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Role of Biotechnology in Water and Wastewater Technology Bruce E. Rittmann Arizona State University, Center for Environmental Biotechnology, 1001 S. McAllister Ave., Tempe, AZ 85287-5701 U.S.A.; Rittmann@asu.edu Abstract. Environmental biotechnology “manages microbial communities to provide services to society.” The key services today include detoxifying contaminated water and soil to reclaim lost resources and converting diffuse energy in biomass to forms easily used by society. Two timely examples are the reduction of oxidized water contaminants (e.g., nitrate, perchlorate, and chlorinated solvents) and the production of methane, hydrogen, electricity, ethanol, and biodiesel. The key science underlying environmental biotechnology is microbial ecology, which has advanced rapidly in the past 20 years through the proliferation of new DNA- and RNA-based techniques to characterize the communities’ structure and function. The molecular methods provide detailed information that helps us understand what aspects of the microbial community need to be managed to ensure that it provides the desired service. Often, we are able to achieve the management goals through partnering the microorganisms with modern materials and physical/chemical processes. Keywords. Bio-reduction, Biotechnology, Environment, Genomics, Microbial Ecology Introduction I define environmental biotechnology as “managing microbial communities to provide services to society.” Most of the services can be broken into two major categories (Rittmann, 2006a): • Microbial communities can detoxify contaminants in water, soils, sediment, and sludge. This allows society to reclaim their resource value. • Microbial communities can convert the energy value in various types of biomass from its diffuse and sometimes hazardous form to energy outputs that are readily used by human society: e.g., methane, hydrogen, electricity, ethanol, and biodiesel. Common to both types of services is that they are based on microbially catalyzed oxidation and reduction reactions. Although oxidation and reduction form the basis for all life, microorganisms possess unparalleled capabilities to do oxidation and reductions reactions that provide them with energy to grow and human society with valuable services. Here, I focus on the value of the reduction products. Environmental biotechnology is a special case of the larger field of biotechnology. One thing that distinguished environmental biotechnology from the other parts of biotechnology is that its science base is the field of microbial ecology (Rittmann and McCarty, 2001; Rittmann et al., 2006). As a science, microbial ecology aims to characterize microbial communities in terms of • what types of microorganisms are present (its phylogenetic structure) • what metabolic reactions these microorganisms carry out (its metabolic function) • how the microorganisms interact with each other and their environment. In most cases, the metabolic reactions constitute the services to society. The past ~20 years have yielded remarkable advancements in DNA- and RNA-based tools that allow us to characterize the communities in these ways, and this has led to the discovery of new microorganisms, new metabolic capabilities, and new biotechnologies (Rittmann et al., 2006).

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We ensure that the communities provide the desired services reliably by managing them. This often involves creating engineered systems that partner the microbial communities with modern materials and physical/chemical processes (Rittmann, 2006a). We are fortunate that materials science and engineering are advancing as rapidly as is molecular microbial ecology. Thus, our expanding understanding of the structure and function of microbial communities can be matched by ever-more sophisticated engineered systems that manage the communities’ structure and function towards social goals. The Value of Reduced Products Historically, environmental biotechnology focused primarily on oxidizing reduced contaminants. Illustrating this is the most famous pollutant in environmental engineering, the biochemical oxygen demand, or BOD. BOD represents the electrons contained in a pollutant by their ability to be removed and transferred to oxygen (O2). For example, BOD can be represented by the removal of electrons from an organic molecule (CH2O) or ammonium (NH4

+): CH2O + H2O CO2 + 4H+ + 4e- NH4

+ + 3H2O NO3- + 10H+ + 8e-

The electrons can be transferred to dissolved oxygen, which consumes or “demands” this essential resource in the aquatic environment: O2 + 4H+ + 4e- 2H2O Traditional wastewater-treatment technologies, such as activated sludge, are means to carry out BOD oxidation and O2 consumption before the wastewater is discharged to a receiving water (Rittmann and McCarty, 2001). This concept of BOD oxidation applies not only to municipal wastewater, but also to treating industrial wastewaters, bioremediating oil spills and leaks, and making drinking water biologically stable. Today, environmental engineers and scientists are coming to realize that many of the greatest challenges for reclaiming water quality lie with oxidized contaminants, or those do not donate electrons, but receive them (Rittmann, 2004, 2006b). The list of oxidized contaminants is long. Some of the most important oxidized contaminants are: • Nitrate (NO3

