Bioconversion of alkylbenzenes by Yarrowia lipolytica
Date
2009-03
Authors
Lind, Aingy Chantel
Journal Title
Journal ISSN
Volume Title
Publisher
Stellenbosch : University of Stellenbosch
Abstract
The abundance of alkane by-products formed in South Africa presents a
feedstock opportunity for the production of a wide range of commercially
important products, such as long-chain dioic acids and alcohols. These
compounds are formed as intermediates through the biological conversion of
alkanes, a route which is particularly attractive when compared with chemical
conversion due to its operation under milder process conditions. Furthermore,
advances in genetic manipulation, which enable the accumulation of a range
of metabolic intermediates, make the biological route remarkably flexible.
From the literature review Yarrowia lipolytica was identified as a promising
organism for use in studying alkane bioconversion because of its ability to
produce large quantities of fatty acids when grown on n-paraffins as a sole
carbon source.
The bioconversion of alkanes will not only depend on the genetic modification
but also on the process conditions to maximise growth and bioconversion.
The overall objective of this project was therefore to investigate the potential
of Y. lipolytica for alkane bioconversion by defining the conditions that
maximise both cell growth and bioconversion. The Y. lipolytica strains
supplied (TVN348, TVN493 and WT), however, were not yet modified to the
extent that accumulation of metabolic intermediates was possible. Use was
therefore made of a model system in which the alkane substrate was
substituted with an even chain alkylbenzene. Since Y. lipolytica is unable to
metabolise the benzene ring, the alkylbenzene is converted to the metabolic
intermediate, phenyl acetic acid (PAA), and the potential for bioconversion
assessed through measuring the accumulation of PAA. The specific
objectives of the project were therefore
1) to define and quantify the parameters for the establishment of an
effective model system in shake flasks with respect to trace elements,
buffering, added nitrogen, oxygen supply, glucose concentration,
alkylbenzene substrate and inducer requirements
2) to use the defined model system to identify the most promising strain
of Y. lipolytica TVN348, TVN493 and WT
3) to use the defined model system and selected strain for evaluation of
the influence of time of substrate addition and glucose concentration
on cell growth and bioconversion of Y. lipolytica under controlled
conditions in an instrumented bioreactor Furthermore, since poor reproducibility in cell growth and bioconversion had
been prevalent in previous studies, it was also aimed to identify and
statistically quantify the reproducibility between duplicate or triplicate samples
in each experiment and between sets of different experiments with respect to
PAA formation and cell concentrations.
Studies were conducted in shake flask cultures to define and quantify the
parameters for the model system. The parameters assessed included trace
elements, buffering, nitrogen concentration, oxygen supply, glucose
concentration, alkylbenzene substrate type and possible inducer
requirements. Trace elements, phosphate buffering and added nitrogen did
not significantly affect the cell growth of Y. lipolytica TVN348. The cell
concentration of Y. lipolytica TVN348 and TVN493 was increased by 65%
and 43% respectively for an increase in oxygen supply by decreasing the
working volume from 150ml to 50ml, while the cell concentration of Y.
lipolytica WT was increased by 41% when oxygen supply was increased by
switching from non-baffled to baffled flasks in 50ml cultures. Bioconversion
was also increased for an increase in oxygen supply: 2.4mM to 29.0mM PAA
(Y. lipolytica TVN348) and 1.2mM to 21.7mM PAA (Y. lipolytica TVN493) for a
decrease in working volume; 10.5mM to 46.6mM PAA (Y. lipolytica WT) when
switching from non-baffled to baffled flasks. These results indicated that
adequate oxygen supply is crucial to both growth and bioconversion, and that
further study should be conducted in 50ml working volumes. Cell
concentrations obtained in 1.6% (wt/v) and 3.2% (wt/v) glucose cultures
(3.95x108cells/ml and 4.03x108cells/ml respectively) indicated that cell growth
was neither enhanced nor inhibited by 3.2% (wt/v) glucose. Of the range of
substrates examined (propylbenzene, butylbenzene, sec-butylbenzene,
hexylbenzene, ethyltoluene and tert-butyltoluene for Y. lipolytica TVN348 and
TVN493; octylbenzene and decylbenzene for Y. lipolytica WT), hexylbenzene
was regarded as the best substrate for bioconversion (14.7mM and 14.1mM
PAA for TVN348 and TVN493 respectively; 42.6mM PAA for WT). Lastly, the
absence of a requirement for an additional inducer such as ethanol or oleic
acid was confirmed when PAA was formed from hexylbenzene in the culture
containing additional glucose (25.0mM). This suggested that when using
hexylbenzene as substrate, bioconversion was induced provided sufficient
glucose was available for cell maintenance.
