Water, Air, and Soil Pollution: Focus (2006) 6: 71–82 DOI: 10.1007/s11267-005-9014-1
C
Springer 2006
MICROBIAL REMOVAL OF ARSENIC
∗
KAUSER JAHAN∗ , PATRICIA MOSTO, CRYSTAL MATTSON, ERIN FREY and LARA DERCHAK Associate Professor of Civil and Environmental Engineering, Rowan University, Glassboro, NJ 08028, USA, Email: ∗
[email protected], Phone: 856-256-5323, Fax: 856-256-5242
(Received 30 December 2004; accepted 31 August 2005)
Abstract. Bangladesh is currently the subject of the world’s largest mass arsenic poisoning in history. Groundwater throughout Bangladesh and West Bengal is contaminated with naturally occurring arsenic from the alluvial and deltaic sediments that form the region’s aquifers. It has been estimated that 75 million people are at risk of developing health effects associated with the ingestion of arsenic. This project focuses on the use of microorganisms such as bacteria and algae to remove arsenic from water. Arsenic in the arsenite form was used in the studies. Experiments were conducted with a common alga and wastewater bacteria. A common green algae Scenedesmus abundans was used for determining arsenic uptake in batch experiments. Results of the experiments indicated that the algae biosorption could be modeled by the conventional Langmuir isotherm model. Algae morphology studies indicated that the algae cells were impacted due to the presence of arsenic as evidenced by clumping or loss of cell clusters. The wastewater bacteria also were capable of high percent of arsenic removal. Results indicate that microbial uptake of arsenic may be a viable method of pretreatment of arsenic contaminated water. However algae and sludge disposal would pose a problem and will have to be dealt with accordingly. Keywords: Arsenic, Algae, Bacteria, Microorganisms
1. Introduction Bangladesh is currently the subject of the “world’s largest mass arsenic poisoning in history” (Harvey et al., 2002). Groundwater throughout Bangladesh and West Bengal is contaminated with naturally occurring arsenic from the alluvial and deltaic sediments that form the region’s aquifers. It has been estimated that 75 million people are at risk of developing health effects associated with the ingestion of arsenic (Karim, 2000). Arsenic was first identified in West Bengal, India, in the 1990s when people started to develop arseniosis, starting with skin rashes and leading to sometimes fatal problems with major organs such as the lungs, kidneys, and bladder (Chowdhury et al., 2000). Similar problems were detected in Bangladesh, which is in close proximity to West Bengal and has a similar land pattern based on alluvial and deltaic sediments. Currently, about 1/3 of the ground water wells in Bangladesh pump up water exceeding the arsenic standard of 50 ppb (parts per billion). In some areas, arsenic levels have been determined to be 2000 ppb, 40 times over the acceptable level for drinking water. Over one-half of the approximately four million wells that constitute the country’s drinking water
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supply have levels of naturally occurring arsenic above the World Health Organization’s standard of 0.01 mg/L, exposing as many as 50 million people to dangerous levels of arsenic in their drinking water. Research indicates that humans are particularly susceptible to arsenic poisoning (Byron et al., 1967). Typically arsenic impacts the major organs and there is now evidence that it is a potent carcinogen (Morton et al., 1976; Duker et al., 2004). In 1996, the US Congress amended the Safe Drinking Water Act of 1974 and directed the Environmental Protection Agency to propose new standards for arsenic in drinking water. In January 2001, the final ruling was published setting the standard at 0.01 mg/L. The toxicity of arsenic is well documented and recognized. Therefore cost effective arsenic removal methods are needed to help poor and developing nations such as Bangladesh. Conventional arsenic treatment technologies include coagulation/filtration, ion exchange, lime softening, adsorption and reverse osmosis (Jekel 1994; Zouboulis and Katsoyiannis, 2002). These technologies are more suitable for the removal of As(V) and typically a preoxidation step is required for As(III) to form As(V). Chemical agents such as ozone, chlorine, hydrogen peroxide are typically used for this preoxidation step (Kim and Nriangu, 2000). However they incur costs and also cause secondary problems with by product formation. In recent years attention has been given to biological arsenic removal as it may have potential as a cost effective solution. 1.1. B ACTERIAL
REMOVAL OF ARSENIC
Microbial arsenate metabolism was first identified by Green (1918). Green isolated an arsenate reducer, Bacterium arenreducens, and an arsenite oxidiser B. arsenoxydans. Since then numerous researchers have reported on isolating arsenate-reducing bacteria from arsenic rich soils and sediments using anaerobic media where As(V) serves as the sole terminal acceptor (Nicholas et al., 2003). Bacteria have been shown to reduce oxyanions of arsenic. Stolz and Oremland (1999) provided the reduction reactions that select bacteria perform on these oxyanions of arsenic. The following equations show the reduction of arsenate and arsenite by the organism C. arsenatis, S. barnesii, D. auripigmentum, B. selenitireducens, and B. arsenicoselenatis. − − + Lactate− + 2HAsO2− 4 + 2H → 2H2 AsO2 + HCO3 4− Acetate− + 2HAsO2− + 5H + → 4H3 AsO3 +HCO− 4 + 2H2 AsO 3