-) and nitrite (NO2-) come from wastewaters and fertilizer run-off; they cause

methemoglobinemia in infants and spurs cultural eutrophication of waters. • Perchlorate (ClO4

-) comes from rocket fuel, propellants, and select Chilean fertilizers; it affects thyroid function and is an endocrine-disrupting chemical. • Selenate (SeO4

2-) comes from coal-fired power plants, oil refineries, metal smelters, and certain irrigated soils; it causes reproductive problems. • Chromate (CrO4

3-) comes from electroplating, mining, and fossil-fuel operations; it causes liver and kidney damage. • Arsenate (H2AsO4

-) is present in certain soils; it causes gastrointestinal damage, cardiac arrest, and cancer. • Chlorinated solvents, such as trichloroethene (TCE), are used as solvents and cleaning agents in industry and commerce; they are known or suspected carcinogens. Reducing the oxidized contaminants produces harmless products (e.g., N2 gas from NO3

- and NO2

-, H2O and Cl- from ClO4-, and ethene and Cl- from TCE) or easily removed solids (e.g., Se°

from SeO42-, Cr(OH)3 from CrO4

3-, and As2S3 from H2AsO4-). Bacteria are able to reduce all of

these oxidized contaminants, provided that a bio-available electron donor is supplied. While other electron donors work for some of the oxidized contaminants, research shows that all of them can be reduced when hydrogen gas (H2) is the electron donor (Banaszak et al. 1999; NRC

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2000; Rittmann, 2004; Nerenberg and Rittmann, 2004; Chung et al. 2006a,b,c; Chung et al., 2007). H2 can be delivered to bacteria indirectly by fermentation of organic compounds or directly by diffusion through a gas-transfer member (Rittmann, 2004). Thus, detoxification of oxidized contaminants that appear in water and wastewater involves their bio-reduction, because the reduced products are harmless or easy to remove. Other reduced products are of high value to society because they are readily used energy carriers. Different microbial communities can convert the energy value of diffuse and sometimes noxious biomass to one of the convenient energy carriers: • Methane gas (CH4) can be combusted to generate electrical energy with relatively low CO2 and NOx emissions per kW-hr. The biochemistry and microbial ecology of methanogenesis from complex organic matter are well studied, and methanogenesis is a proven technology (Rittmann and McCarty, 2001; Speece, 1996) for sludge and high-strength industrial wastewater. The infrastructure for distributing and utilizing CH4, or natural gas, is already in place in many locations. • Hydrogen gas (H2) is an alternate fermentation product that has the advantage, compared to CH4, that it can be utilized in a conventional fuel cell, producing pollution-free electrical energy (Fang et al., 2004; Logan, 2004). • Electricity can be produced directly in a microbial fuel cell, avoiding the need to generate H2 as an intermediate in order to have combustion-free and pollution-free electricity from biomass (Rabaey and Verstraete, 2005; Liu et al., 2004; Logan, 2004). • Fermentation of sugars to ethanol is a long-standing microbial biotechnology that has gained much attention recently as a renewable additive to or substitute for gasoline (Farrell et al., 2006). Downstream processing to produce fuel-grade ethanol is energy expensive, making ethanol a controversial biofuel. • Biodiesel is a promising transportation fuel that can be produced directly from solar energy by using cyanobacteria and algae (Huber et al., 2006). Biodiesel is comprised of C16 to C18 alkanes that are components in the lipids of these microbial phototrophs. The Revolution from Molecular Microbial Ecology Beginning in the last half of the 1980s, molecular tools directed towards the DNA or RNA of microorganisms began to penetrate and then revolutionize environmental biotechnology. The first genomic tools in molecular microbial ecology targeted the small sub-unit (SSU) rRNA, usually directly through hybridization with oligonucleotide probes (Stahl, 1986). The SSU rRNA, also called the 16S rRNA in prokaryotes, is a natural target to identify what types of microorganisms are present, or the phylogentic structure of the community. The advantages of directly targeting the SSU rRNA are that it is present in all independently living microorganisms, is naturally amplified, and has a mixture of conserved and evolved regions. The last feature makes it possible to design oligonucleotide probes that are specific to a single strain or that encompass a range of evolutionarily related strains. The second advantage makes it possible to visualize spatial relationships among different types of bacteria using fluorescent in situ hybridization (FISH) (Amann et al., 1990, 2001). The advent of oligonucleotide probes and FISH began the revolution by enabling researchers to detect and even “see” known types of microorganisms in complex communities, while avoiding the pitfalls of culturing methods. Dissemination of the rRNA-based tools ultimately helped lead to important discoveries about what are important and sometimes essential microorganisms for achieving treatment goals. One

