RESEARCH NEEDS FOR BIOAUGMENTATION - Bioaugmentation for Groundwater Remediation - page 347

activities that may not be present, or may be present at very low levels in the natural environ-
ment, and (2) to select hardy, environmentally adapted microbes for genetic engineering and
introduction, thereby increasing the likelihood of microbial establishment and survival. This
latter consideration is critical in difficult to remediate sites where mixed chemical pollution or
harsh conditions exist. In some cases, the introduced organism may provide an activity (i.e., one
or more enzymatic steps) that bridges the degradative pathways of the indigenous communities
or allows activity under the prevailing site conditions (e.g., anaerobic vs. aerobic conditions).
As the understanding of transformative pathways (such as reductive dehalogenation or dissim-
ilatory iron reduction) improves, it will be possible to construct strains expressing more
efficient enzymes and to identify gaps in degradative pathways that can be filled with designed
microbes.
Other advanced genetic alterations may be at the level of regulation. Altering regulatory
controls can be used to overcome limitations on bioremediation performance, such as those
associated with inhibition from mixed contaminants or particular environmental conditions
(e.g., pH or redox levels). In some cases genetic modifications may involve introduction of
novel transporters allowing uptake of contaminants not otherwise accessible to the cell (Saleem
et al., 2008 ). In addition to genetic modification to obtain strains of interest, improved culturing
methods have been developed which promote adaptive evolution (i.e., mutations producing
desired strain alterations) (Suenaga et al., 2009 ). Strains of interest produced by this method are
highly desirable from a bioaugmentation standpoint as they bypass some of the policy concerns
associated with introduction of organisms into the environment. Furthermore, this natural
adaptation, as opposed to expression of a heterologous activity, may have a lower energy
cost, making these organisms better able to survive and thrive after introduction into the
subsurface environment.
Another application of designer strains has been as biosensors for detecting and
measuring biological activities, contaminant levels, biological oxygen demand or other features
of the environment. Applications on this front have involved introductions of chromosomally
associated detection genes or genes which promote surface-based modifications. In the first
case, fluorescence genes such as luciferase ( lux ) or green fluorescent protein ( gfp ) are fused
downstream of target genes, with activation by appropriate substrates resulting in cell fluores-
cence (Dawson et al., 2008 ). Though not quantitative, this approach can be useful as an
indicator of contaminant presence and/or conversion depending on the choice of physiological
gene. Alternatively, designer microbes that provide surface expressed biological detectors (such
as single chain variable fragments) can be constructed (Dhillon et al., 1999 ). In these cases, the
cell surface functions as a detector of a particular contaminant or transformation product.
Developments in nanoscale biosensors (comments above) may have a profound influence
on both the need and the modifications applied to bacterial strains for application in the
environment.
The ability to synthesize, manipulate and clone large fragments of DNA has led to the
successful cloning of a complete synthetic bacterial genome (Gibson et al., 2010 ). It now seems
possible to eventually generate a whole organism with a specific suite of genes. However, it is
still unknown how well such a designer organism will fare when introduced into a variable and
resource competition driven environment, or which genes to include to ensure survival at a
contaminated site. These are still a long way off.
12.5.3 Bioaugmenting with Genes
The capacity for DNA transfer among bacteria presents the possibility of introducing new
genetic capabilities into environments without the need to specifically engineer strains or