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Research at the Center for Environmental Biotechnology is use-inspired, which means that it is directed toward providing an essential service to make society more sustainable. All research in the center is guided by these three principles:

1. We employ leading-edge research tools – including molecular microbial ecology and modeling – so that we can develop a fundamental understanding of microbial communities and “think like the microorganisms.”
2. We apply modern materials – including membranes and nano-materials – and good engineering strategies to “create systems that work for the microorganisms so that they work for us.”
3. We partner with private and public companies, individual investors and the public sector to commercialize our technologies and get them into the marketplace.

This breadth of activities at the Center of Environmental Biotechnology and its use-inspired focus are what make it unique as a research center. Researchers in the center constantly integrate tools from many disciplines and are stimulated by real-world goals. The center is uniquely able to create new knowledge about microbial communities and apply that knowledge to play a role in creating a sustainable future.

One example of a new biotechnology used to treat lowquality water is the hydrogen-based membrane biofilm reactor (MBfR), an environmental biotechnology that Bruce Rittmann has taken from fundamental research through commercialization activity. In the MBfR, we deliver hydrogen gas (H₂) to microorganisms by allowing it to diffuse through the walls of special membranes that prevent H₂ bubbles from forming. The microbial community grows naturally as a biofilm on the outer wall of the membranes, by removing electrons from H₂ and transferring the electrons to one or more oxidized contaminants in the water. The scientific breakthrough behind the MBfR is the discovery that bacteria that oxidize H₂ are able to reduce almost any oxidized contaminant.The oxidized contaminants include nitrate and many new water contaminants whose harmful effects have recently been discovered. These include perchlorate, chromate, selenate and trichloroethene (TCE). Reducing these contaminants renders them harmless or easily removed from the water by common watertreatment methods. This breakthrough is translated into an effective, reliable, efficient and safe technology.

Biofilms refer to the assemblages of ‘microorganisms and biologically produced macromolecules attached to a surface.’ Biofilms can be found everywhere that moisture is in prolonged contact with a surface.

Biofilms can be categorized as good or bad. Good biofilms are responsible for nutrient cycling in nature and for biodegradation of pollutants in water treatment technology, such as the MBfR. Engineers often wish to develop a dense and firmly anchored biofilm in treatment technologies. Bad biofilms, or ‘biofouling,’ develop on surfaces in which microorganisms are not desired. Biofouling in a water line may interfere with the system’s circulation or may harbor pathogens and is a major source of infection in humans and plants.

During the formation of biofilms, microorganisms usually encase themselves in an extracellular polymeric substances (EPS) or a slime matrix, which improves microbial adhesion to the surface and to other cells. EPS in biofilms protect microbial cells from biocides and also increase tolerance to physical and chemical disturbances. EPS clearly plays a role in making the biofilm firmly attached or easily removed.

The development of biofilms – good or bad, strongly or weakly anchored -- depends on the interactions among microbiological, architectural, and mechanical properties. In the center, these phenomena are studied with state-of-the-art experimental measurements and with mathematical modeling, described in the link to Molecular Microbial Ecology below. Through this integrative research, we can understand how factors like the water-flow velocity, microbial species and substrate availability make the biofilm strong or weak, thick or thin, and simple or complex.

Hydrogen is a proposed “fuel of the future.” The reason for this hope for hydrogen is the development of efficient fuel cells that convert the energy in hydrogen to electricity with high efficiency and zero emissions of air pollutants. However, the future for hydrogen is clouded. Although hydrogen itself is very clean and attractive, almost all of the hydrogen produced at the present time comes from non-renewable fossil sources, such as natural gas and coal. This non-renewable cloud over hydrogen can be lifted by biohydrogen production.

Biohydrogen refers to hydrogen produced by algae, bacteria, or biological components of these organisms. These organisms use renewable biomass or sunlight to produce hydrogen. As our society strives for renewable hydrogen, biohydrogen will be a main alternative. Furthermore, it integrates waste treatment with clean-energy production from renewable sources. For example, biohydrogen can be used to produce electricity in a fuel cell, and it also is a special electron donor for bacteria in treatment processes used for reduced contaminants (such as the hydrogen-based membrane biofilm reactor). Although microorganisms can produce biohydrogen by various processes, fermentation is the simplest process and one we are exploring.

Fermentation is the essential first step in any process to recovery energy from biomass. Biomass is made up of complex organic molecules. Fermentation generates a mixture of simpler molecules: organic acids, alcohols and hydrogen. Thus, a great advantage of fermentation is fast degradation of solids and other complex organics found in wastes and agricultural products. On the other hand, fermentation today converts only about 15 percent of the energy to hydrogen. While fermentation is fast, it is not yet efficient for capturing the energy value of biomass to hydrogen.