Mittal and Ratra (1999) studied the impact of various metal ions on biochemical oxygen demand at various concentrations. The study looked at the effects of metals like aluminum, copper, cobalt, nickel, zinc, mercury, and lead, but did not investigate
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the impact of arsenic on BOD. The results of the study indicated that mercury was the most toxic of all metal ions tested, even at very low concentrations (Mittal and Ratra, 1999). Results of the study show that as metal concentrations are increased, the percentage of growth inhibition increases. Huysmans and Frankenberger (1990) isolated arsenic resistant bacteria in agricultural drainage water and evaporation pond sediments. In their study, a decline in total colony forming units (CFU) was observed when As(III) concentrations were greater than 1 mg/L. There was no effect noted on the CFU when the bacteria was subjected to As(V) concentrations of up to 1000 mg/L. Katsoyiannis et al. (2002) used fixed-bed upflow bioreactors for Arsenic (III) removal from groundwater. The researchers specifically used iron oxidizing bacteria in this study. Arsenic was removed around 80%. This method was reported to be suitable for iron and manganese oxidation along with arsenic removal. Katsoyiannis and Zouboulis (2004) conducted further studies with iron oxidizing bacteria for removing both arsenite and arsenate. The results indicated that both forms of arsenic could be efficiently removed by for the concentration range of interest in drinking water. In addition the oxidation of trivalent arsenic was found to be catalyzed by bacteria leading to an increased arsenic removal because trivalent arsenic cannot be efficiently sorbed to iron oxides. Zouboulis and Katsoyiannis (2005) conducted X-ray photoelectron spectroscopy (XPS) analyses to obtain information for the mechanism of As(III) removal by arsenic oxidizing iron bacteria. Results indicated that As(III) was partially oxidized to As(V) which enabled high arsenic removal efficiency. It is important to note that studies on arsenic removal by bacteria have typically focused on pure culture studies. 1.2. ARSENIC
REMOVAL BY ALGAE
Literature indicates that certain species of algae uptake heavy metals such as copper, cadmium, chromium, lead, and nickel (Qiming et al., 1999). The uptake of metals by algae in aqueous solutions occurs when algae release a protein called metallothioneins. When metallothioneins are released, the alga begins to chemically bind the metal to itself as a defense mechanism to remove the metal from its regular cellular activity (Volesky, 1999). Karna et al. (1999) reported that the noxious effect of metals to algae seems to be related to the production of reactive oxygen species (ROS) and the consequential unbalanced cellular redox status. Algae respond to heavy metals by the synthesis of low molecular weight compounds such as carotenoids and glutathione, and the initiation of several antioxidants, as well as enzymes including superoxide dismutase, catalase, glutathione peroxidase and ascorbate peroxidase. Chlorella sp. and Scenedesmus sp. are the two most common algae species used for metal uptake. The results of a study performed by Suhendrayatna et al. (1999) exposed Chlorella sp. to concentrations of arsenite ranging from 0 to 100 μg As cm−3 the maximum concentration being 100 μg As cm−3 . The cell growth of Chlorella
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sp. was not affected by the arsenite until it was exposed to concentrations higher than 50 μg As cm.−3 At concentrations greater than 50 μg As cm−3 . The cell growth of the species was suppressed. Beceiro-Gonza´lez et al. (2000) concluded that Chlorella sp. retained approximately 50% of arsenite from a solution. During a contact time of up to forty-eight hours it was observed by Taboada-de la Calzada et al. (1999) that there were no improvements in the accumulation of arsenite over a long period of time as most of the biosorption was rapid and occurred in the first fifteen minutes by the Chlorella sp. Harris and Ramelow (1990) studied the use of Chlorella sp. and Scenedesmus sp. for biosorption of gold, copper, cadmium, and zinc. The authors indicated that metal uptake occurs quickly initially due to adsorption or ion exchange. It was found that approximately 90% of sorption took place within fifteen minutes and that the remnants adsorbed at a slower rate. Experiments indicated that the metal binding ability of Scenedesmus sp. was much greater than that of Chlorella sp. Terry and Stone (2002) reported on the biosorption of cadmium and copper contaminated water by Scenedesmus abundans. It was shown that both living and non living S. abundans removed cadmium and copper from water, with better removal rates with the living algae. Algae concentration was not a factor in the metal biosorption process. The study indicated that biosorption by algae could