Regulations Affecting Use or Disposal of Spent Biomass from Biofuel Production Using Modified Organisms

 (or “What do I do with all these dead microorganisms?”)

One common issue facing companies running fermentation processes using microorganisms is how to dispose of, or reuse, the microbial biomass that remains after the production process. In such fermentations, particularly those using modified microorganisms, the spent biomass is typically sterilized or inactivated in some way after the fermentation is completed (e.g. as required under regulations like the EPA TSCA regulations), but something needs to be done with these cells after they are killed.  This entry will discuss some of the regulatory issues pertaining to the possible disposition of such inactivated cell biomass from fermentations of modified microorganisms used for biofuel production. In particular,  I’ll discuss the regulations that may govern the use of inactivated biomass in animal feed – something that has been common for many years in conventional fermentation processes, particularly where the biomass is yeast. Taking this approach for fermentations using modified microorganisms would likely involve regulations of the U.S. Food and Drug Administration, under which animal feed and feed additives are regulated, and which now feature a streamlined procedure under which new substances can be confirmed to have “Generally Recognized as Safe”(GRAS) status. But I’ll also briefly describe regulations that might affect other possible disposal routes for spent microbial biomass, such as disposal into wastewater streams or the application of the biomass to land, either in landfilling or as a soil supplement.

Use of Microbial Biomass as a GRAS Feed Additive

***You can find a more useful, more up-to-date summary of the regulations affecting use of spent biomass in animal feed in a more recent (February 2013) post on the blog***

Companies have talked about using spent biomass from recombinant organism fermentations as animal feed since the early days of the biotech industry, and it is fairly common for fermentation byproducts such as yeast to be used in this manner. Historically, such uses (involving naturally occurring, nonengineered microorganisms) have invoked little or no regulation. However, products intended for human or animal food, as well as food or feed additives, are potentially subject to regulation by the U.S. FDA. This raises the question of how spent biomass from a recombinant or engineered microbe would be covered under FDA rules.

FDA regulation of animal feed is not much different than its regulation of human food, especially for feed intended for use in food-producing animals. Specifically (and contrary to popular belief), FDA does not regulate “food” per se unless a marketed food product is believed to be adulterated, and most existing food products have the equivalent of GRAS status. Instead, FDA regulation is largely directed at new substances proposed for use as human food additives or as animal feed additives. Although some food or feed additives are considered GRAS due to longstanding historical use; most other new substances intended for food use must be approved as an additive through the submission of a Food (or Feed) Additive Petition, which requires considerable safety testing and usually entails a lengthy FDA review.

The alternative has always been to certify, or to ask FDA to certify, that a new food/feed product or additive is GRAS, but in the past the procedures for doing so have been somewhat cumbersome. Under the old system of “GRAS affirmation”, FDA had to ascertain that substances claimed as GRAS were really GRAS, either on its own initiative or in response to a manufacturer’s petition. FDA has attempted to streamline this procedure and has done so by issuing  proposed regulations  in 1997 (which are currently being implemented as an interim policy) that offer a simplified procedure under which applicants can notify FDA of their own determination that the food use of a particular new substance is GRAS. Following FDA’s receipt of such a notification, the Agency evaluates whether the notice provides a sufficient basis to support a GRAS determination or instead whether available information raises issues that lead the agency to question the GRAS status of the substance. The agency issues an opinion letter at the end of its review, and for those notifications that it clears for commercialization, the agency says it has “no questions” about the applicant’s determination of GRAS status but that FDA is not making its own determination of GRAS status. The interim regulations require FDA to acknowledge receipt of each notification within 30 days but it is not clear if there is a specified time period to complete its review and response, and in the notifications I’ve looked at, FDA seems to have taken about 6 months for its review.

FDA has received and reviewed over 300 submissions since the Agency began accepting GRAS notifications in 1998, and most of these raised no questions and so the GRAS status of these substances was established. The great majority of these notices appear to be for human foods and food additives, but the text of FDA’s 1997 Federal Register notice proposing the regulations makes clear that FDA intended the same procedure to apply for GRAS determinations of animal feed or feed additives. Review of FDA’s online database of its GRAS Inventory shows that this process has been used for a number of products comprising or derived from genetically engineered microorganisms – particularly a good number of food additive enzymes produced by recombinant microbes (as well as enzymes manufactured from nonmodified microbial or fungal strains), and has also been used to certify GRAS status for oils isolated from algae or plants, as well as preparations of naturally-occurring microorganisms intended for food use. As far as I can tell, there have only been two notifications declaring GRAS status for food-related uses of genetically engineered microorganisms per se (i.e. as opposed to enzyme preparations or other products purified or extracted from engineered organisms), and these have both been modified yeast strains intended for use in wine-making (GRAS Notifications 000120 and 000175).

Although there doesn’t appear to be a direct precedent for a request for GRAS status of  inactivated biomass of a recombinant organism for animal feed, the path for doing so appears straightforward.  Most applicants have submitted information describing the make-up and construction of the recombinant strain, the manufacturing methods in which the strain would be grown or used, the ways in which the strain (or its products) would be used in food, and the dietary uptake of the microorganism predicted from such uses. Many applicants also included data from toxicological or allergenicity testing or other safety testing, while other applicants had their product reviewed by panels of experts. There appear to be several routes by which a microorganism-based product could be proven to be GRAS, and no doubt consultation with FDA prior to submission would help clarify what might be needed for any specific case. 

***Again, please refer to a more recent (February 2013) post on the blog for a more useful, more up-to-date summary of the regulations affecting use of spent biomass in animal feed.***

Regulation of Other Possible Disposal Options for Spent Biomass

Another possible option for the disposition of spent microbial biomass would simply be to find a lawful way to dispose it, particularly including land disposal (utilizing it as a soil additive or fertilizer) or disposal into wastewater or a public treatment facility. These options have also been used in traditional microbial fermentations. Although there are various regulations governing land or water disposal of certain waste products, there do not appear to be any regulations specific for the disposal of genetically engineered microorganisms,  and generally speaking, once the biomass has been inactivated to meet applicable regulatory standards (e.g. EPA’s 6-log standard under TSCA), the biomass is no longer considered a regulated article subject to the biotechnology rules.

So, it would appear that spent biomass from a recombinant fermentation could be disposed on land or into water without meeting any requirements specific to the biotech origin of the biomass. However, for companies seeking guidance as to “best practices” to be sure such disposal is handled appropriately, we can look to two specific regulations of the U.S. federal government:  the regulations for land disposal of biosolids (sludge); and regulations for wastewater disposal, both of which may offer insights into appropriate practices, even if they do not explicitly cover inactivated biomass from engineered microorganisms.  Although these will be discussed separately below, the common thread appears to be that there are no restrictions on disposal of biomass as long as the item to be disposed has been sufficiently treated to be free of pathogenic bacteria, in particular as shown using the fecal coliform test and other established testing protocols, and is otherwise free of pollutants such as heavy metals. Please note that the following discussion is meant for illustrative purposes only and is not meant to provide legal advice on compliance with any specific regulations, nor is it meant to imply that these regulations formally apply to disposal of biomass from engineered microorganisms – as always, consultation with a knowledgeable attorney or regulatory consultant is recommended.

EPA’s authority to regulate sewage sludge (also known as “biosolids”) arises from the Clean Water Act, with regulations governing the “final use or disposal of sewage sludge generated during the treatment of domestic sewage in a treatment works” found in 40 CFR Part 503. These regulations cover a broad range of provisions, including management practices, operational standards, and pollutant limits. An EPA publication describes the agency’s authority over pollutant limits as follows:

Under Section 405(d), EPA establishes numerical limits and management practices that protect public health and the environment from the reasonably anticipated adverse effects of chemical and microbial pollutants in sewage sludge. On February 19, 1993, EPA promulgated the CFR 40 Part 503 Standards for the Use or Disposal of Sewage Sludge, resulting in numerical standards for ten metals and operational standards for microbial organisms. The 1993 rule established requirements for the final use or disposal of sewage sludge when it is: (1) applied to land as a fertilizer or soil amendment; (2) placed in a surface disposal site, including sewage sludge-only landfills; or (3) incinerated. These requirements apply to publicly and privately owned treatment works that generate or treat domestic sewage sludge and to anyone who uses or disposes of sewage sludge.

Section 503.32 of 40 CFR establishes two categories of sludge, Class A and Class B, depending on the level of pathogenic organisms in the material, and describes specific processes to reduce pathogens to these levels.  The following description is adapted from a website maintained by Siemens Corporation.