Our center’s goal is to increase the biohydrogen yield to around 85 percent. To do this, we are investigating a multi-faceted research agenda that involves two complementary strategies. The first strategy involves controlling the microbial ecology in the fermentation process so that electrons and energy flow to hydrogen, instead of being diverted to other end products. The second strategy involves directing the electrons and energy in the other fermentation end products to biohydrogen in a coupled bioprocess. Each strategy relies on understanding the microbial ecology and using modern materials and good engineering to control the ecology so that the maximum biohydrogen is produced.

In wastewater treatment plants, a complex community of microorganisms degrades the organic solids in sludges and converts the energy value to methane gas (CH₄). Microbiological generation of methane, or methanogenesis, has been used for over a century, but it is not yet efficient enough to use in terms of destroying the organic solids in the sludge or capturing the energy value as methane. Often, the methane gas is “flared,” or simply burned in an open flame, because the amount of methane is not worth the cost to capture, clean and convert to electricity. Research in our center aims to overcome these limitations by increasing production and capturing renewable energy as methane.

One excellent example is a research project investigating the use of forced pulse (FP) technology to pre-treat the sludge in order to increase the yield of methanogenesis and decrease the residual solids requiring disposal. We use a FP unit designed by our partner, a private company called OpenCEL, to lyse cells and break apart biosolids, thereby increasing the energy sources (e.g., sugars) available to the microbial community, including the methanogens. We also partner with the Mesa Northwest Water Reclamation Plant, which hopes to increase the yield of methane from their digested sludge enough that they can afford to convert the methane to electricity, perhaps transforming their wastewater treatment facility from an energy consumer to an energy producer.

Two anaerobic digesters containing waste activated sludge from the Mesa Northwest Water Reclamation Plant are producing methane via microbial transformations. The right reactor contains sludge pre-treated with a Pulsed Electric Field (PEF) process that lyses cells and makes organic material more available for the methanogenic bacteria to digest, thereby increasing the destruction of organic solids and the production of methane.

A bird’s-eye view of the Mesa Northwest Water Reclamation Plant shows the many structures and processes that are involved in treating wastewater. In the center of the photograph, two large domes serve as anaerobic digesters, where methane is produced by bacteria. (photo courtesy of www.cityofmesa.org)

Biological processes are the overwhelming choice for wastewater treatment due to their ease of use, excellent performance, and efficiency. However, when challenged with toxic organic compounds, these processes can be ineffective and in some cases fail, resulting in discharge of harmful pollutants into the environment.

Advanced oxidation processes (AOPs), such as photocatalysis, have wide-ranging applicability, are not susceptible to process upsets from toxic inputs, but generally are not cost-effective nor practical options for many real situations.

By combining these two technologies in a process called photobiocatalysis, we take advantage of the benefits of each, while minimizing their drawbacks. Traditional work on coupled chemical-biological treatment focused on sequentially coupled systems, or those that have the chemical and biological processes in separate stages, but these systems suffer from the indiscriminate nature of advanced oxidation, which results in a large range of products, including those that are too oxidized, toxic themselves, or unavailable for biodegradation. This situation could be improved by combining the two operations into a single-stage, called intimate coupling, whereby bacteria are in close proximity to advanced oxidation, and can therefore remove biodegradable products as they are formed, focusing chemical oxidant on the non-biodegradable fraction.

We achieve intimate coupling by using a photocatalytic circulating-bed biofilm reactor, or PCBBR, shown here, which exploits biofilm carriers to hold and protect the bacteria from harmful advanced oxidation and toxic compounds, but places the bacteria as close as possible to the advanced oxidation so that the biodegradable products are removed as soon as they are produced, focusing the chemical oxidant on the non-biodegradable fraction.

The PCBBR technology offers the potential to efficiently and thoroughly treat many toxic wastewaters including those contaminated with halogenated aromatics, endocrine disrupting compounds, munitions, and a wide range of harmful industrial inputs including pharmaceutical wastes and textile dyes, resulting in a cleaner water being discharged to the environment, a healthier environment, and a healthier world for us to live in.

A revolutionary new environmental biotechnology – the Microbial Fuel Cell (MFC) – turns the treatment of organic wastes into a source of electricity. Bacteria growing as a biofilm on an electrode in a fuel cell oxidize the organic pollutants and transfer the electrons to the electrode, into an electrical circuit, and eventually to oxygen at a second electrode.

The MFC is revolutionary for three reasons. First, it makes the treatment of organic pollutants a direct producer of electricity, not a consumer. Second, it expands fuel-cell technology to use renewable organic materials as a fuel; conventional fuel cells use hydrogen gas, which is today produced from fossil fuels. Furthermore, the MFC can use organic fuels that are wet, the usual form for wastes and fuel crops. Third, the MFC, by operating at ambient temperature, can double to triple the electricity-capture efficiency over combustion, while eliminating all the air pollution that comes from combustion.

The scientific breakthrough leading to the MFC is the recent discovery that some bacteria can transfer electrons into an electrode and create electricity. This breakthrough is translated into a technology by using modern membrane and electrode materials that are compatible with biofilm growth and operation at ambient temperature. To develop economic opportunity for all people around the world, we are in a race to find a nonpolluting bioenergy source of energy. Bioelectric power, using crop or animal waste, could prove to be the solution.

