INVITED PAPER

Environmental Biotechnology in Water

and Wastewater Treatment
Bruce E. Rittmann, M.ASCE1
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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 ence, microbial ecology aims to characterize a microbial commu-

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 vari-
ous types of biomass from its diffuse and sometimes hazard-
ous 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, microorgan-
isms possess unparalleled capabilities to do oxidation and reduc-
tion 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 sci-
1
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:
rittmann@asu.edu
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.
nity 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 commu-
nities in these ways, and this has led to the discovery of new
microorganisms, new metabolic capabilities, and new biotech-
nologies 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 engi-
neered systems that partner the microbial communities with mod-
ern materials and physical/chemical processes 共Rittmann 2006a兲.
We are fortunate that materials science and engineering are ad-
vancing as rapidly as is molecular microbial ecology. Thus, our
expanding understanding of the structure and function of micro-
bial communities can be matched by ever more sophisticated en-
gineered 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 fa-
mous pollutant in environmental engineering, the biochemical

348 / JOURNAL OF ENVIRONMENTAL ENGINEERING © ASCE / APRIL 2010

J. Environ. Eng. 2010.136:348-353.

 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 re-
moval of electrons from an organic molecule 共CH2O兲 or ammo-
nium 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 con-
sumes or “demands” this essential resource in the aquatic envi-
ronment
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O2 + 4H+ + 4e− → 2H2O
Traditional wastewater-treatment technologies, such as activated
sludge, are means to carry out BOD oxidation and O2 consump-
tion 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, propel-
lants, and select Chilean fertilizers, although it can occur natu-
rally; it affects thyroid function and is an endocrine-disrupting
chemical.
• Selenate 共SeO2− 4 兲 comes from coal-fired power plants, oil re-
fineries, 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 gas-
trointestinal 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 prod-
ucts 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
from SeO2− 4 , Cr共OH兲3 from CrO4 , and As2S3 from H2AsO4 兲.
3− −
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 contami-
nants, research shows that all of them can be reduced when hy-
drogen 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 mem-
ber 共Rittmann 2004兲. Thus, detoxification of oxidized contami-
nants that appear in water and wastewater involves their
bioreduction because the reduced products are harmless or easy to
remove.
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J. Environ. Eng. 2010.136:348-353.
Other reduced products are of high value to society because
they are readily useful energy carriers. Different microbial com-
munities 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 in-
dustrial wastewater. The infrastructure for distributing and
using CH4, or natural gas, is already in place in many loca-
tions.
• 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 bio-
mass 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 revolution-
ize environmental biotechnology. The first genomic tools in mo-
lecular 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 con-
served 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 sec-
ond advantage makes it possible to visualize spatial relationships
among different types of bacteria using fluorescent in situ hybrid-
ization 共FISH兲 共Amann et al. 1990, 2001; Sekiguchi et al. 1999兲.
The advent of oligonucleotide probes and FISH began the revo-
lution 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 ex-
ample 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 ex-
ample is the discovery of the now famous Anammox bacteria,
 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 struc-
ture 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 quantifica-
tion. qPCR 共Mackay 2004; Yu et al. 2005兲 overcomes the prob-
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lem 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 microorgan-
isms 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 high-
light three of the several fingerprinting techniques that help us to
achieve this goal for uncharacterized strains.
The first fingerprinting tool is denaturing gradient gel electro-
phoresis 共DGGE兲 共Muyzer 1999; Rittmann et al. 2008a兲. A spe-
cific 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 nega-
tively charged DNA moves toward the positive pole of the elec-
trophoresis apparatus, it encounters stronger denaturant, and it
denatures 共opens up the two DNA strands兲 at a location that de-
pends 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 com-
pared 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 inter-
esting, 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 puta-
tively 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. Pyro-
sequencing 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
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J. Environ. Eng. 2010.136:348-353.
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 struc-
ture, 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 ex-
pressed 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 re-
searchers 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 engi-
neering tools that match our better understanding of the commu-
nities, 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, mem-
branes, 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., pow-
dered activated carbon treatment for treating industrial wastewa-
ter 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 difficult-
to-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 treat-
ment 共Adham and Trussell 2001; Stephenson et al. 2000;
Daigger et al. 2005兲.
• The membrane biofilm reactor 共MBfR兲 delivers H2 gas effi-
ciently and safely to H2-oxidizing biofilm living on the outer
wall of the membrane to remove one or many oxidized con-
taminants 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 bio-
degradable 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 oxi-
dizes organic “fuel” 共often from wastewater, sludge, or other
organic-rich wastes兲 and transfers the electrons to the anode,
 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
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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 mem-
brane 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 bio-
film; 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 respi-
ration 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 mi-
crobial ecology, which has advanced rapidly in the past 20 or so
years through the proliferation of new genomics-based techniques
JOURNAL OF ENVIRONMENTAL ENGINEERING © ASCE / APRIL 2010 / 351
J. Environ. Eng. 2010.136:348-353.
to characterize the communities’ structure and function. The ge-
nomic methods provide detailed information that helps us under-
stand what aspects of the microbial community need to be
managed to ensure that it provides the desired service. The man-
agement 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 par-
ticularly 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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