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Synthetic Biology Toolkit for Bioconversion of Food Waste

Description:

TECHNOLOGY AREA(S): Materials 

OBJECTIVE: Develop a Clostridium molecular toolkit that enable engineering of Clostridium species (spp.) to convert food waste to fuel or other intermediates of value to reduce waste removal costs and to improve sustainability in the field. 

DESCRIPTION: The Army has an urgent need for low-energy, portable solutions for bioconversion in an end-deployment field. Bioconversion can be used to produce fuels and materials, or to remove waste. There is a clear demand for bioconversion: at forward military bases, basic commodities such as gasoline can cost up to $400 per gallon due to delivery costs [1]. In addition, significant waste is generated at forward bases of which 87% are carbon-sources convertible to energy, averaging approximately 7 pounds per day - conversion of this waste would save up to $3500 per year per soldier [1] and significantly reduce waste removal costs. While waste-to-energy technologies are under development to conduct limited bioconversion, these technologies are typically combustion-based and suffer from high equipment needs and significant energy usage. In addition, waste-to-energy technologies are unable to efficiently handle sources of diluted carbon without pretreatment. Specifically, food waste composes the majority of solid waste generated (19%), but also has the highest moisture content (54%) [2]. Microbes can be used for bioconversion of food waste through fermentation technology [3] to either biofuels worth $200-400/ton converted or specialty chemicals and materials such as plastics or enzymes worth $1000/ton converted [4]. Species of Clostridium have been explored for bioconversion such as Clostridium acetobutylicum [3], Clostridium beijerinckii [5], and Clostridium tyrobutyricum [6], among others. Clostridium is a particularly attractive target for its ability to make butanol, hydrogen, and valuable intermediates. However, Clostridium species have been minimally engineered due to the lack of characterization on the organism's regulatory regions and genetics, and the difficulty of growing and genetically engineering the organism. This project seeks to further characterize parts (i.e. biologically functional units) to allow for the synthetic biological engineering of Clostridium, as has been done extensively for model organisms such as E. coli and yeast [7]. The ultimate goal of this project is to develop a toolkit for the engineering of Clostridium for the application of bioconversion of food waste. If successful, these toolkits will allow the construction of efficient Clostridium-powered fermentations. 

PHASE I: Develop assays to isolate and test transcriptional and translational regulatory regions, such as promoters and ribosome binding sites in Clostridium spp. that can produce higher-order compounds (butanol, ethanol, hydrogen, or other valuable intermediates to the Soldier in the field) from carbohydrates (or food waste simulant). The assays should use host derived transcriptional and translational machinery and can be cellular, or cell-free extract based and should be easily extensible to other organisms. The Phase I effort should include a proof-of-principle of the functionality of the assays, and demonstrate expression of non-host derived enzymes using a subset of at least 5 native regulatory elements with differential responses. Native or recombinant multi-enzyme pathways shall be identified where improved transcriptional and translational control will allow for modulation of metabolic output based upon internal and/or external cues. 

PHASE II: Demonstrate the functionality of the assays by producing an expanded parts list of tested functional regulatory regions which are responsive to external and/or internal cues, and construct two of the identified pathways in Phase I in Clostridium spp. using the information obtained from the toolkit. Demonstrate that the genetic elements and circuits function, as designed, in the organism through demonstration of altered protein expression. Demonstrate transcriptional and translational control (via protein expression levels or metabolic output) in the engineered Clostridium using internal or external cues. Demonstrate that engineered Clostridium strains are able to convert food waste into the desired final products more efficiently (>100%) than a non-engineered strain in batch fermentation at an equivalent residence time. Assess scalability and cost-effectiveness of the engineered conversion process and reproducibility as a function of relevant food waste composition and benchmark it against the existing chemical-based technologies. Determine feasibility for ancillary beneficial processes (e.g. generation of potable water and removal of organics from food waste) as a function of the bioconversion process. Assess waste-to-energy conversion in terms of processing parameters (e.g. food waste composition, residence/conversion time, bacterial wash-out/replenishment schedules, etc.). Demonstrate functionality of the engineering toolkit for one or more additional bacteria including but not limited to other Clostridia spp., spore forming bacteria and/or extremophiles. The final deliverable of this effort includes: 1) a list of functional regulator regions and synthetic circuits if used, 2) engineered Clostridium strains, 3) design specifications/parameters for the food waste batch fermenter, 4) scalability and cost analysis, and 5) lab-scale feasibility (as a function of altered protein expression or metabolic outputs) of extending engineering toolkit to at least one other relevant bacterial system. 

PHASE III: The Phase III work will produce a refined genetic engineering toolkit for translation to a host of anaerobic/aerobic bacteria for engineered metabolic outputs based on variable food-derived waste inputs. The toolkit will support the commercially-viable, environmentally responsible design and development of a biologically-based food waste conversion process that can be integrated into an existing or new waste-to-energy conversion system for fielding within forward operating bases to mitigate complications in meeting fuel/energy and water demands. The waste-to-energy system will facilitate a cost-effective, efficient means for the conversion of food waste into higher-order compounds for generation of bioenergy (biofuels, biohydrogen, etc.) to operate generators, lights, vehicles, etc. in addition to other valuable co-products (e.g. potable water). Waste-to-energy systems also have dual-use applications within the civilian sector for efficient municipal waste products. Furthermore, the toolkit would be a basis for engineering other microorganisms, in addition to the novel Clostridium strain, that produce commercially-viable metabolic byproducts. The biologically-derived waste-to-energy solution must be cost effective and commercially competitive compared to existing chemical-based conversion systems. 

REFERENCES: 

1: L. M. Powell, "Converting Army Waste to Fuel: Mobile Integrated Sustainable Energy Recovery," 13th Annual North American Waste-to-Energy Conference pp. 5-6, Jan. 2005.

2: "US Army Central (USARCENT) Area of Responsibility (AOR) Contingency Base Waste Stream Analysis (CBWSA)," pp. 1-53, Apr. 2013.

3: M. D. Servinsky, S. Liu, and E. S. Gerlach, "Fermentation of oxidized hexose derivatives by Clostridium acetobutylicum," Microbial Cell Fact 13 2014.

4: E. Uckun Kiran, A. P. Trzcinski, W. J. Ng, and Y. Liu, "Bioconversion of food waste to energy: A review," Fuel, vol. 134, pp. 389-399, Oct. 2014.

5: H. Huang, V. Singh, and N. Qureshi, "Butanol production from food waste: a novel process for producing sustainable energy and reducing environmental pollution," Biotechnology for Biofuels 2015 8:1, vol. 8, no. 1, p. 1, Sep. 2015.

6: J. JO, D. LEE, D. Park, and J. PARK, "Biological hydrogen production by immobilized cells of Clostridium tyrobutyricum JM1 isolated from a food waste treatment process," Bioresource Technology, vol. 99, no. 14, pp. 6666-6672, Sep. 2008.

7: C. A. Voigt, Synthetic Biology, Part A: Methods for Part/Device Characterization and Chassis Engineering. 2011.

 

KEYWORDS: Clostridium, Food Waste, Bioconversion, Low Energy, Synthetic Biology, Bioengineering, Fermentation, Cell-free 

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