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example is the discovery that Nitrospira usually are the important nitrite-oxidizing bacteria in aerobic nitrification systems (Daims et al., 2000). A closely related example is that the key ammonia–oxidizing bacteria, usually Nitrosomonas, normally live in very dense clusters inside a larger floc or biofilm (Mobarry et al., 1996; Wagner et al., 1998). A third example is the discovery of the now-famous Anammox bacteria, which oxidize ammonia to N2 gas while reducing nitrite (Jetten et al., 1998). The first two examples tell us the characteristics that we want to find in stable, aerobic nitrification. The third example opens up the possibility to oxidize ammonia anaerobically, as long as nitrite can be provided. Molecular microbial ecology continues to develop new tools that expand our abilities beyond the basics of phylogenetic structure. I highlight three new developments that are having a major impact already: quantitative real-time PCR, community fingerprinting, and gene-expression analysis. Quantitative real-time PCR (qRT PCR) is an important new development that is overcoming one of the limitations of the traditional hybridization techniques directed towards the SSU rRNA: poor or cumbersome quantification. qRT PCR (Mackay, 2004) overcomes the problem by monitoring the rate at which a target gene is amplified by PCR. While the gene for the SSU rRNA can be targeted by qRT PCR, other genes can be used to provide better specificity when the SSU rRNA does not discriminate well enough. Experimental and modeling results can be linked to genomic results directly when using qRT PCR. One of the biggest challenges in using molecular techniques in environmental biotechnology is that the important microorganisms often have never been identified, cultured, or sequenced. Thus, methods that rely on targeting a specific sequence of DNA or RNA are not feasible. However, we want to identify and track the “key players,” even if we do not know who they are. I highlight two of the several fingerprinting techniques help us to achieve this goal for uncharacterized strains. The first fingerprinting tool is denaturing gradient gel electrophoresis (DGGE) (Muyzer, 1999). A specific gene (often for the SSU rRNA, but not necessarily so) is amplified by PCR to produce a significant amount of DNA from any member of the community that has the target gene. That DNA is then placed at one end of a special electrophoresis gel that has a gradient of DNA denaturant (urea + formamide). As the negatively charged DNA moves toward the positive pole of the electrophoresis apparatus, it encounters stronger denaturant, and it denatures (opens up the two DNA strands) at a location that depends on the DNA’s C+G content. This yields a series of DNA bands that ought to correspond to a single strain, also known as an operational taxonomic unit (OTU). The banding patterns of DNA obtained over time or from different systems can be compared like fingerprints to assess changes in community structure. An advantage of DGGE is that a band can be excised and its DNA sequenced, giving insight into the phylogeny of the interesting, although unidentified strain. An alternative means to fingerprint a community is to build a clone library (Zhou et al., 1997; Juretschko et al., 2002). As with DGGE, the process begins with extracting the community’s DNA and then amplifying the DNA with a specified primer. The amplified DNA is then separatd by cloning. The separated SSU rRNA genes are screened, amplified, and sequenced. The clone library identifies sequences of putatively important strains and can be used to compare changes over time or across different systems.