Class A biosolids contain minute levels of pathogens, and to achieve Class A certification, biosolids must undergo heating, composting, digestion or increased pH in order to reduce pathogens to below detectable levels.  Once these levels are attained, Class A biosolids can be land-applied without any pathogen-related restrictions at the site.  Class A biosolids can also be bagged and marketed to the public as a fertilizer for application to lawns and gardens.  Class B biosolids have less stringent standards for treatment and contain small but compliant amounts of bacteria.  Class B requirements ensure that pathogens in biosolids have been reduced to levels that protect public health and the environment and include certain restrictions for crop harvesting, grazing animals and public contact.  As is true for Class A biosolids, Class B biosolids must be treated in a wastewater treatment facility and undergo heating, composting, digestion or increased pH processes before leaving the plant.

The Part 503 regulations also specify a number of different testing methods for pathogens and pollutants. The most relevant to the issues considered here include tests for enteric viruses, fecal coliform, Salmonella sp., and bacteria. Finally, Part 503.32 specifies the limits of pathogen concentrations that the sludge must meet before its use or disposal, and specifies several alternative methods to achieve these limits.

If one were to use these biosolids rules as guidance, it would seem that, if spent biomass were treated in one of the methods specified in the regulations (e.g. heating, high pH), if the fecal coliform density and other pathogen levels are lower than the specified regulatory levels, and if the biomass was free of heavy metals and other toxic material, then this biomass could be considered the equivalent of Class A biosolids and could be disposed on land without concern over pathogenic exposure. The types of microbial inactivation procedures typically used on spent recombinant biomass (e.g. as may be required under the TSCA Biotech Rule) might qualify as sufficient treatment under the biosolids regulations, although in some cases it might be necessary or desirable to adopt additional procedures more in line with the biosolids regulations.

EPA also maintains regulations covering the “discharge of pollutants from any point source into waters of the United States”, also established under the authority of the Clean Water Act. Included within these regulations are rules establishing the National Pollutant Discharge Elimination System (NPDES), which EPA administers in cooperation with state environmental agencies.  These regulations are more likely to be applicable to biofuel production facilities, which would likely need to obtain an NPDES permit for the discharge of other liquid wastestreams from the production plant, but here too it does not appear that these regulations contain any specific requirements or impose any unusual obligations for disposal of biomass from microbial fermentations.

Under these regulations, “point sources” include industrial facilities (including manufacturing, mining, oil and gas extraction, and service industries), municipal governments and other government facilities (such as military bases), and some agricultural facilities, such as animal feedlots.  Point sources may not discharge pollutants to surface waters without an NPDES permit. This system is managed by the EPA in partnership with state environmental agencies, 46 of which have been authorized to issue permits directly to the discharging facilities.

“Pollutant” is defined under 40 CFR Part 122.2 to mean: “dredged spoil, solid waste, incinerator residue, filter backwash, sewage, garbage, sewage sludge, munitions, chemical wastes, biological materials, radioactive materials  …, (heat, wrecked or discarded equipment, rock, sand, cellar dirt and industrial, municipal, and agricultural waste discharged into water” (emphasis added), and so this definition potentially includes spent biomass from fermentations.  Part 122.41 of the regulations states that:

(1) The permittee shall comply with effluent standards or prohibitions established under section 307(a) of the Clean Water Act for toxic pollutants and with standards for sewage sludge use or disposal established under section 405(d) of the CWA within the time provided in the regulations that establish these standards or prohibitions or standards for sewage sludge use or disposal, even if the permit has not yet been modified to incorporate the requirement.

Note that the “standards for sewage sludge use or disposal” under Section 405(d) of the CWA are the ones discussed above in the discussion of biosolids regulation. So, it would appear that, to meet discharge standards under an NPDES permit, the effluent must meet the criteria discussed above for maximum levels of coliform bacteria and other potential pathogens, heavy metals and other toxins, and that this should be easily attainable using appropriate microbial inactivation procedures.

Again, the above discussion is presented mostly for guidance as to possible “best practices” for disposing spent biomass in a safe, responsible manner. Although it seems likely that there are no federal regulations affecting disposal of biomass from engineered microorganisms, there might be state or local regulations that would apply in certain locations. Companies should seek more specific guidance from the engineers, consultants or other professionals who might be assisting in designing, building, and obtaining all necessary permits for biofuel production facilities.

D. Glass Associates, Inc. is a consulting company specializing in several fields of biotechnology. David Glass, Ph.D. is a veteran of nearly thirty years in the biotech industry, with expertise in industrial biotechnology regulatory affairs, patents, technology licensing, and market and technology assessments. This blog provides back-up and expanded content to complement a presentation Dr. Glass made at the EUEC 2010 conference on February 2, 2010 entitled “Prospects for the Use of Genetic Engineering in Biofuel Production.” The slides from that presentation, along with more information on D. Glass Associates’ regulatory affairs consulting capabilities, are available at www.slideshare.net/djglass99.

Impact of Biotechnology Regulations on the Use of Genetically Modified Algae for Biofuel Production

In the preceding entry of the blog, I discussed how the regulatory programs of the U.S. Environmental Protection Agency and U.S Department of Agriculture might affect the use of genetically modified algae for biofuel uses. It would appear that the EPA Biotechnology Rule under the Toxic Substances Control Act (TSCA) was more likely to be applicable than the USDA’s biotechnology rules under the Plant Protection Act. But I’ll briefly comment on how algal programs might be handled under each of these programs, and discuss the need to develop a common understanding between regulators and the industry about how these regulations would affect companies developing modified algae for biofuel production and other uses. 

Possible Requirements under EPA Regulations 

As is described in more detail elsewhere in this blog, under the TSCA Biotech Rule, EPA regulates certain industrial uses of “new microorganisms”, which are defined as those that contain coding nucleic acids from more than one taxonomic genus. Although most R&D uses are exempt from reporting under TSCA provided the microorganism is used in a “contained facility”, the use of a new microorganism for a commercial purpose (in an industrial field subject to TSCA) would require the filing of a Microbial Commercial Activity Notification (MCAN) 90 days before the intended commencement of commercial use. (Please refer to that earlier blog entry for a great deal more detail on this regulatory program). 

It is likely that any regulatory review of engineered algae would be subject to the same data and procedural requirements as has been the case for modified bacteria that have so far been subject to TSCA regulation. Specifically, this would include the need to submit a detailed description of the construction of the modified organism, a description of the manufacturing process in which it is intended to be used, description of the controls that would be put into place to minimize possible dissemination of the microbe outside the facility, and whatever data is in the applicant’s possession regarding the possible health or environmental effects of the organism. EPA’s review would focus on balancing the potential risks of the project against the potential benefits, and although one wouldn’t expect most algal strains to pose unusual environmental risks, the issues EPA addresses may be different for algae than they have been for the modified bacteria and fungi that have been the subject of all prior MCANs submitted to date. 

For any algae projects subject to EPA authority under these rules, one potential area of concern would be the design of the bioreactors to be used with the algae. In current practice, algae are often grown in open-air reactors, or in other reactor designs that may differ considerably from traditional bacterial fermentation set-ups. If a reactor was judged by EPA not to be sufficiently “contained” as defined in the regulations, EPA would consider any use of such reactor with live algae to be an outdoor use, triggering the need for regulatory oversight (e.g. requiring submission of a TSCA Environmental Release Application) at the research level and possibly a greater level of scrutiny at commercial scale. Aside from such possible heightened concerns about issues like containment, controlled access to the facility, handling and inactivation of spent biomass and other wastes, one could expect that EPA review and handling of an MCAN for an engineered algae under the TSCA regulations would proceed in much the same way as prior reviews of MCANs for engineered bacteria. With proper planning, advance consultation with the Agency, and given sufficient time to develop the needed data package, algae projects that might fall subject to TSCA should not encounter too much difficulty in being cleared for commercialization. 

Possible Requirements under USDA Regulations 

USDA’s regulation of genetically engineered plants and agricultural microorganisms has been described in detail elsewhere in this blog. Although the great majority of engineered organisms regulated under this rule have been transgenic plants, the current version of the rule is broad enough to cover microorganisms (particularly agricultural microorganisms) that might be plant pests, depending on their genetic make-up and other characteristics. As discussed in the entry preceding this one, it is possible (although somewhat remotely) that certain biofuel algae strains may fall under these regulations, particularly if they contained nucleic acid sequences from a plant pest, or even from the commonly-used Agrobacterium tumefaciens transformation system. 

