RESEARCH NEEDS FOR BIOAUGMENTATION - Bioaugmentation for Groundwater Remediation - page 348

introduce non-native organisms (Cortez et al., 2009 ). In this approach, DNA encoding the genes
required for the reaction of interest (e.g., degradation) is delivered to the contaminated site. The
delivery of this DNA can be accomplished by bioaugmentation with strains harboring donor
DNA, or by injecting DNA encapsulated in delivery materials such as alginate. Through this
approach, new functions could be stably introduced into indigenous populations already
adapted to the environment at hand. Key considerations for application of this approach are
gene degradation rates and the survival times of delivery strains. In some cases, the donor
strains themselves can survive in the introduced environments, promoting continued gene
transfer and presenting an additional active group capable of target degradative ability.
In these cases, bioaugmentation is occurring both through DNA transfer as well as the more
classical introduction of a bacterial population. It is actually highly likely, given that we now
realize that some reductive dehalogenase genes are on mobile genetic elements (Maillard et al.,
2005 ; McMurdie et al., 2011 ), that we may be unintentionally transferring reductive dehalo-
genase genes to native competent species during bioaugmentation with, for example, Dehalo-
coccoides -containing cultures. Such DNA transfer, if it is occurring, is beneficial to

remediation efforts, as it would accelerate the propagation of desired traits.
The less classical approach, injection to the subsurface of encapsulated DNA, requires that
the DNA survive injection, that a cell within the environment take in the released DNA, and that
the DNA is successfully transcribed into an active protein product. This approach may sound
implausible, but the mechanisms for plasmid DNA uptake and regulation of gene expression
within bacteria are becoming better understood, and it is not impossible to foresee this system
becoming industrially applicable. The main advantage of this option is enhanced delivery,
because the encapsulated DNA may be transported in the environment without the additional
need of donor cell survival. DNA encapsulation in synthetic materials allows extended delivery
possibilities in harsh environments where establishment of nonindigenous populations would be
limited.

12.5.4 Bioaugmenting with Viruses

Though not extensively studied, the possibility of using lysogenic bacteriophages to deliver
novel genetic traits has been proposed. In this system, bacteriophages, possibly encased in
biodegradative polyester microspheres, would be delivered to the subsurface. This approach
may present advantages over plasmid DNA delivery (described above in Section 12.5.3 ),
because the bacteriophage DNA is inserted on the chromosome, which provides more stable
maintenance of genetic elements than plasmid insertions.
Considerations with this approach relate to phage specificity and the ability of phages to
attack microbial strains of interest in the subsurface environment. Currently, it is uncertain how
conditions in the environment may modulate prophage switching between lysogenic and lytic
phases, so it is difficult to control or target bacteriophage infections. But the ability of phages to
replicate in the cell and then infect novel hosts offers a promising approach to promoting greater
transfer of desired genetic elements within the subsurface (Sobecky and Coombs, 2009 ).
Development of a viral bioaugmentation assay requires a gene whose function is clearly
defined in terms of activity and substrate range. A fully characterized gene would allow
introduction of a targeted activity to a site, and hence tracking of the success of the bioaug-
mentation. While this seems like a reasonable requirement, the reductive dehalogenases, as a
putative family of interest for this technique, have proven difficult to characterize, and for
many, the substrate range is entirely unknown. This technique also would require an infected
bacterium to properly synthesize the novel gene. This is most likely to be successful if the gene
is introduced to an organism capable of synthesizing similar proteins.