Chapter 10
Bioprocess Development for Mercury Detoxification and Azo-Dye Decolorization Jo-Shu Chang Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan
Biological treatment of environmental pollutants is often very complex and requires intensive control of operation parameters. Fermentation of microorganisms able to degrade or destroy pollutants is a key technology to ensure appropriate cell concentration and populations are maintained in the treatment system. Optimal operation strategies, especially policy for substrates feeding and oxygen supply, need to be developed to enhance the rates of biodegradation or biotransformation for efficient cleanup of the target pollutants. In this chapter, two unique types of biological treatment processes, namely, mercury detoxification and azo-dye decolorization, are introduced as the example to show how fermentation technology and bioreactor approaches are used to develop bioprocesses for environmental applications. Detoxification of mercury is conducted aerobically using wild-type and recombinant strains as the biocatalyst. The performance and stability of fed-batch and chemostat bioreactors for mercury detoxification are greatly correlated to the dosage of the toxic mercury substrate. In contrast, azo dye is less toxic but decolorization of azo dyes requires aerobic/anaerobic sequential environments. Thus, bioreactor design for bacterial decolorization focused on strategies for the supply of oxygen and retention of high cell concentration within the reactors to achieve optimal combinantion of aerobic cell growth and anaerobic decolorization. The effectiveness and feasibility of various bioreactor configurations and strategies are assessed.
© 2004 American Chemical Society
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160 Introduction Environmental Biotechnology has become a promising means to clean up pollutants from the contaminated environments in a more natural and costeffective way. In biological treatment processes, microorganisms capable of degrading or transforming organic or inorganic pollutants into simpler or less toxic forms play a major role. An efficient biological treatment involves effective growth of biodegraders and control of die bioprocess for an efficient biodégradation or biotransformation of the pollutants. Therefore, fermentation technology is obviously a crucial tool to enable an efficient biological waste treatment process. However, it is a great challenge for fermentation technology when it is applied in waste treatment, since the substrates (pollutants) are more complex and often toxic to the microorganisms as compared with those used in food or pharmaceutical industry. In addition, the performance of waste degradation is usually very sensitive to the fermentation conditions (pH, aeration, temperature, food to microorganism (F/M) ratio, nutrients, etc.) and may require aerobic/anaerobic sequential environments (/) for mineralization of some complex pollutants. For instance, biodégradation of chlorinated aromatic compounds requires anaerobic dechlorination step followed by aerobic ring cleavage of the chlorine-free aromatic compounds and subsequent degradation of the resulting fatty acids via β-oxidation steps (2). The other typical example is biological nitrogen removal that converts ammonia to nitrates via an aerobic nitrification step and the nitrates are reduced anaerobically to N with a denitrification step (3). As a result, an appropriate bioprocess design and process control seems to be die key to a successful biological treatment system. This chapter provides examples that demonstrate strategies to develop bioprocesses for mercury detoxification and for azo-dye decolorization. The former is an aerobic process that deals with a toxic substrate. The latter involves an aerobic/anaerobic sequential environment, in which cell growth and degradation of substrate have distinct oxygen requirement. 2
Bioprocess Development for Microbial Mercury Detoxification Mercury and mercurial compounds have been used in a variety of industries, causing severe mercury pollution in aquatic systems and soils (4-6). Conventional mercury-removal methods mainly via physical and chemical routes to either trap and collect mercury from contaminated sites or to remove mercury by chemical precipitation (5,7). The drawback of physical methods has been die requirement for additional treatments, while chemical methods often leave hazardous by-products or residual sludges. Thus, biological methods allowing more natural, efficient and economical cleanup of mercury waste have emerged. Mercury-resistant microorganisms can resist mercury due to their ability to volatilize soluble forms of mercury from die environment via a sequence of enzymatic reactions, which are recognized as mercury detoxification (8-11).
161 The genetic basis of mercury resistance is encoded in mer opérons located on either plasmids or transposable elements (12-14). Organomercurial lyase (merB gene product) catalyzes cleavage of C-Hg bonds in organomercurial compounds to release mercuric ions, which are enzymatically reduced to less toxic and more volatile metallic mercury (Hg°) by merA-product mercuric reductase (12,14-17). Cysteine-rich transport proteins originated from merP and merT genes locate on the periplasmic space and inner membrane, respectively. The MerP and MerT proteins are responsible for the specific delivery of ambient mercuric ions toward mercuric reductase in the cytoplasm for reduction of Hg * to volatile Hg° (16,18). The constitutive mer operon is induced by the subtoxic level of mercuric ions (13,19). The mechanism of mercury detoxification is illustrated in Fig. 1. In contrast to intensive studies and understanding of the molecular genetics of mer operon and the biochemistry of mercury detoxification, (12,14-17), much less effort has been devoted to develop microbial mercury detoxification processes (20-22). Only recently, Saouter et al. (23) reported their preliminary investigation of using Hg -reducing strains to decontaminate a polluted freshwater pond in Tennessee. Other authors (24,25) demonstrated their studies on volatilization of mercury using resting- or immobilized-cell systems. Recent attempts (20,26,27) in our group investigated the kinetic of mercury detoxification by a wild-type mercury-resistant strain (P. aeruginosa PU21) and a recombinant E. coli strain (E. coli PWS1). The two mercury-resistant strains were used to develop batch, fed-batch, and continuous bioreactors to demonstrate operation strategies for efficient and practical detoxification of mercury in wastewater. A mercury vapor recovery device was also designed to recover metallic mercury (Hg°), the end product of mercury detoxification (26). Mercury detoxification occurred when the wild-type or the recombinant strain was cultivated in mercury-containing media. Serial adaptation may be needed to prepare mercury-hyperresistant strain of P. aeruginosa PU21 (26), 2
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while the mercury-reducing activity of the recombinant strain E. coli PWS1 can be turned on by IPTG induction (29). Since mercury is toxic to the mercuryresistant cells when its concentration is higher than some threshold value (28), the loading of mercury into the bioreactor need to be well controlled for a stable mercury detoxification operation. In addition, a sufficient initial cell concentration to start up the bioreactor is also critical to enable total survival of the cells for efficient mercury detoxification (26-28). It was found that P. aeruginosa PU21 (Rip64) is able to detoxify mercury rapidly during its transient growth (20,26). The reduction of mercury with growing cells exhibited a maximal rate (approximately 1.11 χ 10" μg Hg/cell/h) at the substrate concentration of 8 mg Hg/L. Hie mercury detoxification performance of P. aeruginosa PU21 was also correlated to the bacterial growth phases, with cells at lag phase and exponential phase having higher specific detoxification rates. The recombinant strain E. coli PWS1 exhibited better specific mercury detoxification activity man P. aeruginosa PU21 at batch 6
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Fig. 1 Mechanism of bacterial mercury resistance
operations, while the PWS1 strain became unstable during long-term fed-batch operations primarily due to plasmid instability (27). Two fed-batch strategies were used for mercury detoxification operations: step-wisefixed-interval(SWFI) feeding (26) and repeated constant-rate (RCR) feeding (27). For SWFI feeding, mercury detoxification efficiency was 2.9 mg/L/h and 3.3 mg/L/h for 2 and 5 mg Hg/L feeding, respectively. In contrast, for RCR feeding strategy, the mercury detoxification efficiency was closely related to the mercury feeding rate (F ), initial inoculum size (XJ, as well as the initial culture volume (V ) and the best mercury detoxification efficiency was 5.6 mg Hg/L/h, which occurred when F = 16.9 mg Hg/h, X