be a viable mechanism for metal waste treatment and also indicated that the method would work for multicomponent metal systems. The current literature indicates that there are no studies that have investigated a wastewater treatment plant mixed bacteria culture or the alga Scenedesmus abundans for removal of arsenic from water. Thus this study investigates these two types of microorganisms for arsenic removal. The overall objectives of this project were to investigate the feasibility of the use of a mixed culture of aerobic microorganisms for arsenic removal and the use of the alga Scenedesmus abundans to investigate cost effective solutions for poor nations such as Bangladesh. This is one of the first studies investigating the use of common wastewater treatment plant bacteria and the alga Scenedesmus abundans for arsenic removal. Arsenic in the form of arsenite was used in the studies as literature indicates that arsenite As(III) is the more toxic form and more difficult to remove by application of conventional treatment methods (Zouboulis and Katsoyiannis (2005). The objectives of this study were to: • Develop a mixed bacterial culture from a wastewater treatment plant capable of arsenic uptake • Study the impact of arsenite during glucose metabolism by wastewater microorganisms • Determine toxicity and inhibitory impacts on the biodegradation of glucose due to the presence of arsenic, and • Conduct arsenic uptake studies by Scenedesmus abundans and study the impact of arsenic on algae.
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2. Materials and Methods 2.1. WASTEWATER
BACTERIA STUDIES
Wastewater samples were obtained from the aeration tank of a local activated sludge treatment plant in Southern Harrison Township, New Jersey. It was used as a source of microorganisms (seed) for arsenic removal studies without any attempts to classify the different microbial species. Glucose (Dextrose, Fisher Scientific, Pittsburgh, PA) was used as a carbon source at a concentration of 180 mg/L. Arsenic in the form of sodium meta-arsenite (NaAsO2 ) was purchased from Sigma-Aldrich (St. Louis, MO). Arsenic concentrations used varied from 0.05, 0.1, 0.5, 1.0, and 1.5 mg/L in the biodegradation experiments. Nutrient buffer was prepared using the HACH Nutrient Buffer Pillow (HACH, Loveland, CO). All solutions were prepared using deionized distilled water. Experiments were conducted at room temperature 22 ± 0.5 ◦ C in batch reactors using the HACH BODTrak Apparatus (HACH, Loveland, CO). This apparatus monitors oxygen uptake in mg/L with time. All experiments were conducted in duplicates and average values are being reported. Controls containing only seed and glucose with seed were also maintained. Abiotic controls with arsenic and no seed or glucose were also maintained. Arsenic concentrations were monitored at the end of the experiments using a Perkin-Elmer 460 atomic absorption spectrophotometer. Samples were centrifuged at 6500 rpm in a Beckman (Beckman SPINCHRON DLX) centrifuge for 15 minutes. The supernatant was analyzed for total arsenic concentration remaining in the liquid. pH was monitored at the beginning and end of each experiment using an Orion 720 pH Meter. 2.2. ALGAE
STUDIES
The select alga Scenedesmus abundans (UTEX LB 1358) was obtained from the Culture Collection of Algae at the University of Texas and grown on a modified Bristol’s solution (Mosto, 1999) at 16:8 L:D cycle at a temperature of 25 ◦ C. The algae were counted using a Sedgwift-Rafter Counting Chamber under a Leitz Laborlux S-phase-contrast microscope, and. expressed as number of cells per milliliter. Chlorophyll a was determined by filtration of a 50-ml subsample through an acetate filter (Whatman HA 0.45 μm) and extraction using 100% DMSO and determined spectrophotometrically with a Milton Roy Spectronic Genesys 5 Spectrophotometer. Algal biomass as ash-dry biomass was determined by filtering the cultures on pre-ash (550 ◦ C) glass fiber filters (Whatman GF/C) using a Mettler Toledo precision balance. Arsenic removal experiments were conducted in batch tests in 250 ml acid washed Erlenmeyer flasks. Flasks were filled with 200 mL water containing S. abundans at a concentration of 40 mg/L on a dry mass basis. Arsenic was added to the flasks in concentrations of 1, 5, 10, 20, 50 and 100 mg/L. Higher concentrations were used in the algae studies as there is evidence in literature that
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algae can biosorb high concentrations of metals. Flasks were placed in an environmental shaker and samples were obtained at certain time intervals to analyze for residual arsenic concentrations. Residual arsenic concentrations in the water were obtained by filtering the sample through 0.22 mm Millipore filters to remove algae and then acid preserved. Arsenic analyses were carried out in a Perkin-Elmer 460 atomic absorption spectrophotometer. Flasks were tested for residual Chlorophyll a, cell counts and algae biomass. Algae morphology was also observed using a Leitz Laborlux S-phase-contrast microscope. All experiments were conducted in duplicate and average values are presented in the results. Controls were maintained with arsenic only.
