Review of Recent EPA TERAs for Outdoor Research with Modified Microorganisms

In a recent blog post, I summarized submissions to the U.S. Environmental Protection Agency for outdoor field tests of genetically modified microorganisms under the TERA (TSCA Environmental Release Application) provisions of the Agency’s biotechnology regulations under the Toxic Substances Control Act (TSCA). In today’s post, and the ones that will follow, I’ll briefly describe the most recent TERAs, beginning with those filed with EPA since 2014.

As previously described, the commercial use of modified microorganisms for purposes subject to TSCA jurisdiction might be subject to EPA’s biotechnology regulations under TSCA. Commercial use or importation into the U.S. of certain modified microorganisms (i.e., “new organisms” containing coding nucleic acids from more than one taxonomic genus) for purposes that are within TSCA’s jurisdiction require prior submittal to EPA of a Microbial Commercial Activity Notice (MCAN). Applications potentially subject to TSCA jurisdiction include production of industrial chemicals (including biofuels and certain enzymes), as well as uses of microorganisms for environmental purposes such as bioremediation and non-pesticidal agricultural uses. The latter category might include plant inoculants to promote nitrogen fixation, soil amendments, biofertilizers and plant biostimulants. Although R&D activities are largely exempt from TSCA oversight, the biotechnology regulations require that research involving “new microorganisms” in the open environment (outside a “contained structure”) require EPA review before the research can be conducted, through the filing of a TERA.  The TERA process provides an expedited review procedure for small-scale field tests and other outdoor R&D uses of new organisms, with applicants required to submit TERAs at least 60 days in advance of the proposed activity. See my earlier blog post for more background on TERAs, their data requirements, and the review process.

As I reported in the earlier post, there has only been limited experience with TERAs since the biotechnology rule was put into place in 1997.   According to EPA’s websites, there have been about 40 TERAs submitted for open environmental use of engineered microorganisms, and all but four of these proposals were approved. I have reviewed the TERAs filed prior to 2014 in previous posts, and in today’s post I will describe three TERAs filed in 2014 and 2015.

TERAs R-15-0001 and R-15-0002 were submitted by Elemental Enzymes Ag and Turf, LLC. According to the company’s website, Elemental Enzyme creates “enzyme, peptide and natural solutions that improve plant health, performance and yield” by applying cross-disciplinary scientific solutions to complex agricultural problems. Their products and processes are “applied through seed treatment, in-furrow soil treatments, foliar applications, tree injection, fertilizer impregnation and fertigation”. These TERAs proposed a series of field tests of a technology in which select beneficial enzymes were expressed on the outermost layer (the exosporium) of spores of Bacillus thuringiensis subsp. Israelensis. The company was developing this technology in the expectation that such spores would be an efficacious way to deliver enzymes directly to crop plants. B. thuringiensis is of course a species well-known for different subspecies that express specific insect toxins, and which have been used as natural biopesticides against lepidopteran and coleopteran pests for many years. However, Elemental Enzymes was not making use of that trait, and in fact their strain of B. thuringiensis subsp. Israelensis was missing the genes needed to encode the insect toxin.

The company created multiple strains of modified B. thuringiensis subsp. Israelensis, each expressing a different plasmid-encoded enzyme. The eight enzymes expressed by these strains were endo-1,4-β-glucanase, β-1,3 glucanase, phytase, chitosanase, protease 2, alkaline phosphatase 4, lipase EstA,  and phosphatidylcholine phospholipase. The strains were to be administered either directly to seed or in-furrow, and field tests were planned in 14 states on a variety of crops including corn, squash, tomatoes and cucumber. The purpose of the trials was to determine if the enzymes imparted beneficial growth-promoting properties to any of the crop plants; monitoring of the modified strains was also contemplated.

It is not known in the public record if these tests were carried out and what the result may have been, and there were no follow-up TERAs submitted for these strains. Today, the company is selling or developing a number of products for agriculture, but spore-delivered growth-promoting enzymes do not appear to be among them.

