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United States Department of Agriculture

Agricultural Research Service

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Research Project: Selection and Testing of Heterotrophic Algae and Fungal Strains for Growth on Papaya, Glycerol, Albizia, and Sugarcane Bagasse

Location: Tropical Plant Genetic Resources and Disease Research

2013 Annual Report


1a.Objectives (from AD-416):
PBARC is implementing the ‘zero waste’ concept in which we make use of the agricultural and other waste feedstock to produce biofuels, feeds, and other value added products. In this specific cooperative agreement, we focus on using culled papaya fruit, glycerol, albizia wood, and sugarcane bagasse as the carbon source for heterotrophic oil-producing algae and fungi, and subsequently harvest the algae and fungi to extract oil and high protein meal. The oil would be the source for producing biodiesel or aviation fuel, and the meal as a component of animal feed. This project solely focuses on getting products to the oil and meal stage. The most important starting element of the project is to obtain algae or fungi that effectively use the above selected feedstock as carbon sources. To accomplish this phase, we will collaborate with BioTork, LLC which has a patented ‘evolugate’ process to rapidly select heterotrophic algae and fungi that can grow efficiently on targeted feedstock. These algae/fungi would be transferred to PBARC where personnel will determine the optimal conditions for growing the selected algae/fungi on the selected feedstock, characterize the oil and meal for their suitability as biofuel or animal feeds, and analyze the economics of producing the selected algae/fungi under mini-pilot scale.


1b.Approach (from AD-416):
PBARC does not have the expertise or process for rapidly selecting algae or fungi to feed on the identified feedstock. This agreement will continue the very successful collaboration between PBARC and BioTork that was started in a previous SCA. BioTork will collaborate with PBARC to select heterotrophic algae/fungal strains that grow on the feedstock, do preliminary characterization of these strains, such as rate growth on the feedstock, and then send the strains to PBARC. PBARC will grow the strains on quantities of feedstock, harvest the algae/fungi and extract and characterize the resulting oil and meal. The oil will be subsequently tested by a third party for their potential to produce biodiesel and advanced fuels. Another third party will test the meal in animal feeding studies. PBARC will scale up the production and analyze the economic feasibility for large scale production of algae/fungi oil and meal. PBARC and BioTork will continue their close collaboration to ensure the highest chance of success for this specific cooperative agreement.


3.Progress Report:

The goal of this cooperative agreement is to implement zero-waste concept to use agricultural and other waste feedstock to produce biofuels, feeds, and other value-added products, and contributes to objective 2 of the in-house project 5320-21610-001-00D that this project was originally established under (expired in FY2011). This is the final report for this project.

In collaboration with the Pacific Basin Agricultural Research Center (PBARC), BioTork, LLC, has been developing new strains of oil-producing microbes (algae, fungi, etc.) that are highly adapted for growth on agricultural and agroindustrial wastes. In the first phase of the contract, we focused on strains that are adapted for converting culled papaya fruit into renewable oil and high-protein animal feed. The purpose of this second phase of the collaboration is to expand the variety of Hawaiian agroindustrial wastes that can be effectively converted into oil-rich microbial biomass. Agroindustrial targets include biodiesel-derived crude glycerol from a biorefinery on the Big Island and lignocellulosic biomass from Albizia. PBARC and BioTork jointly decided that the first target would be biodiesel-derived crude glycerol for several reasons. First, BioTork has extensive experience adapting strains for crude glycerol and already has microbial strains that represent excellent starting points in collection. Second, the Pacific Biodiesel biorefinery is close to the papaya packingsheds near Hilo, meaning that our two feedstocks (papaya cull and crude glycerol) are already collected in close proximity to each other. The concept behind this phase of the proposal is to take an existing BioTork strain that has already been pre-adapted to a sample crude glycerol from a different biodiesel facility, adapt it for crude glycerol produced by Pacific Biodiesel in Hawaii and develop fermentation processes for converting this waste glycerol into renewable oil and high-protein animal feed. The renewable oil produced in this process can be directly plugged back into the Pacific Biodiesel facility to produce more biodiesel, or it can be converted into other advanced drop-in fuels like green diesel, green gasoline or green jet fuel with catalytic hydrocracking technology. Pacific Biodiesel produces ~2800MT of crude glycerol in Hawaii. From this feedstock, we anticipate that we can produce up to 160,000 gallons of renewable oil and 760 MT of high-protein feed.