3. Results and Discussion Both the wastewater bacteria and the alga studies gave positive results. Both bacteria and alga were capable of arsenic removal even at high arsenic concentrations. The impact of arsenic on wastewater bacteria was studied by observing oxygen uptake during glucose metabolization by a mixed culture obtained from a local wastewater treatment plant. Common wastewater bacteria found in aerobic activated sludge systems include a wide variety of bacterial species such as Pseudomonas, Alcaligenes, Bacillus, Flavobacterium, Micrococcus and Achromobacter (Grady et al., 1999). Figure 1 represents the results of the oxygen uptake for arsenic concentrations varying from 0.05 mg/L to 1.5 mg/L. Oxygen uptake for the controls with seed only demonstrated negligible oxygen uptake and have not been included in the graph. Abiotic results indicated that sorption of arsenic to the glass was negligible over the duration of the experiment. It is evident that this range of arsenite (0.05–1.5 mg/L) concentrations tested did not significantly impact the oxygen uptake due to glucose biodegradation during the first twenty four hours of the testing for all arsenic concentrations. Pronounced impact on oxygen uptake was noted at 1.0 mg/L of arsenite. Statistical data analyses were conducted to determine if the data for oxygen uptake in the presence of arsenic were statistically different from the control data. Data for the 1–10 day duration was fitted by linear regression for each curve to obtain the slopes of the regression lines. The significance of the difference of the slopes between the control and the arsenic reactors was tested using a paired t-test (Crow et al., 1960). A null hypothesis that states that the difference between the slopes of the regression lines is zero was tested by comparing the “t” value obtained to the critical “t” value selected at a significance level of 2%. If “t” experimental exceeded the critical “t” value, the null hypothesis is rejected and the difference between the data is significant at the 2% significance level. All five reactor data in the presence of arsenic were statistically different when compared to the control results. Glucose metabolism continued in all reactors in the presence of arsenic indicating that the arsenic concentrations were not
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200 180
Oxygen Uptake (mg/L)
160 140 120 100 Control
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As 0.05 mg/L
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Time (Days) Figure 1. Oxygen Uptake in mg/L for Glucose Biodegradation in the presence of Arsenic
toxic enough to totally suppress bacterial metabolism. However suppressed oxygen concentrations in comparison to the control indicate that glucose metabolization was being inhibited by the presence of arsenic. The pH concentrations did not fluctuate from the original average concentration of 7.4, indicating that the system was well buffered. Arsenic concentrations were measured in the supernatant at the end of the experiments after centrifuging the sample to remove the bacterial mass. The percentage of arsenic removed in each reactor was calculated and plotted. Figure 2 indicates the percentage of arsenic removal by the bacteria. The figure indicates that arsenic was removed by the bacteria significantly. Removals on an average were typically 63%. This removal is encouraging from a treatment standpoint. It is apparent that low concentrations of arsenic as arsenite maybe removed by wastewater microorganisms. Katsoyiannis and Zouboulis (2004) indicated that trivalent arsenic is oxidized by bacteria under aerobic conditions. The researchers indicated that this oxidation of arsenic led to higher arsenic removal as pentavalent arsenic can be readily adsorbed by bacteria cell surface or other adsorbents such as iron oxides. In these experiments the adsorption of arsenic to the glass surface or volatilization via the abiotic controls was negligible. As such the arsenic could either remain in the water phase or be associated with the cell mass. Alga experiments were conducted with live alga only. Taboada-de la Calzada et al. (1999) indicated that Arsenic (III) uptake by an alga species Chlorella
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Figure 2. Percent Arsenic Removal by Wastewater Microorganisms
