and microbial activity during treating acid Harper, S.H.T., Lynch, J.M., 1981. The chemical components and decomposition of
mine drainage. Seven passive biochemical reactors used in the wheat straw leaves, internodes and nodes. J. Sci. Food Agric 32, 1057e1062.
present study were efcient for increasing pH and alkalinity and Hiibel, S., Pereyra, L., Riquelme, M.V., Reisman, D., Reardon, K., Pruden, A., 2011.
Effect of organic substrate on the microbial community structure in pilot-scale
removing sulfate (>60%) and metals (85e99% for Fe2 and 70e100% sulfate-reducing biochemical reactors treating mine drainage. Environ. Eng. Sci.
for Zn2), without signicant differences between the three 28, 563e572.
retention times. The 4-day HRT generated higher alkalinity and Hu, X.F., Jiang, Y., Shu, Y., Hu, X., Liu, L., Luo, F., 2014. Effects of mining wastewater
discharges on heavy metal pollution and soil enzyme activity of the paddy
excess residual suldes in the efuent. The 1-day HRT washed out elds. J. Geochem. Explor. 147, 139e150.
biomass and increased input of dissolved oxygen in the reactors, IBM, 2010. IBM SPSS Statistics for Windows, Version 19.0. IBM, Armonk, NY.
leading to higher ORP, which decreased metal removal efciency. Lewis, A.E., 2010. Review of metal sulphide precipitation. Hydrometallurgy 104,
Hydraulic retention time had signicant effects on the reactive Lynd, L.R., Weimer, P.J., van Zyl, W.H., Pretorius, I.S., 2002. Microbial cellulose uti-
mixture composition. Low ow in 4-day HRT reactors favored lization: fundamentals and biotechnology. Microbiol. Mol. Biol. Rev. 66,
growth of microorganisms that rapidly consumed organic nitrogen 506e577.
Mariner, R., Johnson, D.B., Hallberg, K.B., 2008. Characterization of an attenuation
and organic carbon in the reactive mixture. High ow in 1-day HRT system for the remediation of Mn(II) contaminated waters. Hydrometallurgy
reactors decreased the pH (<6.3) at week 36, especially at the 94, 100e104.
bottom layer, probably from depletion of neutralizing agents McCauley, C., O'Sullivan, A.D., Milke, M.W., Weber, P., Trumm, D., 2009. Sulfate and
metal removal in bioreactors treating acid mine drainage dominated with iron
caused by constant AMD input and upward ow in the reactors.
and aluminum. Water Res. 43, 961e970.
Removal of Fe and Zn, probably in the form of suldes, took place Mikanova, O., 2006. Effects of heavy metals on some soil biological parameters.
mainly in the bottom layers of all reactors, mainly in the 2-day HRT. J. Geochem. Explor. 88, 220e223.
Neculita, C.M., Zagury, G.J., Bussie re, B., 2008a. Effectiveness of sulfate-reducing
This led to accumulation of metal suldes and eventually produced
passive bioreactors for treating highly contaminated acid mine drainage: I.
toxicity in the reactive mixture, leading to lower SRB counts and Effect of hydraulic retention time. Appl. Geochem. 23, 3442e3451.
dehydrogenase activity. The HRT strongly inuenced performance Neculita, C.M., Zagury, G.J., Bussie re, B., 2008b. Effectiveness of sulfate-reducing
of reactors and resulted in physical and biological inhibitions of SRB passive bioreactors for treating highly contaminated acid mine drainage: II.
Metal removal mechanisms and potential mobility. Appl. Geochem. 23,
and failures in the reactors. As a result, HRT must be a balance 3545e3560.
between rate and removal efciency. Neculita, C.M., Zagury, G.J., Bussie re, B., 2007. Passive treatment of acid mine
drainage in bioreactors using sulfate-reducing bacteria e critical review and
research needs. J. Environ. Qual. 36, 1e16.
Acknowledgements Nordstrom, K., Blowes, D., Ptacek, C., 2015. Hydrogeochemistry and microbiology of
mine drainage: an update. Appl. Geochem. 57, 3e16.
This research was funded by the Ponticia Universidad Javeriana Okabe, S., Santegoeds, C.M., De Beer, D., 2003. Effect of nitrite and nitrate on in situ
sulde production in an activated sludge immobilized agar gel lm as deter-
(ID. 5177) and the Department of Natural Sciences of the Uni- mined by use of microelectrodes. Biotechnol. Bioeng. 81, 570e577.
versidad Central. We thank Carlos Diaz for assistance with labora- Postgate, J.R., 1984. The Sulphate-reducing Bacteria. Cambridge University Press,
tory analysis. Cambridge, UK.
Prasad, D., Wai, M., Berube, P., Henry, J.G., 1999. Evaluating substrates in the
treatment of acid mine drainage. Environ. Technol 20, 449e459.
References Pruden, A., Messner, N., Pereyra, L., Hanson, R.E., Hiibel, S.R., Reardon, K.F., 2007. The
effect of inoculum on the performance of sulfate-reducing columns treating
Adams, B., Anderson, R., Bless, D., Butler, B., Conway, B., Dailey, A., Zownir, A., 2014. heavy metal contaminated water. Water Res. 41, 904e914.
Reference Guide to Treatment Technologies for Mining-inuenced Water. vol. Robinson-Lora, M.A., Brennan, R.A., 2011. Anaerobic precipitation of manganese and
542-R-14-001. US Environmental Protection Agency-OSRTI, Washington, DC. co-existing metals in mine impacted water treated with crab shell-associated
APHA, AWWA, WEF, 2005. In: Clesceri, L.S., Greenberg, A.E., Eaton, A.D. (Eds.), minerals. Appl. Geochem 26, 853e862.