These TERAs are unique in several ways. They are likely the only submissions to a federal agency for a field test involving spores of genetically modified microorganisms. And the proposed experiments were somewhat unusual in the use of a species known for its pesticidal properties for a purpose unrelated to such properties. These TERAs also appear to have been the first for testing of soil inocula since the nitrogen-fixing experiments under TERAs in 1999-2000, which is of interest given the recent increase in commercial activity towards developing improved seed and soil inoculants to improve crop growth and yield.

TERA R-15-0003 was submitted by the National Institute of Standards and Technology (NIST) on April 30, 2015 for a proposed field test of a modified strain of Saccharomyces cerevisiae. This proposal is also one that is unlike any other TERA that has yet been filed with EPA. The purpose of the proposed field test was to test the ability of a modified yeast strain to serve as a “reference material to support first responder training and workflow evaluation for on-site biological assessment technologies”.  In response to a 2011 report from the Department of Homeland Security, researchers at NIST were developing a program to train first responders how to deal with “suspicious white powder incidents” that might arise from terrorist activity. The goal of this project was to evaluate whether this modified S. cerevisiae strain could be used “as a qualitative and quantitative reference material for microbial abundance measurements” under conditions mimicking a field response to a white powder incident.

The NIST team modified a wild type S. cerevisiae strain to contain a 438 bp fragment from a heterologous organism, Methanocaldococcus jannaschii, a noncoding sequence selected to have minimal homology to the S. cerevisiae sequences, which could be used to detect the microorganism in PCR assays. In the proposed test under the TERA, cells of the modified strain were to be lyophilized and released to the environment in two ways: either fixed on metal coupons which would be placed at different locations around the test site, or released to the atmosphere in a plume at the site. In the first case, the plan was for the researchers to retrieve the metal coupons and obtain microbial samples using swabs, and the samples would then be quantified using qPCR and viable-cell counting. In the second case, clean metal coupons would be used to collect the microorganisms from the plume, and the coupons used as in the first experiment to obtain microbial samples for qPCR and plate counting.

NIST conducted the test in July 2015 and subsequently filed a report with EPA describing the results. The air plume experiment was not carried out due to unforeseen circumstances, but the first experiment with the metal coupons was conducted. The report concluded “Overall, the use of the yeast was a success. Of the 4 detection sites, only 3 sites reported results. One site did not report results due to technical difficulties at the time of testing. All the reporting sites successfully detected the yeast from the samples collected from coupons with yeast”.

These two proposals from several years ago are indicative of the breadth of R&D activity in exploring the uses of modified microorganisms for beneficial uses in the environment. As noted above, the Elemental Enzyme TERAs can be seen as an early harbinger of renewed commercial interest in developing improved microorganisms for non-pesticidal agricultural purposes, and the NIST proposal is an example of how the techniques of modern biotechnology can be applied in innovative and perhaps unexpected ways.

In the blog post that will follow in a few days, I’ll describe another batch of TERAs, including three successful proposals for open-pond field testing of genetically modified algae.

To conclude, I’ll reiterate my earlier comments that the TERA process is well-suited to allow the assessment of the potential risks of proposed environmental uses of modified organisms. The TERA process allows outdoor uses of modified microorganisms to take place in a stepwise fashion under appropriate monitoring and agency oversight, to enable legitimate scientific issues of environmental risk assessment to be addressed with data from actual controlled small-scale environmental use, thus facilitating subsequent risk assessments for larger-scale uses.

D. Glass Associates, Inc. is a consulting company specializing in government and regulatory affairs support for renewable fuels and industrial biotechnology. David Glass, Ph.D. is a veteran of over thirty-five years in the biotechnology industry, with expertise in industrial biotechnology regulatory affairs, U.S. and international renewable fuels regulation, patents, technology licensing, and market and technology assessments. More information on D. Glass Associates’ regulatory affairs consulting capabilities, and links to some of Dr. Glass’s prior presentations on biofuels and biotechnology regulation, are available at The views expressed in this blog are those of Dr. Glass and D. Glass Associates and do not represent the views of any other organization with which Dr. Glass is affiliated.