vulagaris was not different for live or dead alga. The authors indicated that slightly higher arsenic removals were obtained with the live alga as binding of arsenic not only occurs with adsorption to the cell surface but also transport through the cell membrane via biological activity. Alga experiments indicated that the selected freshwater alga was also capable of removing arsenic. Removal appeared to be rapid and within the first hour of the experiment. Figure 3 shows typical arsenic concentrations remaining with time for select arsenic tests with S. abundans. The rapid uptake of arsenic by the algae in the first hour is consistent with other literature (Terry and Stone 2002; Taboadade la Calzada, 1999). Arsenic could either be bound with the alga or be in the water phase as the biotic and abiotic controls showed negligible loss of arsenic. The data also indicated increases in arsenic concentration with time, indicating that desorption processes may occur if the contact time with algae is too long. This was also evidenced in studies reported by Beceiro-Gonz´alez et al. (2000). Algae biomass at the end of the experiment with S. abundans is presented in Figure 4. The algae biomass did not vary significantly with increasing arsenic concentrations. Chlorophyll a results indicated that higher concentrations of arsenic did not significantly impact the chlorophyll a concentration. Morphology studies however indicated cell clumping with S abundans losing its four cell configuration. This was consistent for all arsenic concentrations. Visual inspection of the flasks during the experiments indicated alga clumping with pronounced effects. At the end of the experiments 20 ml of the algae suspension from each flask was centrifuged and the centrifuged algae pellet was resuspended in growth media without arsenic.
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MICROBIAL REMOVAL OF ARSENIC 6000
Arsenic Concentration Remaining (ppb)
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4000 As 1 mg/L As 5 mg/L
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Time (Hours) Figure 3. Arsenic Concentration with Time for Algae Experiments
Algae B iomass (mg/L)
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ro l
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Figure 4. Algae Biomass in mg/L (dry weight) at the end of the Arsenic Uptake Experiments
The algae from all flasks was able to regrow without any indications of permanent toxic effects of arsenic. The data was further plotted to determine if a conventional adsorption model could be used to represent the data. The data could be represented using the Langmuir isotherm model typically used for common adsorbents such as activated carbon. Figure 5 indicates the Langmuir isotherm plot for studies with S abundans.
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3
q mmol/g
2.5 2 1.5 1 0.5 0 0
0.5
1
1.5
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Equilibrium Concentration Ce (mM)
Figure 5. Langmuir Isotherm for Arsenic Soprtion to Algae
The Langmuir equation used for data analyses is given below: q=
bqmax .c 1 + bc
where
c (mM) and q(mmol/g) are the equilibrium concentrations of arsenic in the solution and biomass phases, resepectively, and b and q max are the Langmuir constants. The value of q max obtained was 3.78 mmol/g and b was 0.053 (1/mM). The adsorptive capacities ranges (q max ) reported for lead, copper and cadmium are in the range of 1.0–1.6, 1.0–1.2 and 0.8–1.2 mmol/g respectively for various marine macro algal species (Qiming et al., 1999). Overall results indicate that both the algae and wastewater bacteria are capable of arsenic removal. This removal is probably a combination of the oxidation of As(III) to As(V) and biosorption of arsenic to the microorganisms. The select alga species demonstrated high arsenic removal percentages at very high arsenic concentrations.
4. Conclusions This study has focused on the feasibility of microbial removal of arsenic from water as a viable mechanism of arsenic treatment. Experiments conducted with mixed bacterial cultures from a wastewater treatment plant and a common freshwater alga S abundans for arsenic removal indicated that microbes can remove arsenic as much as by 70%. Toxic and inhibitory effects were not seen during the bacterial studies. Alga morphology changed in presence of arsenic. Sorption of arsenic with algae could be modeled by the conventional Langmuir isotherm. The isotherm constants indicate a high adsorptive capacity of the select alga for arsenic. Current waste
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removal practices for arsenic or other metals typically include expensive, maintenance intensive treatment processes such as chemical precipitation, ion exchange or membrane processes. Biological removal of arsenic may have potential from a cost alternative and environmentally friendly process standpoint.
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