There does not appear to ever have been any formal submissions to USDA for permits for interstate movement or field testing of any genetically modified algae strain. It would therefore be hard to speculate what approach USDA might take to define a class of algae that it would consider to be subject to the biotech regulations, although for any projects that might fall under these regulations, one would expect USDA to consider the same sorts of issues and require the same sorts of data as has been the case for those few modified microorganisms that have historically been  subject to these regulations. Specifically, that would likely include the requirement to submit information describing the construction of the organism, and assessing the possibility that the engineered organism might have plant pest characteristics or other potential environmental impacts.  However, it may turn out that USDA may determine that biofuel applications of genetically engineered algae are outside their core agricultural mission and so the agency may well decline to assert its regulations over such applications.  

Impact of Biotechnology Regulations on the Use of Modified Algae for Biofuel Production 

It is quite important for any uncertainty about the regulatory path for modified algae to be cleared up, so that companies developing such organisms can understand the path to regulatory approval and clearance for commercialization, and so that the general public can be assured that such uses are subject to proper, scientifically-sound, regulation. In fact, this is an issue that has recently been targeted by the Technical Standards Committee of the Algal Biomass Organization (ABO), in the draft of one of its Technical Standards Descriptions. The Committee notes the divisiveness that has often arisen over proposals for uses of GMOs, and hopes that standardization of regulatory requirements can go a long way towards preventing that from occurring in the future. The Committee hopes to develop standards for regulatory review of different categories of modified algae based on the genetic engineering method used in their creation (e.g. differentiating strains in which a single or a small number of exogenous genes are expressed from strains created by directed evolution or synthetic biology), and to develop a predictable, standardized “roadmap” for permitting and regulatory approval of projects involving modified algae. For example, the Technical Standards Descriptions talks of developing “the set of rules for operating each class of engineered organism in lab settings, outdoor pilot plants, and scaled up farm facilities” and “developing a permitting roadmap to encourage uniform, reasonable and affordable permitting processes to be developed in every national and international permitting jurisdiction that is pertinent to algal farming”. 

These goals are laudable, and arguably critical for the ultimate success of the algal biofuel industry.  In fact, such roadmaps have emerged for applications such as agricultural biotechnology that have used more traditional microbial or plant hosts, and the experience that industry and governments have gained with those prior biotechnology efforts should go a long way towards seeing a favorable outcome for algae as well. For example, it would be very useful to develop and articulate the universal principles that could define safe, commercially appropriate procedures for the large-scale growth of industrial algal strains, with the goal of having these principles implemented and adopted across regulatory programs in the U.S. and internationally. Although such principles could reach across various aspects of the R&D and production process, they might be most valuable in specifying what hardware, procedures, and other safeguards could be put in place to ensure that novel algal strains could be grown under conditions to minimize or prevent both contamination from outside algal species as well as release of the production strain from the facility, in a manner that would satisfy existing regulatory requirements for “containment”. As noted above, I expect this will be a major point of discussion companies will face with regulatory agencies for commercial use of GMO algae strains.   

However, it may be somewhat unreasonable to hope that one could separately define different categories of organism based on the technology used to create the genetic modification, as ABO’s Technical Committee hopes, so that the different categories might be regulated differently. This would certainly be difficult in the U.S., where the definitions specifying which organisms fall under the regulations and which are not covered have been fixed in regulation or legislation for many years. In fact, a cornerstone of the early years of policy development of biotech regulations in the U.S. (approx. 1983 to 1991) was that regulations should not be “process based” (i.e. based on the method used for the genetic modification), but that the level of regulatory oversight be determined by the inherent potential risks of the organism regardless of the method by which it was constructed. So, for example, under the TSCA rule, a microorganism is covered if it is “intergeneric”, regardless of how it got that way; and under the USDA rule, an organism is regulated solely on its potential to exhibit plant pest characteristics, regardless of the method of construction. Under the current regulatory scheme it would be up to the applicable regulatory agency to determine if, for example, a strain created by the precise engineering of synthetic biology posed any greater or lesser risk than a strain created by mutation or by older methods of recombinant DNA genetic engineering. The same is likely to be true overseas – I hope to summarize some of the applicable European Union standards in a future blog entry, but for now I’ll say that my understanding of these standards is that levels of regulatory oversight in the EU are determined by the inherent risk of the organism without undue regard for the method by which it was constructed, and that manufacturers using engineered organisms in commercial production are allowed to determine on their own what procedures and safeguards are appropriate to ensure “containment” of the production strain. 

In the meantime, under the current U.S. regulatory regime, I can offer the following comments and recommendations for companies contemplating the use of modified algae in biofuel projects. 

  • Well in advance of the projected commercial start date, seek guidance from a regulatory attorney or consultant about the regulatory status of the production strain(s), and possibly plan an early presubmission meeting with agency staff.
  • Although use of a genetically modified organism in research or in a pilot plant would ordinarily be exempt from oversight by either EPA or USDA, in the case of algae this would be dependent upon the nature of the reactor in which the organisms would be used, and if an open-air algal reactor is not be judged to be sufficiently “contained”, this could cause even research use to be subject to agency oversight.
  • Because there may be uncertainty over federal agency jurisdiction, companies should be alert to the possibility of state or local government involvement in projects involving modified algae. In certain situations it might be desirable to involve such local agencies in early discussions, so that there are no surprises if they are brought into the process later on.

I expect that many of these issues will be discussed in the near future, as more companies begin to approach commercial stage with modified biofuel algae strains. As is often the case, it will likely be the “early adopters” who are first to propose such projects to the regulatory agencies that will not only blaze the trail but also bear the brunt of resolving any regulatory uncertainty there may be over how modified algae would be regulated. 

D. Glass Associates, Inc. is a consulting company specializing in several fields of biotechnology. David Glass, Ph.D. is a veteran of nearly thirty years in the biotech industry, with expertise in industrial biotechnology regulatory affairs, patents, technology licensing, and market and technology assessments. This blog provides back-up and expanded content to complement a presentation Dr. Glass made at the EUEC 2010 conference on February 2, 2010 entitled “Prospects for the Use of Genetic Engineering in Biofuel Production.” The slides from that presentation, along with more information on D. Glass Associates’ regulatory affairs consulting capabilities, are available at www.slideshare.net/djglass99 or at www.dglassassociates.com.

Regulations Affecting the Use of Genetically Modified Algae for Biofuel Production

Earlier blog entries have discussed the regulatory schemes of the U.S. EPA and U.S Department of Agriculture that might affect the use of genetically modified microorganisms and plants for biofuel uses. I’ll now turn my attention to how genetically engineered algae might be regulated under these or other regulatory programs. From a technological standpoint, programs for genetic modification of biofuel algae strains have lagged behind similar efforts for microbes and plants (but see the excellent recent review by Radakovits et al. of progress in engineering algae for biofuel use), and so there have been few, if any, companies that have reached or approached the point where they might have to worry about how commercial use of modified algae might be regulated. And for the same reason, it seems that the agencies themselves have not given this question much thought, or at least that the agencies have made few if any public statements about the applicability of their programs to engineered algae. However, uncertainty about the regulatory path that might await modified biofuel algae could become a factor holding back the industry’s development, as evidenced by the concern over this issue expressed by the Algal Biomass Organization’s Technical Standards Committee (which I’ll discuss in more detail in the next entry of the blog). In this entry, I’ll discuss whether engineered algae might fall under EPA’s TSCA Biotech Rule or USDA’s biotechnology regulatory program, and in the entry to follow, I’ll discuss some of the issues raised by the ABO committee, including the possible impact of these regulations on algal biofuel programs, along with some thoughts about how companies can successfully navigate the applicable regulatory requirements. 

Applicability of EPA Biotech Regulations on Use of Modified Algae in Biofuels Projects 

My earlier blog entries have described EPA’s program of regulating certain genetically modified microorganisms under the Toxic Substances Control Act (TSCA), when used for certain industrial purposes not regulated by other federal agencies. All the new microorganisms that have been subject to EPA oversight under this rule have been modified versions of host organisms that were either bacteria, yeast or fungi. So the question would be whether algae strains genetically modified to contain coding nucleic acid sequences from another taxonomic genus (i.e., “intergeneric”, or “new” under the Biotech Rule) would fall under EPA jurisdiction under the TSCA Rule. 