Results from duplicate or triplicate flasks in each individual shake flask
experiment were reproducible and conclusions were based solely on results
which showed 95% confidence intervals. However, reproducibility problems
were experienced with results between different sets of experiments carried
out under the same conditions. The model system was therefore defined by: 1) no addition of trace elements,
additional buffering or added nitrogen, 2) cultures grown in 50ml volumes to
supply an adequate amount of oxygen crucial for growth and bioconversion,
3) 3.2% (wt/v) glucose and 4) addition of 1% (v/v) hexylbenzene at 24h with
no inducer requirements.
Use of the model system in shake flask cultures to identify the most promising
of the three strains of Y. lipolytica supplied demonstrated that there was no
significant difference in cell growth or bioconversion between these strains. Y.
lipolytica WT (which has no genetic modifications) was therefore used for
further investigation until an appropriate strain could be substituted when it
became available.
The growth and bioconversion of Y. lipolytica WT was further investigated
under controlled conditions in a bioreactor. The influence of time of substrate
addition (11h, 24h, 48h) and glucose concentration (3.2% and 6.4% (wt/v)) on
growth and bioconversion was examined.
When hexylbenzene was added at 48h, cell growth was increased
(8.90x108cells/ml) when compared to two of the triplicate cultures with
hexylbenzene addition at 24h (4.74x108cells/ml and 3.92x108cells/ml) and the
culture with hexylbenzene addition at 11h (2.82x108cells/ml). The third of the
triplicate cultures with hexylbenzene addition at 24h, on the other hand,
exhibited the strongest growth (2.23x109cells/ml). The poor reproducibility
between the triplicate cultures with hexylbenzene addition as 24h made it
difficult to determine whether hexylbenzene addition at 24h or 48h maximised
cell growth. Furthermore, the cell growth was not significantly improved when
the glucose concentration was increased from 3.2% (wt/v) to 6.4% (wt/v)
(7.47x108cells/ml for 6.4% glucose culture), however it was also not inhibited.
The highest amount of specific PAA formed by Y. lipolytica WT was found
when hexylbenzene was added at 11h (7.4x10-11mmol PAA/cell), however the
highest accumulated PAA was produced in the culture that exhibited the
strongest growth with hexylbenzene addition at 24h (41.4mM). This
suggested that the bioconversion of hexylbenzene was maximised when it
was added during the active growth phase. It is therefore recommended to
conduct fed-batch experiments in future to maintain the active growth phase.
Accumulated PAA was increased in 6.4% (wt/v) glucose culture (15.2mM
PAA) when compared with two of the 3.2% (wt/v) glucose cultures (5.4mM
and 4.3mM PAA). These results indicated that the increased glucose
concentration did not inhibit the bioconversion. Furthermore, PAA was formed
when 5% (wt/v) residual glucose was observed in the culture, suggesting that
the bioconversion of hexylbenzene was not inhibited at glucose concentrations as high as 5.0% (wt/v). If future work were to be conducted in
bioreactor culture where glucose is added in fed-batch operation, glucose
concentrations in cultures of up to 5% (wt/v) could be considered for initial
studies.
During bioconversion by Y. lipolytica, the PAA measured after hexylbenzene
exhaustion did not, however, correspond to 100% conversion. Further, poor
reproducibility was found in the bioreactor cultures. The disappearance of
hexylbenzene without a corresponding accumulation of PAA and poor
reproducibility was investigated by determining whether PAA was further
degraded or alternatively, whether other metabolic intermediates were being
formed and accumulated from the hexylbenzene. However, substitution of the
hexylbenzene with PAA as substrate confirmed that PAA could not be
metabolised. Further, NMR analyses of both the aqueous and organic phases
of the culture did not identify any additional metabolic intermediates. It is
recommended that additional analyses be conducted on the aqueous and
organic phases to further assess the possible accumulation of intermediates.
The development of the model system in shake flask cultures demonstrated
the importance of adequate oxygen supply for both cell growth and
bioconversion. It was also shown that no inducer was needed because
hexylbenzene acted as its own substrate inducer. Furthermore, comparison of
Y. lipolytica strains TVN348, TVN493 and WT under the defined conditions of
the model system revealed that the genetically modified strains (TVN348,
TVN493) did not exhibit enhanced bioconversion. Bioreactor cultures using
the model system under controlled conditions further showed that
bioconversion was not inhibited at a 5% (wt/v) residual glucose concentration
and suggested that bioconversion was maximised when hexylbenzene was
added during active growth phase. This informs on future work, suggesting
fed-batch operation in order to extend the active growth phase, where
glucose concentrations in the bioreactor of up to 5% (wt/v) can be considered.
Description
Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2009.
Keywords
Yarrowia lipolytica, Bioconversion, Dissertations -- Process engineering, Theses -- Process engineering