Standard Methods for the Examination of Water and Wastewater, twenty-rst ez-Navarrete, C., Zamorano, A., Ferrada, C., Rodrguez, L., 2009. Sulphate reduc-
ed. American Public Health Association, Washington, DC. tion and biomass growth rates for Desulfobacterium autotrophicum in yeast
ASTM, 1995a. Standard test method for pH of soils. In: Annual Book of ASTM extract: supplemented media at 38  C. Desalination 248, 377e383.
Standards, Section D4972-95a. ASTM International, West Conshohocken, PA, Schumacher, B.A., 2002. Methods for the Determination of Total Organic Carbon
pp. 27e28. (TOC) in Soils and Sediments. NCEA-C-1282, EMASC-001. Ecological Risk
ASTM, 1995b. Standard test method for laboratory determination of water (mois- Assessment Support Center, Ofce of Research and Development, U.S. Envi-
ture) content of soil and rock. In: Annual Book of ASTM Standards, Section ronmental Protection Agency, Las Vegas.
D2216-92. ASTM International, West Conshohocken, PA, pp. 178e181. Serra-Wittling, C., Houot, S., Barriuso, E., 1995. Soil enzymatic response to addition
ASTM, 2009. Standard methods for sulfate-reducing bacteria in water and water- of municipal solid-waste compost. Biol. Fert. Soils 20, 226e236.
formed deposits. In: Annual Book of ASTM Standards, Section D4412-84. Song, H., Yim, G.J., Ji, S.W., Nam, I.H., Neculita, C.M., Lee, G., 2012. Performance of
ASTM International, West Conshohocken, PA, pp. 108e122. mixed organic substrates during treatment of acidic and moderate mine
Chang, I., Shin, P., Kim, B., 2000. Biological treatment of acid mine drainage under drainage in column bioreactors. J. Environ. Eng. 138, 1077e1084.
sulphate-reducing conditions with solid waste materials as substrate. Water USEPA, 2001. Flame Atomic Absorption Spectrophotometry. Method 7000b. US
Res. 34, 1269e1277. Environmental Protection Agency, Washington, DC.
Cheong, Y., Das, B., Roy, A., Bhattacharya, J., 2010. Performance of a SAPS-based Utgikar, V.P., Harmon, S.M., Chaudhary, N., Tabak, H.H., Govind, R., Haines, J.R., 2002.
chemo-bioreactor treating acid mine drainage using low-DOC spent mush- Inhibition of sulfate-reducing bacteria by metal sulde formation in bioreme-
room compost, and limestone as substrate. Mine Water Environ. 29 (3), diation of acid mine drainage. Environ. Toxicol. Chem. 17, 40e48.
217e224. Vasquez, Y., Escobar, M.C., Neculita, C.M., Arbely, Z., Roldan, F., 2016. Selection of
Chu, H., Lin, X., Fujii, T., Morimoto, S., Yagi, K., Hu, J., Zhang, J., 2007. Soil microbial reactive mixture for biochemical passive treatment of acid mine drainage. En-
biomass, dehydrogenase activity, bacterial community structure in response to viron. Earth Sci.
long-term fertilizer management. Soil Biol. Biochem 39, 2971e2976. Waybrant, K.R., Ptacek, C.J., Blowes, D.W., 2002. Treatment of mine drainage using
Dische, Z., 1962. Color reactions of carbohydrates. In: Whistler, R.L., Wolfram, M.L. permeable reactive barriers: column experiments. Environ. Sci. Technol. 36,
(Eds.), Methods in Carbohydrate Chemistry. Academic Press, New York, 1349e1356.
pp. 475e514. Yoo, K., Sasaki, K., Hiroyoshi, N., Tsunekawa, M., 2004. Fundamental study on the
Di Toro, D.M., McGrath, J.A., Hansen, D.J., Berry, W.J., Paquin, P.R., Mathew, R., removal of Mn2 in acid mine drainage using sulfate reducing bacteria. Mater.
Wu, K.U., Santore, R.C., 2005. Predicting sediment metal toxicity using a sedi- Trans. 45, 2422e2428.
ment biotic ligand model: methodology and initial application. Environ. Toxicol. Younger, P.L., Banwart, S.A., Hedin, R.S. (Eds.), 2002. Mine Water, Hydrogeology,
Chem. 24, 2410e2427. Pollution, Remediation. Kluwer Academic Publishers, Dordrecht, The
Dvorak, D.H., Hedin, R.S., Edenborn, H.M., McIntire, P.E., 1992. Treatment of metal- Netherlands.
contaminated water using bacterial sulfate reduction: results from pilot-scale Zagury, G.J., Kulnieks, V.I., Neculita, C.M., 2006. Characterization and reactivity
Y. Vasquez et al. / Chemosphere 153 (2016) 244e253 253

assessment of organic substrates for sulphate-reducing bacteria in acid mine Rock Drainage. Cairns, QLD, Australia, pp. 845e853.
drainage treatment. Chemosphere 64, 944e954. Zhang, M., Wang, H., 2014. Organic wastes as carbon sources to promote sulfate
Zaluski, M.H., Trudnowski, J.M., Harrington-Baker, M.A., Bless, D.R., 2003. Post- reducing bacterial activity for biological remediation of acid mine drainage.
mortem ndings on the performance of engineered SRB eld- bioreactors for Miner. Eng. 69, 81e90.
acid mine drainage control. In: Proc. Sixth International Conference on Acid