There appears to be a basis in the TSCA regulations for certain algal strains to be subject to EPA oversight. Section 725.3 of the TSCA regulations define the term “microorganism” as encompassing “those organisms classified in the kingdoms Monera (or Procaryotae), Protista, and Fungi, the Chlorophyta and the Rhodophyta of the Plantae, and viruses and virus-like particles” . This definition is further explained in the preamble to the 1997 Federal Register notice (emphasis added): 

In the proposed rule (59 FR 45550-51), EPA defined  “microorganisms” in Sec. 725.3 as those organisms classified under the 5-kingdom system of Whittacker … in the kingdoms Monera (or Procaryotae), Protista, and Fungi, the Chlorophyta and the Rhodophyta of the Plantae, and viruses and virus-like particles. Therefore, this definition includes, but is not limited to, bacteria, protozoa, fungi, mycoplasmas, mycoplasma-like organisms, spiroplasmas, microphytoplanktons, green and red algae, viruses, and virus-like particles (e.g., viroids, satellites, and virusoids). …

Commenters thought the proposed definition of “microorganism” was reasonable and included the appropriate organisms. Thus, EPA will retain the definition of “microorganism” as discussed in the proposed rule and found in the regulatory text in Sec. 725.3.

In addition, in its Regulatory Impact Analysis accompanying the 1997 rule, EPA stated the following (emphasis added):  

The microorganisms potentially affected by the rule are those for which the corresponding chemical use would be subject to TSCA jurisdiction. By statute, the Toxic Substances Control Act (TSCA) regulates all chemical applications not specifically exempted in the Act. Language in the Act has been interpreted to include living microorganisms (i.e., microscopic living cells such as bacteria, fungi, protozoa, microscopic algae, and viruses).

Neither the preamble language nor the Regulatory Impact Analysis language carries the force of law, but this language makes it clear that the Agency considers that it has the authority  to claim oversight responsibility over certain uses of genetically modified algal species. 

Applicability of USDA Biotech Regulations on Use of Modified Algae in Biofuels Projects 

As described in earlier blog entries, the U.S. Department of Agriculture regulates most transgenic plants used for agricultural or other research and commercial purposes using its biotechnology regulations under the Plant Protection Act. Although many algal species are photosynthetic, it is not clear whether any algal species would be considered to be potentially subject to USDA’s existing biotechnology regulations. In the current regulations, the term “Plant” is defined as: “Any living stage or form of any member of the plant kingdom including, but not limited to, eukaryotic algae, mosses, club mosses, ferns, angiosperms, gymnosperms, and lichens (which contain algae) including any parts (e.g. pollen, seeds, cells, tubers, stems) thereof, and any cellular components (e.g. plasmids, ribosomes, etc.) thereof”. But under the biotechnology rule, APHIS does not regulate “plants”, but instead “plant pests”, and so the fact that “eukaryotic algae” are included in the definition of “plant” may have little bearing on the question. “Regulated articles” under the biotech regulations include only “organisms that are or contain plant pests”, and in practice this has largely resulted in the regulation of those genetically engineered plants  and some agricultural microorganisms which contained sequences from specific microbial, plant and animal genera that contain species that are potential plant pests. The list of known or potential plant pest species is contained in 7 CFR Part 340.2. This list does not appear to include the names of any of the genera of algae that have been suggested for biofuel use. 

Although the regulations give APHIS leeway to determine that other organisms are “potentially” plant pests, generally speaking, if an engineered algae is not from one of the genera shown on the list in Part 340.2, it would not a priori be subject to regulation under the existing rules. However, many transgenic plants have been captured by the regulations because the most common vectors for plant transformation include sequences from the genus Agrobacterium, a genus found on the list of potential plant pests. So, some engineered algae may become subject to the regulations if the vectors used in the genetic engineering include “plant pest” sequences, and (as summarized in the recent review by Radakovits et al. 2010) it is true that Agrobacterium-mediated transformation is one method that has been used in genetic engineering of algae, although it is not likely to be the most common method used today. 

At least one online reference has reported that, as part of its ongoing rulemaking process, APHIS has considered making its jurisdiction over algae more explicit, but I’ve reviewed the 2008 Proposed Regulations, and I could not find any evidence of such intent. The proposed rule defines the scope of organisms that would be potentially subject to regulation much differently than the current rule. Proposed Section 340.0 of the proposed regulations defines two categories of organisms as requiring permits: 

(b) Genetically engineered organisms whose importation, interstate movement, or release into the environment is subject to the regulations in this part are: 

(1) Genetically engineered plants if:
(i) The unmodified parent plant from which the GE plant was derived is a plant pest or noxious weed, or
(ii) The trait introduced by genetic engineering could increase the potential for the GE plant to be a plant pest or noxious weed, or
(iii) The risk that the GE plant poses as a plant pest or noxious weed is unknown, or
(iv) The Administrator determines that the GE plant poses a plant pest or noxious weed risk. 

(2) Genetically engineered non-plant, non-vertebrate organisms if:
(i) The recipient organism can directly or indirectly injure, cause damage to, or cause disease in plants or plant products; or
(ii) The GE organism has been engineered in such a way that it may increase the potential for it to be a plant pest: or
(iii) The risk that the GE organism poses as a plant pest is unknown, or
(iv) The Administrator determines that the GE organism poses a plant pest risk.

The proposed rule’s definition of “plant” is far more general than that of the current rule, and it is not clear whether an algal strain could be considered a “genetically engineered plant” under subclause (a) of the above definition. However, the Agency could use the discretionary powers of subclauses (b)(2)(ii), (iii) or (iv) to determine that a genetically engineered algae might require regulation under these proposed rules, particularly if there are any vector sequences or other introduced genes that might have arisen from a “plant pest” organism or a noxious weed. However, it does not appear that any algal strain, either modified or naturally occurring, has been regulated by USDA under any of its regulations, including its longstanding plant pest regulations under 7 CFR Part 330. 

In my view it is unlikely that USDA would assert regulatory authority over a proposed biofuel use of a modified algal strain unless it was a fairly large-scale commercial use of the strain, and only if there were some clear link, such as a possible plant pest risk, to agriculture or to a particular region or sector of U.S. agriculture. And further, it’s unlikely that the agency would claim jurisdiction over applications where the novel strain was used in a contained reactor, because their regulations cover only interstate movement or outdoor uses of engineered organisms (see discussion below). So, my sense is that USDA regulation of engineered algae is unlikely.

Possible precedents for regulation of engineered algae 

There are two prior cases that illustrate how challenging it might be for genetically modified algae to fit neatly within the purview of one federal regulatory program. There is one publicly-known situation where a company requested a USDA ruling on whether a specific engineered algal species would be subject to the biotechnology regulations. The company is Coastal BioMarine of Bridgewater, Conn. (www.coastalbiomarine.com), an aquaculture company dedicated to growing shellfish and algae (algae being a food source for shellfish). Coastal contacted USDA in April 2008 to request clarification of the regulatory status of three marine algal species engineered with a gene encoding a glucose transporter protein. USDA responded in a letter dated May 19, 2008, and their response, although limited to this specific situation and not related to biofuel applications at all, provides useful insight into how the agency might regulate algae for a biofuel application.

The key points made by USDA in the letter are as follows: 

  • Based on the information  provided, the engineered algal strains would not be covered by the USDA biotech regulations, because neither the recipient (“host”) species nor the source of the glucose transporter gene are listed on the “plant pest” list in Part 340.2 of the regulations. However, this determination would change if the vector sequences or selectable markers used in the genetic engineering arose from “plant pest” organisms. 
  • Even if the species were subject to the regulations, USDA oversight would not be needed to use the organisms in a contained reactor. This is important because USDA’s biotechnology regulations cover only outdoor use and interstate movement of potential plant pests. 
  • Large-scale use of these organisms, particularly for use in a biofuel application, might raise environmental issues that would trigger a need for USDA oversight. The letter suggested that in order to progress to large-scale commercial use of such species, an applicant might need to pursue a “delisting” petition to establish non-regulated status for the organisms. 
  • USDA suggests that other agencies, notably FDA and EPA, might be involved in regulating such species.

This decision by USDA is interesting, and it is consistent with some of the circumstances regarding the proposal by Mera Pharmaceuticals for algal pharmaceutical production that is discussed below. In particular, the decision that USDA lacks regulatory authority for small-scale tests if there is no “plant pest” risk, and the uncertainty about other agencies’ jurisdiction, are both features also seen in the Mera story. 