Among the most toxic chemicals of global concern are the radionuclides found in contaminated waste streams and groundwater. For example, former nuclear-weapons facilities are severely contaminated with plutonium, uranium, and neptunium because of past practices that released mixtures of the radionuclides and other organic and inorganic wastes into adjacent soil and groundwater. The greatest potential for cleanup of such sites is in situ bioremediation, which exploits the reactions of microorganisms to directly or indirectly alter the chemical form of the radionuclides, rendering them immobile and less toxic. The microorganisms interact directly with the radionuclides by catalyzing chemical redox transformations. The microorganisms act indirectly by producing acids, bases and complexing ligands that react with the radionuclides.

Radionuclide bioremediation is a complicated situation, since the fate of radionuclide depends on so many different reactions, which proceed through multiple steps and at vastly different rates. Such a complex scenario can be understood and controlled only by using mathematical modeling. Such a unique biogeochemical mathematical model, CCBATCH, was developed by Bruce Rittmann and colleagues to connect all the different types of reactions that control the fate of radionuclides and a large range of metals. Current research is focused on applying CCBATCH towards bioremediation of plutonium, one of the most toxic materials.

To create a clean environment for all the people around the world, it is necessary to mitigate waste problems left behind as legacies of dangerous past activities, such as making nuclear weapons. When understood well, such as with CCBATCH, in situ bioremediation has great promise to deal with one of the most difficult challenges -- groundwater and soil contamination by radionuclides, such as plutonium.

Environmental bioremediation, the concept of harnessing the metabolic capabilities of microbes to biodegrade environmental contaminants, is in widespread use today. Medical Bioremediation is the new proposal to apply this successful concept to a novel waste problem: the accumulation of pathogenic materials inside the human body with aging. Important diseases associated with an “age-related waste problem” are heart disease (cholesterol and its derivatives in the artery wall), diabetes (protein modifications due to exposure of the tissues to high sugar levels), Alzheimer?s disease (beta-amyloid plaques and paired helical filaments (PHF)tau in the brain), and age-related macular degeneration (lipofuscin of the retinal pigment epithelium). As many as two thirds of all deaths in the U.S. can be associated with some sort of age-related ?waste problem,? and, as the population ages, this fraction will increase further.

To obtain a proof of concept of medical bioremediation, we have isolated bacteria that degrade 7-ketocholesterol, a major pathological cholesterol derivative accumulating in our coronary arteries. 7-ketocholesterol can poison our immune cells and keep them from cleaning out arterial cholesterol deposits. Using classical biochemical and innovative forward-genetics methods, we are now identifying the enzymes that the bacteria use to break down 7-ketocholesterol. Eventually, one or a small number of these enzymes could be administered to the human bloodstream in a manner similar to insulin injections. Hopefully, they would degrade the 7-ketocholesterol and allow our immune cells to remediate arterial cholesterol plaques.

We are also investigating several other target compounds underlying the age-related diseases noted above.

The scientific core of environmental biotechnology is microbial ecology, a scientific discipline dedicated to understanding complex communities of microorganisms: what microorganisms are present, what is their metabolic potential, what part of the potential are they realizing, and how they interact with each other and their environment. Fundamental scientific research in microbial ecology provides us with a deep understanding of how the complex communities work. In summary, the Center for Environmental Biotechnology employs a range of tools from molecular microbial ecology so that it can understand complex microbial ecosystems, allowing us to “think like the microorganisms.”

Fortunately, we have powerful new tools to help us analyze these intriguing organisms and their community organization. Among the tools we can use are molecular methods that probe the genetic information of the microorganisms in microbial communities. By targeting genomic DNA, we can identify which microorganisms are present and what reactions they can perform; through the use of RNA-based techniques we can identify the members in the community that are actively growing and what reactions they are performing. Using in-situ techniques, we can also investigate the metabolic interactions that take place among microorganisms in a mixed community. Applying these molecular tools to understand microbial communities is called molecular microbial ecology, a critical research strength of our c.

In particular, the center has exceptional capabilities to investigate changes in microbial structure through the use of molecular techniques that target the 16S rRNA gene and the 16S rRNA, such as denaturing gradient gel electrophoresis (DGGE), real-time PCR, and fluorescent in situ hybridization (FISH). DGGE is especially powerful for doing “detective work” to identify important, but uncharacterized strains. Real-time PCR is powerful for quantifying the different types of microorganisms. FISH allows us to do 3-Dimensional visualization of the communities and understand the interactions among different strains.

The center also targets specific metabolic genes and their expression to investigate the role of critical enzymes in detoxification and energy-generating reactions occurring in natural or engineered techniques including reverse transcriptase PCR, RNA or cDNA microarrays, and real-time PCR to quantify over- and under-expression of specific genes and for microarray validation. Identification of specific genetargets will allow us to investigate critical factors affecting the performance of natural or engineered microbial systems. The expression of such critical genes can be used to monitor and evaluate the success of engineered processes.