INVITED PAPER
Environmental Biotechnology in Water and Wastewater Treatment Bruce E. Rittmann, M.ASCE1 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 共e.g., nitrate, perchlorate, selenate, and chlorinated solvents兲 and the production of methane, hydrogen, and electricity. The key science underlying environmental biotechnology is microbial ecology, which has advanced rapidly in the past 20 or so years through the proliferation of new genomics-based techniques to characterize the communities’ structure and function. The genomic 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 achieve the management goals through partnering the microorganisms with modern materials and physical/chemical processes. The membrane biofilm reactor and microbial fuel cells offer excellent examples of exciting new technologies that come directly from this kind of partnering. DOI: 10.1061/共ASCE兲EE.1943-7870.0000140 CE Database subject headings: Biological processes; Ecosystems; Water treatment; Wastewater management. Author keywords: Bioreduction; Bioenergy; Biotechnology; 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 共Rittmann 2006a兲: 1. Microbial communities can detoxify contaminants in water, soils, sediment, and sludge. This allows society to reclaim their resource value. 2. 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, and electricity. 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 reduction reactions that provide them with energy to grow and human society with valuable services. It is the ideal “win-win” situation. Environmental biotechnology is a special case of the larger field of biotechnology. One thing that distinguishes environmental biotechnology from the other parts of biotechnology is that its science base is the field of microbial ecology 共Rittmann and McCarty 2001; Rittmann et al. 2006; Rittmann 2006a兲. As a sci1
Regents’ Professor of Environmental Engineering and Director of the Center for Environmental Biotechnology, Biodesign Institute, Arizona State Univ., P.O. Box 875701, Tempe, AZ 85287-5701. E-mail:
[email protected] Note. This manuscript was submitted on May 15, 2009; approved on July 23, 2009; published online on August 15, 2009. Discussion period open until September 1, 2010; separate discussions must be submitted for individual papers. This paper is part of the Journal of Environmental Engineering, Vol. 136, No. 4, April 1, 2010. ©ASCE, ISSN 0733-9372/ 2010/4-348–353/$25.00.
ence, microbial ecology aims to characterize a microbial community in terms of • What types of microorganisms are present 共the community’s phylogenetic structure兲; • What metabolic reactions these microorganisms carry out 共its metabolic function兲; and • How the microorganisms interact with each other and their environment. In most cases, the metabolic reactions constitute the services to society. The past 20 or so years have yielded remarkable advancements in genome-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 共Rittmann et al. 2006, 2008a兲. This spawned a field that is now called molecular microbial ecology. 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 共Rittmann 2006a兲. 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 toward social goals.
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
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oxygen demand 共BOD兲. BOD represents the electrons contained in a pollutant by their ability to be removed and transferred to oxygen 共O2兲. For example, BOD can be represented by the removal of electrons from an organic molecule 共CH2O兲 or ammonium 共NH+4 兲 CH2O + H2O → CO2 + 4H+ + 4e− NH+4 + 3H2O → NO−3 + 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 its receiving water 共Rittmann and McCarty 2001兲. 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 共Rittmann 2004, 2006b, 2007兲. The list of oxidized contaminants is long. Some of the most important oxidized contaminants are: • Nitrate 共NO−3 兲 and nitrite 共NO−2 兲 come from wastewaters and fertilizer run off; they cause methemoglobinemia in infants and spur cultural eutrophication and hypoxia in waters. • Perchlorate 共ClO−4 兲 mainly comes from rocket fuel, propellants, and select Chilean fertilizers, although it can occur naturally; it affects thyroid function and is an endocrine-disrupting chemical. • Selenate 共SeO2− 4 兲 comes from coal-fired power plants, oil refineries, metal smelters, and certain irrigated soils; it causes reproductive problems. • Chromate 共CrO3− 4 兲 comes from electroplating, mining, and fossil-fuel operations; it causes liver and kidney damage. • Arsenate 共H2AsO−4 兲 is present in certain soils; it causes gastrointestinal damage, cardiac arrest, and cancer. • Chlorinated solvents, such as trichloroethene 共TCE兲, are used as solvents and cleaning agents in industry and commerce; they are known or suspected carcinogens. Reducing the oxidized contaminants produces harmless products 共e.g., N2 gas from NO−3 and NO−2 , H2O and Cl− from ClO−4 , and ethene and Cl− from TCE兲 or easily removed solids 共e.g., Se0 3− − from SeO2− 4 , Cr共OH兲3 from CrO4 , and As2S3 from H2AsO4 兲. Bacteria are able to reduce all of these oxidized contaminants, provided that a bioavailable 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 共H2兲 is the electron donor 共Banaszak et al. 1999; National Research Council 共NRC兲 2000; Rittmann 2004, 2007; Nerenberg and Rittmann 2004; Chung et al. 2006a,b,c, 2007兲. H2 can be delivered to bacteria indirectly by fermentation of organic compounds or directly by diffusion through a gas-transfer member 共Rittmann 2004兲. Thus, detoxification of oxidized contaminants that appear in water and wastewater involves their bioreduction because the reduced products are harmless or easy to remove.