Mera’s 2005 proposal to grow genetically modified algae in Hawaii for the purpose of pharmaceutical production is illustrative of how local concerns or opposition could lead to a state or local government assuming control over a project, especially in the absence of federal oversight. As is well documented on the Internet (e.g. http://archives.foodsafety.ksu.edu/agnet/2005/8-2005/agnet_aug_3.htm#story2 or  http://www.ibiblio.org/ecolandtech/SoilWiki/message-archives/JoeCummins/msg00519.htm), Mera proposed moving as many as eight strains of Chlamydomonas reinhardtii from California into Hawaii, for the purpose of growing them to produce pharmaceuticals including antibodies and interleukins. According to these Web accounts, the three federal agencies that oversee almost all commercial biotech activities, FDA, USDA and EPA all waived oversight of these field trials, opening the door to state regulation. The Hawaii Department of Agriculture (DOA) eventually took responsibility for permitting the trials, because Chlamydomonas is listed on the state Department of Agriculture’s “list of restricted organisms” under its quarantine laws. Further, DOA staff determined that the biopharm algae posed an “above moderate risk,” which means that the BOA needed to approve the project. The DOA made this determination based on the lack of federal oversight, the DOA’s lack of experience with engineered algae, concerns regarding large-scale production outdoors, and the “unknown effects on the environment if accidentally released.” This project drew a great deal of public criticism and opposition, and although it appears that the Hawaii DOA eventually granted permits for to allow at least some portions of the trial, a local citizen’s group filed a lawsuit that resulted in a judicial ruling that an environmental assessment would be needed before the trials could proceed. This decision was ultimately affirmed by an appeals court, and it appears that Mera’s field tests of these engineered algae have never taken place. 

Although this is a cautionary tale for algae biofuel projects, there are unique aspects of the Mera story that may not make it completely applicable to biofuels. Specifically, the fact that the algae were to be used in R&D to develop a pharmaceutical production process may have led to the lack of federal oversight. In a nutshell, both EPA and USDA may have decided that, because the project involved pharmaceutical manufacture, each agency would yield to FDA authority. In EPA’s case, not only would TSCA jurisdiction be ruled out due to FDA jurisdiction but perhaps also because it was a research project; in USDA’s case the Agency probably decided there was no plant pest risk for a small-scale project (as they did for Coastal BioMarine, discussed above). However, to my knowledge, the FDA does not regulate pharmaceutical manufacturing processes until the manufacturer is ready to have the final, commercially-ready process certified, and so FDA would not have the infrastructure or the regulatory authority to exercise any control over a small-scale project. So, this may be viewed as an isolated incident, but it should serve as a reminder that (as was often the case in the early days of agricultural biotechnology) state or local governments, not to mention concerned citizens, will often jump in where there is a perceived federal regulatory vacuum, so that developers of engineered biofuel algae need to pay attention not only to regulatory agencies at all governmental levels, but also to good community relations practices and principles. 

So, it may prove that there might be conflicting agency oversight over projects involving modified algae, or more correctly that certain projects may fall through the cracks between differing agency authorities. And as the Mera story and other situations throughout the history of biotechnology have shown, when there is a vacuum in federal regulatory authority, the states or local governments may step in with stricter regulations than would have been faced under a federal program. 

In the next entry of the blog, I’ll discuss how these regulations may impact plans to use modified algae for biofuel production and comment not only on how individual companies should plan for such regulation but also about the efforts by the algal biofuel industry to have a greater influence on the regulations that may affect commercialization efforts. 

D. Glass Associates, Inc. is a consulting company specializing in several fields of biotechnology. David Glass, Ph.D. is a veteran of nearly thirty years in the biotech industry, with expertise in industrial biotechnology regulatory affairs, patents, technology licensing, and market and technology assessments. This blog provides back-up and expanded content to complement a presentation Dr. Glass made at the EUEC 2010 conference on February 2, 2010 entitled “Prospects for the Use of Genetic Engineering in Biofuel Production.” The slides from that presentation, along with more information on D. Glass Associates’ regulatory affairs consulting capabilities, are available at www.slideshare.net/djglass99 or at www.dglassassociates.com.

Impact of USDA Regulations on the Use of Genetically Modified Plants as Biofuel Feedstocks (Part 2)

The previous entry of this blog listed the various permit applications and notifications that the U.S. Department of Agriculture (USDA) has received for the field use of genetically engineered plants and trees intended for use as biofuel feedstocks. These field test requests have been reviewed by the Biotechnology Regulatory Service (BRS) division of USDA’s Animal and Plant Health Inspection Service (APHIS) under USDA’s biotechnology regulations, found in 7 CFR Part 340 of the Code of Federal Regulations. In this entry, I’ll discuss the issues that have arisen in USDA’s review of those requests, particularly two applications from Arborgen and Syngenta requesting approval for activities beyond small-scale research,  and their possible implications for future applicants, and I’ll also make some general comments about the impact of the USDA regulations on development of transgenic biofuel feedstocks. 

Although the need to submit notifications or obtain permits from USDA can be seen as one more hurdle to be surmounted in the development of improved biofuel feedstocks, in reality the biotech regulation poses only a minor regulatory barrier at the early stages of product development. Agbiotech and seed companies have been living under this regulation for over 20 years, and they have found USDA APHIS easy to deal with, and that the agency’s Biotechnology Regulatory Services branch applies sound scientific principles to its review of notifications and permit applications. Many observers consider the USDA regulatory regime to be one of the reasons why the agricultural biotechnology industry has ultimately been successful in introducing into U.S. agricultural markets transgenic seeds for improved varieties of many important crop species, in that the regulations provided a predictable, scientifically-sound path to approvals of field tests and commercial use of engineered plants. 

That being said, the presence of the regulations does impose an added requirement on developers of transgenic energy crops that is not borne by companies using classical plant breeding or other traditional techniques to improve biofuel feedstocks. Notifications need to be submitted 30 days in advance of any planned outdoor planting of a transgenic plant, regardless of the size of the field plot, and permit applications must be submitted 120 days in advance (although in recent practice USDA has been approving many permit applications far quicker than this stated timeframe).  Although notifications require only a minimal amount of information, permit applications require compilation and submission of considerable data and information about the make-up of the plants to be tested, the size and location of the test plot and the procedures to be followed in the test, and consideration of the potential environmental impacts of the test. In addition, it is likely that, for the earliest field tests of any given transgenic variety, it will be necessary to prevent or control the release of pollen from the engineered plants, and possibly to ensure that the test plot is sufficiently isolated from growing regions of the same species as to make unwanted horizontal gene flow unlikely. Once a given variety has been used in the field after successfully navigating the regulatory process, not only will approval of subsequent permit applications become easier, but it should also be easier to prepare permit applications, since much of the needed information will already be in hand from the earlier applications. 

With the exception of familiar species such as corn and others, it is likely that most engineered biofuel crops will require permits for outdoor testing, rather than be allowed to proceed under notifications. Changes may come if USDA eventually issues new regulations based on the proposed revised rule that was published in 2008. Under that new proposal, notifications would be abolished in favor of a multi-tiered permit scheme, and presumably those field tests that qualified for notifications in the past would be subject to the least-stringent of the different tiers of permits. The impact of this change on transgenic energy crops is not clear, but given USDA’s interest in seeing “nontraditional” (e.g. industrial or pharmaceutical) uses of transgenic plants handled under permits rather than notifications, it may turn out that field uses of engineered energy crops will require one of the more stringent levels of permit. 

Although small-scale research field tests are more or less routinely reviewed and approved, the same may not be true of expanded field use, and is certainly not true when companies need to seek approvals for unlimited commercial use and sale of a given variety. The recent experiences of Arborgen, in its applications for large-scale testing of transgenic Eucalyptus, and Syngenta, in its petition to delist (i.e. deregulate) its amylase-expressing corn, may give some indication of what companies may face, particularly in the early days when USDA is still gaining familiarity with whatever issues may need to be addressed for the widespread use of transgenic energy crops. 

As described in the previous entry of this blog, Arborgen is a leading forestry biotechnology company that has been developing improved hardwood trees for use as energy crops, and which has received many APHIS permits for small-scale field tests of transgenic Eucalyptus engineered for this purpose. USDA’s recent decision to grant two particular ArborGen permit applications involved the company’s request to continue research on transgenic Eucalyptus trees that had already been planted at over two dozen sites under a prior APHIS permit (Number 08-039-102rm), to allow the planting of additional trees and to allow the transgenic Eucalyptus trees to flower at the various field site locations. These plants are a clone known as EH1 that was derived from a hybrid of Eucalyptus grandis x Eucalyptus urophylla and which have been genetically engineered with different constructs. The purpose of the these field tests is for ArborGen to assess the effectiveness over several years of growth of gene constructs which are intended to confer cold tolerance; to test the efficacy of a gene introduced to alter lignin biosynthesis; and to test the efficacy of a gene designed to alter fertility. This latter gene, barnase, is expressed to create male sterility, in order to limit possible gene flow from the transgenic trees. 

