Minh Dao | ALES Graduate Seminar

Event details: A graduate exam seminar is a presentation of the student’s final research project for their degree.
This is an ALES MSc Final Exam Seminar by Minh Dao. This seminar is open to the general public to attend.

MSc with Dr. David Bressler.

Thesis Topic: The Microbial Conversion of Methanol to Lipids

Abstract: 

In recent times, the global economy has been facing growing food and energy demands,
largely due to fast population growth, and an increasingly unpredictable climate. While several
multifaceted endeavors are being undertaken to alleviate these demands, this thesis proposes a
biorefinery approach that contributes to the collective solution, where methanol is converted to
lipids via microbial pathways. Having long been a common industrial feedstock, methanol is
becoming even more appealing due to technologies that allow the sustainable and efficient
production thereof. On the other hand, along with those extracted from animals and plants,
microbial fatty acids are a great source of lipids that has not been fully exploited. Lipids are a
versatile precursor for many essential commodities, and creating new pathways for its production
can help satisfy the ever-increasing demand for food and energy. As such, this research investigated
methods to bring methanol and microbial lipids together in an effort to uplift their industrial
potential, as well as to contribute to the scientific understanding of the oleaginicity in
methylotrophic microorganisms.

In the first stage of this project, several known microorganisms were screened to find the
ideal candidate to perform the methanol-to-lipid conversion. With no suitable hosts identified, the
second stage undertook a bioprospecting approach, where microorganisms were isolated from
several promising habitats that could potentially harbor oleaginous methylotrophs. A strain
collection of 82 isolates was obtained, among which 70 showed signs of intracellular carbon
accumulation, using a lipid-binding dye Sudan Black B. Almost all isolates that showed dye
retention were obtained from wetlands or oil sands-related environments, both of which are known
to contain a disproportionately higher concentrations of carbon sources, such as methanol,
methane, hydrocarbons, and carbohydrates, compared to other nutrients, therefore making them
ideal environments for carbon-accumulating methylotrophs. The major genera found were
Hyphomicrobium and Ancylobacter, both belonging to the Alphaproteobacteria class. Gas
chromatography analysis of selected isolates revealed that Methylorubrum extorquens OSD20,
Hyphomicrobium spp. SdF61, OSCF32, and OSFP11 can accumulate approximately 20% or more
of their fatty acids as cell dry weight, making them oleaginous methylotrophs. Hyphomicrobium
sp. OSFP11 is a potential production host for methanol-to-lipids conversion, thanks to its high
proportion of long-chain fatty acids and low short-chain fatty acid quantity, which are considered
undesirable products in this research (89% and 11% of total fatty acids, respectively). M.
extorquens OSD20 and Hyphomicrobium sp. SdF61, on the other hand, produced significant levels
of this undesirable compound (30% and 17% of total fatty acids, respectively).

As the final stage of this project, the effects of culture conditions on isolates’ fatty acid
profiles were examined. Compared to solid culture on agar, Hyphomicrobium sp. OSFP11’s
butenoic acid (C4:1) proportion in its fatty acid profile was reduced from 11.1% to almost zero,
while its long-chain fatty acid level increase from 67.6% on agar to 82.4% in liquid culture.
Hyphomicrobium sp. OSCF32 saw a reverse trend, with more C4:1 and less long-chain fatty acids
in liquid culture compared to agar culture. This isolate was also able to produce a substantial level
of dihydrosterculic acid, a cyclopropane fatty acid on agar (23% of fatty acid profile), which
however, was reduced to 5% in liquid culture. Cold (5 °C) and warm (45 °C) incubation
temperatures were found to significantly alter isolates’ fatty acid profiles. Compared to 30.8% at
the 30 °C control, M. extorquens OSD20 saw a reduction in its short-chain fatty acid proportion to
22.5% at both 5 °C and 45 °C, and an increase in long-chain fatty acids (from 58.8% at 30 °C to
70.6% at 5 °C, and 67.2% at 45 °C). Overall, this research identified and characterized various
potentially novel methylotrophic bacteria that can accumulate significant levels of lipids as well
as polyhydroxyalkanoates. At the same time, it revealed further knowledge about methylotrophic
populations in understudied environments, thereby contributing to a strong foundation for further
research on native methylotrophic microorganisms and their applications.