Sulfate reducing bacteria are obligate anaerobes and members of a heterogeneous group of eubacteria and archaebacteria which are characterized by their use of sulfate as a terminal electron acceptor during anaerobic respiration (Hansen, 1994; Akagi 1995; Colleran et al., 1995; Cypionka, 1995; Hamilton, 1998). SRB form a group of sulfate reducing prokaryotes and the main genus is Desulfovibrio. Desulfovibrio desulfuricans is often used to immobilize dissolved heavy metals as metallic sulfides.
Although many bacteria can produce sulfide, only a few are capable of producing sulfide at a rate that is sufficient for large-scale applications. These rapid sulfide generating bacteria are able to conserve energy by the reduction of sulfate (Widdel and Hansen, 1992), and they are generally termed as SRB. Sulfate is activated by means of adenosine triphosphate (ATP). The enzyme ATP sulfurylase catalyzes the sulfate to phosphate of ATP, leading to the formation of adenosine 5'-phosphosulfate (APS) as shown in Fig. 2.2 (Madigan et al., 2003). Even though ATP gets hydrolyzed, end products formed are to be removed for the completion of the reaction since the reaction is energy requiring. Pyrophosphate (PPi) can be hydrolyzed to phosphate by pyrophosphate (Cypionka, 1995).
Figure 2. 2 Dissimilative and assimilative patways of sulfate reduction. The enzymes catalyzing the reactions include (1) ATP sulfurlyase, (2) APS reductase, (3) sulfite reductase, (4) trithionate reductase, (5) thiosulfate reductase, and (6) APS kinase (Akagi, 1995; Madigan et al., 2003).
The sulfate moiety of APS is reduced directly to sulfite (SO32-
) by the enzyme APS reductase with the release of adenosine monophosphate (AMP) in dissimilative sulfate reduction. Whereas in assimilative reduction, another phosphate group is added to APS to
form phosphoadenosine 5'-phosphosulfate (PAPS), where sulfate moiety gets reduced to sulfite with the release of phosphoadenosine 5'-phosphte (PAP) (Madigan et al., 2003). Two hypotheses are proposed for the reduction of sulfite to different forms of sulfide; one hypothesis is that a direct six electron reduction of sulfite to sulfide ocurs with the aid of the enzyme, sulfite reductase without the formation of any ioslable intermediate compounds (Akagi, 1995).
In the second hypothesis, two intermediates, trithionate and thiosulfate are formed in which trithionate pathway involves a recycling mechanism of sulfite that is released during the reduction of trithionate to thiosulfate by the trithionate reductase, and the reduction of thiosulfate to sulfide by the thiosulfate reductase (Akagi, 1995). Hydrogen sulfide is excreted into the environment in the case of dissimilative sulfate reduction, whereas in case of the assimilative reduction, H2S formed is immediately converted into organic sulfur compounds, such as amino acids (Madigan et al., 2003)
The SRB are broadly categorized into two types depending on their oxidative capability: the genera in the first group (Desulfovibrio, Desulfomonas, Desulfotomaculum and Desulfobulbus) consume lactate, pyruvate, ethanol and certain fatty acids as carbon source but are not capable of oxidizing acetate to carbon dioxide (CO2). The genera in the second group (Desulfobacter, Desulfococcus, Desulfosarcina and Desulfonema) are specialized in the oxiding short chain fatty acids, particularly acetate. SRB are capable of surviving in a wide range of pH conditions, more particularly their growth is optimum between pH 5 and 9 (Postgate, 1984). Sulfate reducing bacteria populations have been found to occur at temperatures ranging from the psychrophilic to the hyper thermophilic range (Kolmert, 1999).
2.6.1 Classification of SRB
The SRB represents a group of chemoorganotrophic and strictly anaerobic bacteria, which include representatives of the genera Desulfovibrio, Desulfomicrobium, Desulfobacter, Desulfosarcina, Desulfotomaculum, Thermodesulfobacterium, etc. (Odom and Singleton, 1993). Generally, SRB are classified into different groups depending on their applications as detailed further:
Extremophilic SRB: Among the diversity of sulfate reducing prokaryotes, the acidophilic, thermophilic and psychrotolerant bacteria are extremophiles that could improve the performance of existing treatment systems.