What distinguished these two permit applications from the many others that Arborgen has received over the years was the request to allow large numbers of the engineered trees to flower without the need for measures to restrict or prevent flowering. In most small-scale field trials of transgenic plants, it is necessary for the applicant to propose measures to prevent pollen set and/or flowering of the experimental plants, so as to prevent outcrossing and gene flow to native plants of the same or related species. Field trials of new varieties of trees usually need to be conducted over multiple years, and in order to accurately assess the performance of the trees over time, they must be permitted to maintain their normal lifestyle, which would include flowering, and so this request was a necessary step in Arborgen’s commercialization process. But because the request entailed allowing a large number of trees to flower at multiple field sites, APHIS decided to perform a full Environmental Assessment (EA) before deciding whether to issue the permits.  On June 3, 2009 APHIS announced the availability of a draft EA for public review and comment, but then on January 19, 2010 the Agency announced the availability of an amended EA that included consideration of one previously-unavailable document, at which time the review and comment period was extended until  February 18, 2010. In its decision on May 12, 2010 to grant the permits, APHIS made the final Environmental Assessment available to the public. 

The EA considered a broad range of potential issues and potential environmental impacts of the test, including those listed in the Table below. In spite of the male sterility engineered into the trees, the EA discussed the possibility for gene flow via pollen spreading from the trees, but concluded this was unlikely, given the male sterility of the trees, the expected limited range of pollen from  Eucalyptus, a tree which is ordinarily pollinated by insects rather than wind, and the fact that there are no native species of Eucalyptus in the United States and no established E. grandis populations in any location close to the proposed field tests. It seems clear that the chief concern, and the reason for compiling the EA before granting the permits, was the need to fully review the impact of allowing such a large number of trees to flower. Although this addresses a legitimate environmental issue, and it is certainly wise for other developers of engineered trees as biofuel feedstocks to consider this issue for their own activities, I’d stress that this concern is somewhat unique to perennial species, particularly trees, that will need to be grown over several years to reach maturity before harvesting for fuel use. For those energy crops that would be sown, grown and harvested during a single growing season (the way food crops and other agricultural commodities are typically grown), the issue of flowering over a multiple year lifespan may be less relevant, or even irrelevant. However, other issues may arise for annual species, especially food crops, used for biofuel production, as we’ll see in the Syngenta story. 

Table 1. Select Issues Considered in Arborgen Environmental Assessment. (Source: USDA Environmental Assessment)

  • Alteration in Susceptibility to Disease or Insects – Potential of the Eucalyptus to Harbor Plant Pests
  • Expression of the Gene Products, New Enzymes, or Changes to Plant Metabolism – Risk of the Gene Products on the Environment
  • Alteration in Weediness characteristics – Potential of the Engineered Eucalyptus to be Invasive
  • Possibility of Gene Flow Within the Field Test
  • Possibility of Gene Flow Outside of the Field Test
  • Possibility of Vegetative Propagation / Persistence Outside of the Field Test
  • Potential of the Eucalyptus in the Field Tests to Become an Invasive Species that Threatens Native Plant and Animal Communities
  • Transfer of Genetic Information to Organisms with which it Cannot Interbreed – Horizontal Gene Transfer to Other Organisms
  • Risks to Threatened and Endangered Species

As described in the previous entry, in 2005 Syngenta Seeds, a major multinational seed and agbiotech company, submitted a petition asking USDA for a determination of “nonregulated status” for a corn variety (known as “Event 3272”) genetically engineered to produce a microbial amylase enzyme that facilitates ethanol production by enhancing starch degradation in the preprocessing step. In this petition, Syngenta requested that APHIS make a determination that these corn plants should no longer be considered regulated articles under 7 CFR part 340. This petition process is the route under the regulations by which transgenic plants have been reviewed and cleared for widespread commercial use, and as of this writing, APHIS has approved 78 such “delisting” petitions for commercialization of engineered crop varieties. However, because Syngenta’s request would have led to the first determination of “nonregulated status” for an industrial use of a transgenic plant variety, APHIS conducted its review very carefully, and decided to prepare an Environmental Assessment before announcing its decision on the petition. 

APHIS published a notice in the Federal Register on November 19, 2008 announcing the availability of the Syngenta petition and a draft environmental assessment for public comment. APHIS solicited comments on the petition, on whether the engineered corn is likely to pose a plant pest risk, and on the draft EA. APHIS received over 13,000 comments on the petition, the draft EA, and the plant pest risk assessment by the close of the 60-day comment period, which ended on January 20, 2009, and in response to these comments, APHIS extended the comment period until July 6, 2009.  As of this writing APHIS has not announced any further decision on this petition. 

The EA itself addressed issues such as those shown in Table 2.  However, as explained in the June 4, 2009 Federal Register notice extending the comment period, there were several issues raised by commenters that APHIS felt justified further review. One issue was the appropriateness of the Agency’s determination that the amylase-expressing corn was not a plant pest – several comments argued that the alpha-amylase enzyme engineered into Event 3272 corn may cause damage (degradation of corn starch products) to manufactured or processed plant products if Event 3272 corn became inadvertently included in the manufacturing and processing of corn starch products. The commenters claimed that this type of damage comes within the definition of a plant pest (a position with which APHIS disagrees). Some of the commenters were also concerned about what they felt were specific food safety concerns, such as the potential for Event 3272 corn to be allergenic, as well as concerns surrounding the potential economic and manufacturing issues if Event 3272 corn were to become present in corn wet-milling processes. Comments were also received questioning the appropriateness for U.S. energy policy of using corn to produce ethanol, an issue with APHIS determined was outside their purview under the regulations. 

Table 2. Select Issues Considered in Syngenta Environmental Assessment. (Source: USDA Draft Environmental Assessment)

Management Considerations:

  • Corn Production
  • Cropping Practices
  • Impact to Other Specialty Corn Products
  • Ethanol Production

Public Health Considerations

  • Human Health
  • Worker Safety

Environmental Considerations

  • Gene Movement (Pollen flow)
  • Water Use in Ethanol Production
  • Effects on Animals
  • Effects on Plants
  • Effects on Soil

In my view, the major issues that USDA has faced in this review have all had to do with the fact that corn is a food crop. Some of the negative comments from the public have focused on the usual “food vs. fuel” arguments, with the commenters maintaining that it is not in the best interests of the U.S. to produce ethanol from a plant species that is so important for the nation’s and the world’s food supply. But other comments on the draft EA have followed familiar lines as has been seen in other applications for commercial use of engineered agricultural crops, including concerns over what is called “adventitious presence”.  This term refers to the inadvertent mixing of trace amounts of one type of seed, grain or food product with another, particularly when the first type of seed or grain is a genetically engineered plant or plant product. Many transgenic plant products are intended for a specialized use and often must be grown, harvested and transported separately from other varieties of the same species; in some cases the segregation is required because the transgenic variety has not been approved for food use. Although the harvested crop is segregated, some observers are concerned that the transgenic plant material could be accidentally mixed with nontransgenic material, or that nontransgenic corn could acquire the transgene through pollen-mediated gene flow. Some members of the public are concerned about the potential for such commingling and concerned that this could lead to contamination of the food supply with materials not approved for food use. This is an issue that has been known to agbiotech and seed companies for many years, and although the actual risks from low levels of commingling are likely to be minimal, strategies have been developed to isolate transgenic crops by a suitable distance from growing regions of nonmodified plants, to ensure segregation of harvested grain or seed, and also to reliably test food products for such inadvertent mixing. Although these issues were all addressed in the Draft EA, it may be that such issues have been the major reason Syngenta has not received its U.S. approval for commercial sale of Event 3272 corn. 

It is somewhat troublesome to the industry to think that this application, the first that would allow commercial sale and widespread production of a transgenic plant for a biofuel purpose, has been pending for almost 5 years without resolution. However, it has been a fairly consistent (although bothersome) trend in the history of biotechnology regulation that the earliest application(s) of any given type have taken far longer to approve than would become the norm for later applications. So to some extent, a lengthy review for the Syngenta petition was almost inevitable, and one can expect subsequent reviews to be quicker and more efficient. 

In addition, I think that many of the issues being considered in USDA’s review of the Syngenta petition are specific to any proposed use of a food crop, particularly one as economically important as corn, for an industrial purpose. Today, corn is the most abundant and most cost-effective feedstock for ethanol production in the U.S., but  most of the ethanol and biofuels industry see corn as a transition to “advanced biofuels” produced from cellulosic feedstocks and other non-food-use crops such as specialty grasses or trees. And so, while proposed commercial use of feedstocks like transgenic switchgrass, Jatropha or poplar would certainly require careful consideration of the possible environmental impacts, such proposals would most likely not engender any concerns over commingling, adventitious presence, or the alleged “contamination” of the food supply, and this alone could make agency review of the applications far more straightforward.   