Biodiesel-derived crude glycerol contains a variety of compounds that inhibit the growth of microorganisms. Thus, microbes that are capable of growing on refined glycerol often grow very poorly on crude glycerol. Moreover, every sample of biodiesel-derived crude glycerol is fundamentally different in terms of chemical composition, which is determined not only by the oil feedstock, but also by the transesterification methodology. This means that microbes that can grow on one sample of crude glycerol may not be capable of growing on another. BioTork has developed a library of microorganisms capable of growing on biodiesel-derived crude glycerol from various biodiesel facilities. In particular, we developed a strain of heterotrophic algae that was adapted for a sample of waste glycerol from a biodiesel facility whose feedstock is mainly soybean oil. While the parent strain of this adapted strain was capable of growing on refined glycerol, it was incapable of growing on even low concentrations of soybean biodiesel-derived crude glycerol. We began by culturing the parent strain on refined glycerol and, over time, replaced the refined glycerol with increasing concentrations of soybean biodiesel-derived crude glycerol. Eventually, the only source of carbon and energy was crude glycerol. After that, we began adapting the strain for increasing concentrations of crude glycerol, up to 20% (v/v). The adapted strain accumulates lipids to approximately 55% of its cell dry weight, yielding 10-fold higher lipid titers than the parent strain on medium containing 5% (v/v) crude glycerol. This strain is capable of yielding 64 gallons or renewable oil and ~200 kg of high protein algae meal per MT of crude glycerol.

For the most part, Pacific Biodiesel uses animal fat and waste cooking oil as the feedstock for biodiesel production in Hawaii. The use of this feedstock is likely to have a large impact on the palatability of the crude glycerol for our adapted strain. On 2/25/13, BioTork requested a sample of crude glycerol from the Pacific Biodiesel facility and received the first sample on 3/11/13. We determined that the maximum concentration of crude glycerol upon which detectable growth of our strain could be demonstrated was 3% (v/v). We then put our strain into the Evolugator™ evolution machine on 3/26/13 on medium containing 3% (v/v) crude glycerol. However, we found it impossible to maintain continuous cultures on this substrate due to its high toxicity. It is not uncommon to see good growth in batch culture, and poor growth in continuous culture, especially when toxic compounds are present. Viable cultures were not recoverable from the machine after 4/16/13. The high level of toxicity caused us to analyze the sample. It was discovered that this sample contained only 20% glycerol and 19% methanol. Thus, this sample was taken from the wrong step in the biodiesel process, prior to the recovery of methanol. We stop attempting to evolve our strain on this substrate on 5/3/13.

On 4/6/13, BioTork requested a second sample of crude glycerol that arrived on 5/17/13. Analysis of this sample confirmed that it was 95% glycerol with no detectable methanol. As before, we set out to determine the maximum concentration of Pacific Biodiesel crude glycerol upon which we could detect growth of EVG45. Despite the fact that the Pacific Biodiesel crude glycerol was ostensibly more 'pure' than the soybean biodiesel-derived crude glycerol, we saw no growth of our strain on 50 g/L and 25 g/L Pacific Biodiesel crude glycerol. This could be explained by the presence of low concentrations of toxic compounds specific to the animal fat/waste cooking oil feedstock. We found that the maximum concentration of crude glycerol upon which growth of our strain could be demonstrated was 1% (v/v). Our strain was reinoculated into the Evolugator™ evolution machine on medium containing 1% (v/v) crude glycerol. After demonstrating that continuous culture could be maintained, the concentration was increased to 1.5% (v/v) crude glycerol. The strain is growing, albeit slowly, which is to be expected considering the toxicity of the substrate. The adaptation process is continuing.


Last Modified: 9/21/2014
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