Other reduced products are of high value to society because they are readily useful 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 共CH4兲 can be combusted to generate electrical energy with relatively low CO2 and NOx emissions per kilowatt-hour. The biochemistry and microbial ecology of methanogenesis from complex organic matter are well studied, and methanogenesis is a proven technology 共Rittmann and McCarty 2001; Speece 1996兲 for sludge and high-strength industrial wastewater. The infrastructure for distributing and using CH4, or natural gas, is already in place in many locations. • Hydrogen gas 共H2兲 is an alternate fermentation product that has the advantage, compared to CH4, that it can be used in a chemical fuel cell, producing pollution-free electrical energy 共Fang et al. 2004; Logan 2004; Rittmann 2008兲. • Electricity can be produced directly in a microbial fuel cell 共MFC兲, avoiding the need to generate H2 as an intermediate to have combustion-free and pollution-free electricity from biomass 共Rabaey and Verstraete 2005; Liu et al. 2004; Logan 2004; Rittmann 2008兲.
Revolution from Molecular Microbial Ecology Beginning in the last half of the 1980s, genomic tools directed toward the deoxyribonucleic acid 共DNA兲 or ribonucleic acid 共RNA兲 of microorganisms began to penetrate and then revolutionize environmental biotechnology. The first genomic tools in molecular microbial ecology targeted the small subunit 共SSU兲 ribosomal RNA 共rRNA兲, usually directly through hybridization with oligonucleotide probes 共Stahl 1986兲. The SSU rRNA, also called the 16S rRNA in prokaryotes, is a natural target to identify what types of microorganisms are present, or the phylogenetic 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 variable 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 共FISH兲 共Amann et al. 1990, 2001; Sekiguchi et al. 1999兲. 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 共e.g., only microorganisms that can grow rapidly in the selective medium can be cultured兲. 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 example is the discovery that Nitrospira usually are the important nitrite-oxidizing bacteria in aerobic nitrification systems 共Daims et al. 2000; Siripong and Rittmann 2007兲. This finding overthrew the prevailing dogma that the important nitrite-oxidizing bacteria are Nitrobacter. Now, we are able to look for the truly important nitrite oxidizers. 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 共Mobarry et al. 1996; Wagner et al. 1998兲. We do not know why this is the case, but it is common in nitrifying activated sludge. A third example is the discovery of the now famous Anammox bacteria,
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which oxidize ammonia to N2 gas while reducing nitrite 共Jetten et al. 1998兲. 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 共Rittmann et al. 2008a兲. I highlight three new developments that are having a major impact already: quantitative polymerase chain reaction 共qPCR兲, fingerprinting, and gene expression. qPCR is an important new development that is overcoming one of the limitations of the traditional hybridization techniques directed toward the SSU rRNA: poor or cumbersome quantification. qPCR 共Mackay 2004; Yu et al. 2005兲 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 qPCR, 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 qPCR. 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 three of the several fingerprinting techniques that help us to achieve this goal for uncharacterized strains. The first fingerprinting tool is denaturing gradient gel electrophoresis 共DGGE兲 共Muyzer 1999; Rittmann et al. 2008a兲. A specific gene 共often for the SSU rRNA, but not necessarily so兲 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 共urea+ formamide兲. As the negatively charged DNA moves toward the positive pole of the electrophoresis apparatus, it encounters stronger denaturant, and it denatures 共opens up the two DNA strands兲 at a location that depends on the DNA’s guanine cytosine content. This yields a series of DNA bands that may correspond to a single strain, also known as an operational taxonomic unit 共OTU兲. 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. A second means to fingerprint a community is to build a clone library 共Zhou et al. 1997; Juretschko et al. 2002; Rittmann et al. 2008a兲. As with DGGE, the process begins with extracting the community’s DNA 共most often, 16S ribosomal DNA兲 and then amplifying the DNA with a specified primer set. The amplified DNA is then separated by cloning into competent Escherichia coli cells. 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. The third fingerprinting technique is pyrosequencing 共Nyren et al. 1993; Ronaghi et al. 1996; MacLean et al. 2009兲, which is one of the ”next generation” sequencing methods. Pyrosequencing avoids the cloning step by going directly from PCR amplification to sequencing. It provides very high throughput, allowing an entire bacterial genome to be sequenced in a few days 共MacLean et al. 2009兲. Pyrosequencing has opened up the
