(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.