Are there lessons that the industry can draw from this small (N=2) sample of lengthy regulatory reviews for expanded uses of transgenic biofuel species? I think there are, although some are fairly obvious and not at all surprising. For example: 

  • The earliest proposals under any relatively new regulation will always face the toughest scrutiny and, at least in the biotech world, often will undergo inordinately long review. However obvious this may be, it does argue that the companies that hope to be next in line behind Arborgen and Syngenta plan their proposals carefully, and devote sufficient attention to presubmission meetings and discussions with USDA APHIS staff (something that is always recommended for regulatory submissions of any kind).
  • The issues that have likely led to the length of these reviews were ones that could be predicted, yet are ones that might not affect every transgenic biofuel proposal. The concern in the Arborgen proposal about allowing large numbers of transgenic trees to flower at multiple locations would likely be applicable only to those energy crop species where multiple years of growth would be needed before the plants could be harvested for fuel use, and even for those crops, concern over this issue is likely to abate as more experience is gained with the field use of these transgenic species. And the concerns in the Syngenta review are likely to mostly be specific to the use of a food crop in biofuel production, and most other proposed transgenic biofuel crops would not need to address such issues.
  • Finally, although the nearly 25 years of experience we’ve had with the growth of transgenic plants in the open field, including tens of thousands of field tests worldwide, have made the regulatory path for small-scale testing quite straightforward, the same is not true for applications that go beyond limited-scale field tests. Some of these proposals do in fact pose environmental issues that regulatory agencies still must contend with, but these larger-scale proposals betray the fact that the small but vocal group of biotech opponents have not gone away, and in some places (like Europe) their impact is felt beyond their numbers, especially with regard to engineering of any food crop. Biofuel companies should count on the fact that there will always be opponents to any proposed large-scale or commercial use of transgenic plants, even for projects with clear environmental benefits or that address public policy imperatives. Although the concerns raised by such critics should not, in the end, be sufficient to derail any well-planned proposal, companies do need to take these concerns seriously and plan accordingly. 

D. Glass Associates, Inc. is a consulting company specializing in several fields of biotechnology. David Glass, Ph.D. is a veteran of nearly thirty years in the biotech industry, with expertise in industrial biotechnology regulatory affairs, patents, technology licensing, and market and technology assessments. This blog provides back-up and expanded content to complement a presentation Dr. Glass made at the EUEC 2010 conference on February 2, 2010 entitled “Prospects for the Use of Genetic Engineering in Biofuel Production.” The slides from that presentation, along with more information on D. Glass Associates’ regulatory affairs consulting capabilities, are available at www.slideshare.net/djglass99 or at www.dglassassociates.com.

Impact of USDA Regulations on the Use of Genetically Modified Plants as Biofuel Feedstocks (Part 1)

As noted in a previous entry in this blog, the use of certain genetically modified plants as feedstocks for biofuel production may be subject to regulations promulgated by the U.S. Department of Agriculture (USDA) that have been used for over two decades to regulate the agricultural biotechnology industry. In this posting I will discuss the growing number of transgenic plant varieties intended for biofuel use that have already been submitted for USDA review and used in field trials under this rule, and in the posting that will follow, I’ll consider the impact these regulations may have on the development of improved fuel feedstocks using advanced biotechnology.   

USDA’s biotechnology regulations, found in 7 CFR Part 340 of the Code of Federal Regulations, have been the major U.S. government rules that have covered uses of transgenic plants in agriculture and more recently the increasing interest in using plants for other industrial purposes, such as production of pharmaceuticals, industrial products, and phytoremediation.  The regulations are administered by the division of USDA known as the Animal and Plant Health Inspection Service (APHIS), through a dedicated office within APHIS called Biotechnology Regulatory Services (BRS). Today, many research field tests involving the most commonly studied species can be conducted merely upon 30 days advance notice to the agency, but certain uses of transgenic plants for less-familiar industrial uses would likely require submission of a permit application 120 days in advance of a proposed field test, with USDA approval of the permit needed before the test could begin. Field tests conducted to date of improved energy crops have made use of both the notification and the permit systems, depending on the crop species to which the genetic modifications have been made. To obtain approval for unlimited commercial sale of a transgenic plant variety, the applicant must submit a petition to have USDA “delist” that variety, to have it removed from the need for regulation, thus allowing anyone to grow the plant without a permit, a process that at least one company has initiated for a new energy crop.   

Examples of Approved Field Uses of Modified Plants as Biofuel Feedstocks   

As of this writing in May/June 2010, there have been a number of transgenic plant varieties engineered for improved biofuel production that have been reviewed by APHIS Biotechnology Regulatory Services. These projects have been at several different stages of commercial development, ranging from early field tests to at least one application for commercial approval via the “delisting” process. The following is a summary of those applications that have been submitted by for-profit companies, grouped in a company-by-company manner (expanded company profiles can be found in earlier entries of this blog). There have also been several applications from academic institutions for field use of plants that appear to have been engineered for improved biofuel production, but those are not listed here. More information about the permits and notifications listed here can be found at the field release database search site administered for APHIS by Virginia Tech University. Please also note that there have also been numerous field trials of transgenic biofuel crops that have taken place in Canada, principally by Agrisoma and Targeted Growth, Inc. These will be described in a future entry in this blog.   

ArborGen has been the industry leader in improving trees through advanced genetics, and in recent years the company has diversified from its roots in the forestry industry to begin developing purpose grown trees to produce cellulosic ethanol. ArborGen is particularly targeting trees such as U.S. plantation hardwoods, and among the potential bioenergy products the company is developing are cold tolerant eucalyptus; short rotation hardwoods; and short rotation pine varieties. ArborGen has conducted numerous field tests of engineered trees – from 2001 through May 2010, the company had submitted about 280 permit applications and notifications to APHIS for field tests of engineered trees of several different species, almost all of which have progressed to field trials. More recently, Arborgen has obtained USDA clearance for field testing of Eucalyptus hybrids and clones genetically modified with traits such as cold tolerance and altered lignin biosynthesis being developed as improved energy crops: for example, just since 2008, the company has made the following 19 submissions all involving Eucalyptus modified with such traits.   

Permits (all for Eucalyptus)  

  • 10-112-101r, for planting in Alabama, submitted 04/22/10,  Pending.
  • 08-039-102rm, for planting in 7 states, submitted 02/03/10,  issued 04/23/08 (note: 172.7 acres)
  • 09-070-101rm, for planting in Florida and South Carolina, submitted  03/11/09, issued 04/29/09
  • 06-325-111r, for planting in Alabama, submitted 06/03/08,  issued 06/27/07
  • 08-151-101r, for planting in Florida, submitted 05/30/08,  issued 06/12/08
  • 08-014-101rm, for planting in Florida and South Carolina, submitted 01/14/08,  issued 05/12/10  (note: 130.5 acres)*
  • 08-011-106rm, for planting in 6 states, submitted 01/11/08,  issued 05/12/10 (note: 197.2 acres)*

Notifications (all for Eucalyptus)  

  • 09-208-103n,  for planting in South Carolina,  submitted 07/27/09
  • 09-182-101n,  for planting in South Carolina,  submitted 07/01/09
  • 09-023-106n,  for planting in South Carolina,  submitted 01/23/09
  • 08-175-101n,  for planting in Florida,  submitted 06/23/08
  • 08-157-102n,  for planting in Alabama, Louisiana, Mississippi and South Carolina,  submitted 06/05/08
  • 08-157-101n,  for planting in South Carolina,  submitted 06/05/08
  • 08-148-101n,  for planting in South Carolina,  submitted 05/27/08
  • 08-144-104n,  for planting in Alabama and South Carolina,  submitted 05/23/08
  • 08-134-103n,  for planting in Alabama, Florida, Louisiana, and South Carolina,  submitted 05/13/08
  • 08-095-104n,  for planting in South Carolina,  submitted 04/04/08
  • 08-092-115n,  for planting in Alabama and South Carolina,  submitted 04/01/08
  • 08-063-115n,  for planting in South Carolina,  submitted 03/03/08

*The two permits listed last above (Numbers 08-014-101rm and 08-011-106rm) were only recently (May 12, 2010) issued by APHIS upon the completion of an Environmental Assessment and a finding of no significant risk. This will be discussed in more detail in the next entry of this blog.  