field of metagenomics 共Dinsdale et al. 2008兲, in which targeted DNA of an entire community can be sequenced in parallel, providing a very deep phylogenetic fingerprint of complex communities. 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 共mRNA兲 or final protein products. The production of mRNA is measured by reverse-transcriptase 共RT兲 PCR, in which the expressed mRNA is extracted, converted to complementary DNA by reverse transcription, and then amplified by PCR 共Freeman et al. 1999; Rittmann et al. 2008a兲. It also is possible to do qPCR on the mRNA transcripts, or RT-qPCR. Final protein products also can be analyzed by the proteomic techniques of matrix-assisted laser desorption/ionization 共MALDI兲 mass spectroscopy 共Halden et al. 2005; Rittmann et al. 2008a兲. The combination of genomic and proteomic 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, nanoparticles, conductors, and semiconductors—and physical/chemical processes are being adopted to expand the scope or reliability of environmental biotechnologies 共Rittmann 2006b兲. Two examples have long-standing histories: e.g., powdered activated carbon treatment 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 共Pitkat and Berndt 1981兲 and biofiltration after ozonation is used to remove difficultto-biodegrade compounds and produce a biologically stable drinking water 共Brunet et al. 1982; Sontheimer 1978; Nerenberg et al. 2000兲. Here are a few newer examples of exciting “hybrid” systems: • The membrane bioreactor uses membrane filtration instead of sedimentation to achieve more reliable activated sludge treatment 共Adham and Trussell 2001; Stephenson et al. 2000; Daigger et al. 2005兲. • The membrane biofilm reactor 共MBfR兲 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 共Lee and Rittmann 2002; Nerenberg et al. 2002; Rittmann et al. 2004; Rittmann 2007兲. • Nanoscale TiO2 and ultraviolet light are used for advanced oxidation as a pretreatment to make recalcitrant organics biodegradable 共Ollis 2001; Pulgarin et al. 1999; Rodriguez et al. 2002; Marsolek et al. 2008兲. • In a MFC, a biofilm living on the anode of a fuel cell oxidizes organic “fuel” 共often from wastewater, sludge, or other organic-rich wastes兲 and transfers the electrons to the anode,
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instead of directly into a soluble electron acceptor 共Liu et al. 2004; Rabaey and Verstraete 2005; Rittmann 2006a; Rittmann et al. 2006; Marcus et al. 2007兲. The electrons are transferred through an electrical circuit, where they reduce O2 to form H2O. The output is renewable electricity produced directly from the “biomass fuel” and without combustion. • A microbial electrolysis cell 共MEC兲 is a variation on the MFC in which O2 is excluded from the cathode. Then, the electrons that reach the cathode reduce H+ to form H2 gas 共Liu et al. 2005; Rittmann 2008; Lee et al. 2009兲. The MEC is a means to produce a high yield of renewable H2 gas from biomass. 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 共Rittmann 2006b, 2007兲, including that it should allow reliable reduction of many oxidized contaminants 共noted above兲. 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. 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 biofilm, making it possible to supply H2 directly to the bacteria and resulting in a 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 共noted above兲 from drinking water or groundwater 共Nerenberg et al. 2002; Nerenberg and Rittmann 2004; Chung et al. 2006a,b,c, 2007; Rittmann 2007; Ziv-El and Rittmann 2009兲; • Removal of one or several of the oxidized contaminants from industrial wastewater; and • Advanced N removal in wastewater treatment 共Cowman et al. 2005; Hasar et al. 2008兲. Marrying a biofilm to the anode in an MEC or MFC is a second example of how understanding microbial ecology can lead to a major technological advance. The key to success in an MFC or MEC is selecting for bacteria that are able to transfer electrons to the anode, which is a solid conductor, not a soluble molecule. Only recently have researchers realized that some bacteria are able to transfer the electrons from an electron carrier in their outer membrane to an anode located 10– 100s of micrometer away from the bacteria. These bacteria are best called anode-respiring bacteria 共ARB兲, since they gain energy for their growth by respiration of electrons to the anode surface 共Rittmann et al. 2008b兲. The ARB grow as a biofilm on the anode surface, where they construct a conductive matrix to transfer the electrons rapidly to the anode and in response to the electrical potential at the anode 共Torres et al. 2008兲. Thus, providing sufficient anode surface and controlling the anode potential are key management tools to have an efficient biofilm anode.
Conclusions The key science underlying environmental biotechnology is microbial ecology, which has advanced rapidly in the past 20 or so years through the proliferation of new genomics-based techniques
to characterize the communities’ structure and function. The genomic 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. The management goals are achieved through exploiting modern materials and physical/chemical processes to create an environment that selects for the right microorganisms and ensures that they carry out the desired services. The MBfR, MFC, and MEC offer particularly good examples of new technologies that come directly from this kind of management approach.
Acknowledgments Dr. Rittmann presented this paper as the Simon W. Freese Lecture on May 19, 2009 at the EWRI Annual Conference in Kansas City, Mo.
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