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All of the methods mentioned so far address community structure, or “who is there.” Gene-expression analyses give insight into community function, or “what the community is doing,” by assessing what genes are expressed to produce messenger RNA (mRNA) or final protein products. The production of mRNA is measured by reverse-transcriptase PCR, in which the expressed mRNA is extracted, converted to DNA by reverse transcription, and then amplified by PCR (Freeman et al., 1999). Final protein products can be analyzed by the proteomic techniques of matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy (Halden et al., 2005). The combination of genomics and proteomics tools offer researchers and practitioners of environmental biotechnology the chance to understand the fine details of how microbial ecosystems work to provide us with desired services. The molecular tools help us know where to focus our community-management skills to ensure reliable and cost-effective processes. Managing Microbial Communities Effective community management demands that we have engineering tools that match our better understanding of the communities, as well as the rising expectations of society. Part of the management comes from the traditional and always essential tools of mass balance, kinetics, and modeling. Fortunately, we also can take advantage of a revolution occurring in materials engineering and science. Modern materials – for example, membranes, nano-particles, conductors, and semi-conductors – and physical/chemical processes are being adopted to expand the scope or reliability of environmental biotechnologies (Rittmann, 2006b). A few examples have long-standing histories: e.g., Powdered Activated Carbon Treatment (PACT®) for treating industrial wastewater having recalcitrant organic contaminants involves adding powdered activated carbon to activated sludge to adsorb very difficult-to-biodegrade and often toxic organics (Pitkat and Berndt, 1981); biofiltration after ozonation is used to remove difficult-to-biodegrade compounds and produce a biologically stable drinking water (Brunet et al., 1982; Sontheimer, 1978; Nerenberg et al., 2000). Here are a few newer examples of exciting “hybrid” systems. • The Membrane BioReactor (MBR) uses membrane filtration instead of sedimentation to achieve more reliable activated sludge treatment (Adham and Trussell, 2001; Stephenson et al., 2000; Daigger et al., 2005). • The Membrane Biofilm Reactor (MBfR) delivers H2 gas efficiently and safely to H2-oxidizing biofilm living on the outer wall of the membrane to remove one or many oxidized contaminants (Lee and Rittmann, 2002; Nerenberg et al., 2002; Rittmann et al., 2004; Rittmann et al., 2005). • Nano-scale TiO2 and UV light are used for advanced oxidation as a pre-treatment to make recalcitrant organics biodegradable (Ollis, 2001; Pulgarin et al., 1999; Rodriguez et al., 2002). • In a microbial fuel cell (MFC), a biofilm living on the anode of a fuel cell oxidizes organic “fuel” and transfers the electrons to the anode, instead of directly to a soluble electron acceptor (Liu et al, 2004; Rabaey and Verstraete, 2005; Rittmann, 2006a; Rittmann et al., 2006). The MBfR is an excellent example of the marriage of modern materials with the understanding of microbial communities. H2 has many advantages as an electron donor for driving microbial reduction reactions (Rittmann, 2006b), including that it should allow reliable reduction of many oxidized contaminants (noted above). Even so, H2 has not been used as an electron donor in the past, because it could not be delivered to bacteria efficiently and safely (Rittmann 2006b). Delivery by diffusion through the wall of gas-transfer membrane in an MBfR is the breakthrough that overcomes the roadblock. Success is achieved by matching a gas-transfer membrane with

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biofilm, making is possible to supply H2 directly to the bacteria and resulting in rapid and nearly 100% utilization of H2. As an added benefit, the supply rate of H2 to the biofilm is self-regulated by the loading of oxidized contaminant to the biofilm; the biofilm demands only as much H2 as it needs to reduce the oxidized contaminants. The capacity to supply H2 is easily controlled by adjusting the H2 pressure inside the membrane, thus making operation simple and almost foolproof. Marrying a H2-oxidizing biofilm to a gas-transfer membrane creates a platform technology. Thus, the MBfR can be used in many settings that involve one or more oxidized contaminants: • Removal of one or several of the oxidized contaminants (noted above) from drinking water or groundwater (Nerenberg et al., 2002; Nerenberg and Rittmann, 2004; Chung et al., 2006a,b,c; Chung et al. 2007) • Removal of one or several of the oxidized contaminants from industrial wastewater • Advanced N removal in wastewater treatment (Rittmann et al., 2005; Cowman et al., 2005). References Adham S. and Trussell R.S. (2001). Membrane Bioreactors: Feasibility and Use in Water

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