“A recently completed pilot study between BioTork and BASF demonstrated that these engineered microorganisms could be further optimized for maximal industrial performance using adaptive evolution,” says Tom Lyons, chief scientific officer of BioTork. “Laboratory results confirm a synergy effect between the metabolic modifications engineered by BASF and the proprietary adaptive evolution technology used by BioTork.” BioTork is the holder of an exclusive, worldwide license to use the adaptive evolution technology of Evolugate, LLC in the fields of bio-based chemical and biofuel production.

1. BioTork Announces Collaboration with
BASF
By BioTork LLC | June 15, 2012
BioTork LLC announces collaboration with BASF to optimize certain microbial strains for
the industrial production of bio-based polymers and green chemicals. After six months of
collaboration, BASF and BioTork came to the conclusion that a combination of
their complementary approaches to strain development can improve the efficiency and
resulting economics of biochemical production processes. The financial terms of the
partnership have not been disclosed.
BASF has been conducting intensive research on the use of microorganisms for the
production of proteins, enzymes, vitamins and other high value and low cost chemicals. In
their natural environment, microorganisms generally synthesize these chemicals only to meet
their own requirements for survival. The challenge faced by chemical companies is to push
these microorganisms to produce these chemicals faster, in much larger quantities, and under
industrial conditions that are different from the microorganisms’ natural environment. This is
the only way to use microorganisms for commercially viable production of chemical
products. To achieve this goal, BASF is successfully using metabolic engineering techniques
to make targeted modifications to certain microorganisms in order to optimize bioconversion
capability, increase yield and eliminate bottlenecks.
“A recently completed pilot study between BioTork and BASF demonstrated that these
engineered microorganisms could be further optimized for maximal industrial performance
using adaptive evolution,” says Tom Lyons, chief scientific officer of BioTork. “Laboratory
results confirm a synergy effect between the metabolic modifications engineered by BASF
and the proprietary adaptive evolution technology used by BioTork.” BioTork is the holder
of an exclusive, worldwide license to use the adaptive evolution technology of Evolugate,
LLC in the fields of bio-based chemical and biofuel production.
The Evolugate adaptive evolution technology is a continuous culture apparatus that selects
the fittest genetic variants from among a population under controlled environmental
conditions that mimic those needed for the most economical industrial process. After rounds
of selection—which can achieve success in a matter of weeks or months—the
microorganisms acquire capabilities that were absent from the original strain. These new
capabilities provide the microbe with the potential to enhance industrial performance and
thereby improve the economics of the process. Examples of these newly acquired properties
could be a faster growth rate, the ability to grow at non-optimal temperatures, resistance
to inhibitors or growth under nutrient limiting conditions.
“We are extremely pleased with the capacity of our technology to complement the
metabolic modifications that are engineered by BASF,” says Marc Penicaud, BioTork vice
president for business development in Europe. “We will dedicate all the necessary resources

2. to ensure BASF achieves major advancements in the production of high value bio-based
chemicals.”
At BioTork, we believe that microorganisms are the key to breaking the global addiction to an ever
dwindling supply of fossil fuels. Relying on our expertise in accelerated evolution we develop strains
of yeasts, algae, fungi, archaea and other types of microorganism capable of converting different
types of biomass into high value bio-based chemical commodities traditionally derived from
petroleum.
MISSION STATEMENT
Our mission is to develop microbial strains with improved industrial performance capable of
producing bio-based chemical commodities with the most cost effective production
processes.
VISION
Our vision is to use the power of biochemistry to replace all petro-chemical products with
quality, cost competitive and green biomass derived alternatives.
Technologies
Micro-organisms can naturally perform many tasks that are useful to producers of bio-based
chemical commodities. However, several variables under industrial conditions are totally
different than the natural environment where the micro-organisms are usually found.
Temperature, carbon sources, inhibitors and other variables are all elements that the microorganisms must be adapted to for efficient industrial performance.
Using continuous culture, we adaptively evolve microbial strains under conditions that mimic
those targeted production processes, and select for the fittest naturally occurring variants.
After rounds of selection that can take place in matter of weeks or months, the strains acquire
improved properties allowing them to perform industrial tasks, which the original strain was
incapable of doing.
BioTork develops microorganism to convert papaya waste to fuel
By Erin Voegele | January 05, 2012
A developmental research project being completed in collaboration with Florida-based
BioTork LLC, Hawaii-based Rivertop Solutions LLC and the USDA Pacific Basin
Agricultural Research Center has successfully demonstrated a method to turn Hawaiian
papaya waste into renewable oil that can then be converted into biofuels.
According to BioTork Chief Scientific Officer Tom Lyons, Hawaii’s unique economy and
infrastructure offers several challenges. As much as 90 percent of the state’s fuel is imported,
at relatively high cost, which causes energy challenges. In addition, livestock producers and
fisheries within the state are in need of locally sourced feed materials. The state also has
significant challenges in the arena of waste disposal. A solution made available through
BioTork’s unique technology can address all three of these issues by diverting waste biomass

3. from landfills and using that material to produce oil for conversion into fuel, as well as a feed
product suitable for use by Hawaii’s livestock producers and fisheries.
The process being developed by the group currently addresses Hawaii’s papaya waste, but
could be tailored to deal with other types of agricultural wastes in the future. Hawaii produces
a lot of papaya, Lyons said. In a down year in a down economy, he estimates that as much as
50 percent of the state’s papaya harvest is disposed of by packing houses because of
imperfections in the fruit that make them unsalable to the market. In fact, Lyons estimates
that more than 15 million pounds of the fruit is currently disposed of each year.
The developmental research project BioTork is participating in seeks to convert that waste
papaya into microbial biomass that is rich in oil. According to Lyons, the oil can be converted
into biofuels, with the remaining biomass serving as feed.
BioTork has developed a new technology that allows microorganisms to be cultured
continuously, in essence allowing for accelerated breeding. “We start with naturally
occurring microorganisms,” Lyons said. In the case of the papaya waste project, the process
features two different types of microorganisms; and algae strain and a single-celled yeast-like
mushroom.
“The process we use is essentially evolutionary optimization,” Lyons explained. “[These
microorganisms] are kept growing in a constant state of active growth. Over time they adapt
to the conditions where they are growing—in this case, adapting to grow on puréed papaya.
Over time they will learn to eat more of [the material] that is in the papaya faster because the
use of evolutionary optimization forces the fittest, fastest progeny to take over. In essence,
what we are doing is accelerated breeding.”
BioTork’s contribution to the developmental research project stems primarily from its ability
to complete this type of evolutionary optimization on microorganisms. In turn, PBARC is
scaling up the process. To date, the organization has evaluated the technology on a 15-liter
scale. According to Lyons, there are near-term plans to scale that production level up to the
100-liter and 500-liter scales. A pilot project of 2,000 liters is also in the works.
To complete the conversion, puréed papaya is introduced into a standard fermentation tank,
where it is suspended in a growth media. The algae and mushroom cells are cultured in the
fermentation tank where they feed on the material, converting it into oil. The resulting oil can
then be pressed out of the cells and converted into fuel. According to Lyons, the
microorganisms his company has developed for the project have already achieved an oil yield
of approximately 30 percent of the dry weight of the cells.
Lyons adds that the group is interested in developing similar microorganism mixes to deal
with other agricultural waste materials in Hawaii. The consortium is also currently working to
form partnerships with end-users of fuel within the state. This includes the U.S. Department
of Defense and the U.S. Navy, which is Hawaii’s single largest fuel user.
BioTork Mastering the Art of Microbiology
for Bio-based Chemical Commodities