Acidophilic SRB: Oxygen normally enters the deep geological environments during mining activities and aids in chemical and biological oxidation processes. Hydrogen ions and sulfate are produced which lower the pH significantly in the range of 2-3 (Kolmert, 1999;
Madigan et al., 2000). At present, biological AMD treatment uses mostly the neutrophilic SRB that are highly sensitive to acidic conditions (Jong and Parry, 2006). In these type of systems, treatment is carried out in two stages; first the SRB grow in separate tanks where hydrogen sulfide is produced and the sulfide is transferred to a second reactor containing the metal contaminated water which results in precipitation of metal sulfides. Acidophilic or acid-tolerant bacteria are capable of growing in acidic environment, and the use of these type of extremophiles will simplify the system and keep the process economics low (Kolmert et al., 2001; Kimura et al., 2006).
Thermophilic SRB: Some industries typically discharge wastewater at high temperatures of 50 to 70 ºC and even above 90 ºC. The thermophilic SRB are capable of sustaining particularly at high temperatures and these can be directly applied for treating wastewater at high temperatures. It eliminates cooling of the process water and allows direct use of the treated water without the need of any additional reheating. These systems normally produce less sludge and are capable of treating high organic loading rates with high removal efficiency (Vallero, 2003; Pender et al., 2004).
Cold-adapted SRB: Treatment of industrial wastewater in countries with cold environments is found to be different from other countries and it is recognized that psychrophilic SRB generally have an optimum growth temperature of 18 ºC, whereas the optimum temperature for sulfate reduction is 28-30 ºC. However, bacteria reducing sulfate below 4 °C have been identified. A low reaction rate of the sulfate reduction process at low temperatures could be compensated by an increased number of bacteria (Knoblauch et al., 1999; Sahm et al., 1999).
2.6.2 Sulfate removal mechanism and its significance to heavy metal removal from wastewater
Generally, SRB are capable of converting sulfate or sulfite (SO32-
) to different forms of sulfide (S2-/HS-/H2S) by using electron donating substrates in the form of COD already present in wastewater or by using low-cost externally added substrates. In this process, sulfate acts as an electron acceptor to support anaerobic respiration as shown in the equation
2.1. The substrates are either partially oxidized (e.g. to acetate) or fully oxidized to CO2
based on the type of bacteria.
2 2
- - 2 2
4 COD S /HS/H S CO
SO 2.1
Factors, such as availability of growth nutrients, age and physiological state of bacterial cells, environmental conditions (pH, ionic strength and temperature), presence of competitive ions and concentration of the biomass can influence the sulfate removal mechanism. Once sulfate-reducing conditions are established, sulfide precipitation becomes the predominant mechanism of metal removal from AMD or metallic wastewater (Machemer and Wildeman, 1992; Bechard et al., 1994; Kaksonen and Puhakka, 2007).
Biogenic sulfide generated by SRB can form highly insoluble metal precipitates. Thus, the sulfides can precipitate soluble heavy metals in wastewater streams or polluted groundwater to insoluble sulfide precipitates, thereby decreasing the bioavailability of toxic metals (Mizuno et al., 1994), as represented in the equation 2.2. Since the metals ions are highly concentrated in the precipitate as insoluble form, the resulting metal sulfide precipitates can be removed and recycled back to industry for reuse. Extensive care should be taken in handling the hydrogen sulfide produced and separate provision should be provided for the recovery of metals from the insoluble precipitates formed.
M MS
S2- n 2.2
where, M is metal, n is the valency and MS is metal sulfide precipitation.
In view of existing methods for the removal of heavy metals and sulfate from wastewater, biological processes exhibit some key advantages over the conventional treatment methods, such as (i) metal specific, (ii) efficient in terms of very low residual metal concentration compared to other common physico-chemical processes, (iii) less intensive in terms of energy and materials consumption, and (iv) very low production of secondary sludge.
The key advantages of the use of SRB in biological heavy metal removal by sulfate reduction can be summarized as follows:
1. Overall low treatment costs together with a very high treatment efficiency 2. Reduction or elimination of costs associated with metal sludge disposal
3. The elimination of wastewater treatment sludge avoids the geotechnical costs of pond construction and the costs associated with providing valuable land to sludge ponds (Bratty et al., 2006).
Although the potential use of microbial sulfate reduction for treating heavy metal loaded wastewater has been reported as early as 1969, development of SRB based on passive and active treatment system is quite recent.