Ceres, Inc. develops and markets low-carbon, non-food grasses for advanced biofuel and biopower uses. The company is already selling high-yielding switchgrass cultivars and high-biomass sorghum hybrids under the Blade Energy Crops brand, and Ceres plans to use marker-assisted breeding and other technology platforms to introduce a additional enhancements to these crops, including further increases in biomass yield and other agronomic and compositional improvements. Ceres is also carrying out an advanced trait development project to increase biomass yields of energy grasses by as much as 40% in coming years, while simultaneously decreasing the use of inputs such as nitrogen fertilizers, in a project which has received funding from the Advanced Research Projects Agency – Energy. On May 20, 2010, Ceres announced that it had entered into a research collaboration with Novozymes, the world’s largest enzyme provider, to co-develop customized plant varieties and enzyme cocktails for the production of cellulosic biofuel. The companies will initially work to determine the best enzyme cocktails for the biorefining of Ceres’ commercial switchgrass seed products. The partners will also begin similar evaluations of sweet sorghum, and Ceres’ researchers plan to develop customized plant varieties that can be degraded more easily by Novozymes’ enzymes.  

Ceres has obtained three USDA permits since 2008 for field testing of drought tolerant or sterile switchgrass, and currently has three pending permit applications for engineered switchgrass that appear to correspond to to the ARPA-funded project, as follows.  

  • 10-130-103rm, Switchgrass with increased nitrogen utilization, for planting in Georgia, submitted 05/10/10, application pending 
  • 10-116-105rm, Switchgrass with increased nitrogen utilization (also increased drought tolerance and sterility), for planting in California, submitted 04/26/10, application pending 
  • 10-054-102rm, Switchgrass with increased nitrogen utilization, for planting in Tennessee, submitted 02/23/10, application pending   

Edenspace Systems Corporation ia plant biotechnology company that is developing innovative new energy crops. Responding to the market need for lower-cost ways to produce cellulosic biofuels such as ethanol and butanol, Edenspace is developing Energy Corn™ and other enhanced energy crops such as poplar and sorghum. On May 19, 2010, Edenspace announced a collaborative agreement with Syngenta Ventures for the development of new energy crops. Under the terms of the collaboration, Syngenta will be licensing Edenspace access to intellectual property and expertise relating to crop technology, in return for Edenspace equity and certain rights to commercialize Edenspace-developed technology. Edenspace will utilize Syngenta’s technology in its development of new traits in corn, sorghum, switchgrass, and other energy crops.   

Edenspace has made several USDA submissions for field uses of plants engineered for  “improved digestibility”. Details of the genetic modifications were not part of the public record, but likely involve modifications to lignin, or expression of heterologous enzymes that could enhance the digestibility of the cellulosic components of the crop. One other notification was for poplar trees that had been engineered with altered cell walls. The company’s submissions include the following tests conducted under notifications: 

  • 10-098-103n, Corn with digestibility improved, for planting in Kansas, submitted  04/08/10
  • 09-310-102n, Corn with digestibility improved, for planting in Puerto Rico, submitted  11/06/09 
  • 09-308-106n, Corn with digestibility improved, for planting in Puerto Rico, submitted  11/04/09 
  • 09-175-101n, Poplar with cell wall altered, for planting in Kansas, submitted 07/24/09.
  • 09-074-101n, Corn with digestibility improved, for planting in Kansas, submitted  03/15/09

In addition, Edenspace has had one permit application approved for a biofuel crop:    

  • Permit No. 09-054-105r, for field use of Grey Poplar in Kansas, submitted 03/10/09, issued 05/06/09.

Infinite Enzymes is using a plant biotechnology platform to produce commercially available enzymes for converting cellulosic biomass to ethanol. The technology utilizes the transgenic maize production system—producing enzymes in the embryo, or germ, of the corn seed. Infinite Enzymes has applied for and obtained the following permits for field use of its transgenic corn plants expressing cellulase enzymes. The most recent permit would encompass a planting of 15 acres in Arkansas, larger than any of the previous submissions.   

  • 10-056-103rm, Corn expressing a cellulase, for planting in Arkansas, submitted  02/25/10, issued  03/12/10
  • 09-225-105rm, Corn expressing a cellulase, for planting in Puerto Rico, submitted  08/13/09, issued 10/15/09
  • 09-053-101rm, Corn expressing a cellulase, for planting in Illinois, submitted 02/22/09, issued 03/12/09
  • 09-034-101rm, Corn expressing a cellulase, for planting in Arkansas, submitted  02/03/09, issued 03/31/09
  • 08-216-101rm, Corn expressing a cellulase, for planting in Puerto Rico, submitted  08/03/08, issued 09/29/08

 Syngenta is a world-leading, multinational agribusiness company. Its two main commercial areas  are the sale of seeds and crop protection products. Syngenta’s major activity in biofuels is a line of transgenic corn expressing an engineered thermostable amylase for enhanced ethanol production. This corn variety, which the company refers to as its “output trait corn amylase”, is sold under the brand name Enogen. Originally known by the internal product name “Corn [Maize] event 3272”, this line was developed using recombinant DNA technology to introduce into corn the amy797E gene and the pmi marker gene. In 2005, after many years of field trials, Syngenta submitted a petition to USDA to have this corn variety deregulated to permit commercial sale of the seed. Although USDA issued an Environmental Assessment in June 2009 stating their intention to grant the petition, the matter was opened for public comment and USDA has not, to date, made a decision on the deregulation petition. This will be discussed in more detail in the next entry of this blog.  

Syngenta has conducted numerous field trials of engineered corn and other crops over the years. According to a 2007 regulatory filing with USDA, the following are the field trials for Event 3272 corn which had been conducted through the date of the 2005 deregulation petition.  

Year   USDA Notification or Permit No.   Trial Sites by State  
2002   02-022-02r/m   HI  
2003   03-021-01r/m   FL,IA,IL,MN,PR,SD,WI  
    03-021-02r/m   HI  
2004   04-051-08n   IA  
    04-064-04n   FL,HI,IA,ID,IL,IN,KY,MN,NE,PA,PR,SD,WI  
    04-082-03n   IA  
    04-126-03n   NE  
    04-203-03n   PR  
    04-216-02n   HI  
2005   05-042-09n   HI, NE  
    05-049-10n   CO,FL,HI,IA,ID,IL,IN,KY,MN,MO,NE,PR,SD,WI  
    05-102-02n   IL  
    05-104-08n   HI  
    05-255-01n   HI  

 Targeted Growth, Inc. (TGI) is a crop biotechnology company focused on developing products with enhanced yield and improved quality for the agriculture and energy industries. Founded in 1998, TGI has developed a technology portfolio based on the principle that regulating cell cycle processes can directly and significantly enhance plant yields.  The company is using conventional breeding and biotechnology to enhance the suitability of select energy crops for use as fuels, by improving sugar, starch and oil profiles, as well as characteristics of cell wall formation. For ethanol production TGI is focused on corn and sorghum; for biodiesel, soybean, canola, and Camelina. The company has made over 50 submissions to USDA for field test approvals since 2002: the following are the ones among these submissions that appear most relevant to the development of biofuel feedstocks. These include the following tests conducted under notifications:  

  • 10-119-104n, Corn with increased sugar content, for planting in Iowa, submitted 04/29/10
  • 10-081-107n,  Corn with increased sugar content and yield increase, for planting in Iowa, Illinois submitted 03/22/10
  • 10-029-101n,  Corn with increased sugar content, yield increase, increased digestibility, for planting in Iowa, Illinois submitted 01/29/10
  • 09-140-101n, Corn with enhanced digestibility of plant wall, increased sugar content and yield increase, for planting in Indiana
  • 09-075-113n, Corn with increased digestibility, for planting in Indiana, Iowa, submitted 03/16/09  

In addition, TGI has had one permit application approved for a biofuel crop:  

  • 08-154-102r , Camelina engineered for increased yield, for planting in California, Montana and Washington State, submitted 06/16/09, issued 05/05/09

The experience of these companies in going through the USDA regulatory process has mostly been positive, and approval for most research field trials has been fairly easy to obtain. However, the experiences of Arborgen and Syngenta in requesting USDA approval for activities beyond small-scale research may offer a cautionary note to other developers of transgenic energy crops. In the next blog entry, I’ll discuss the issues that have arisen in USDA’s review of those requests and their possible implications for future applicants, and I’ll also make some general comments about the impact of the USDA regulations on development of transgenic biofuel feedstocks.  

D. Glass Associates, Inc. is a consulting company specializing in several fields of biotechnology. David Glass, Ph.D. is a veteran of nearly thirty years in the biotech industry, with expertise in industrial biotechnology regulatory affairs, patents, technology licensing, and market and technology assessments. This blog provides back-up and expanded content to complement a presentation Dr. Glass made at the EUEC 2010 conference on February 2, 2010 entitled “Prospects for the Use of Genetic Engineering in Biofuel Production.” The slides from that presentation, along with more information on D. Glass Associates’ regulatory affairs consulting capabilities, are available at www.slideshare.net/djglass99 or at www.dglassassociates.com.