Biotechnological production of vanillin using microbial cells
Author(s)
Brunetti, Lorenza
Date Issued
May 27, 2013
Type
Doctoral Thesis
Abstract
This PhD research project focused on the optimization of the biotechnological process for vanillin
production from ferulic acid using recombinant Escherichia coli cells.
Nowadays flavours market covers about one fourth of the global food additives market with a 25
million dollar turnover and a 5.5% growth rate. Flavouring compounds are generally produced by
chemical synthesis or extractive methods from natural sources. Flavours produced by chemical
synthesis, are classified as “artificial flavours”; also these processes are damaging for environment
and cause some problems with unwanted compounds, with reduction of the process efficiency and
rise of the product recovery costs. On the other hand, extraction processes from plants are often
more expensive because of the low concentrations of the molecules target in the raw material.
Moreover cost of aromatic compounds extracted from plants depends on uncontrollable factors such
as plant diseases and weather conditions. The drawbacks of both methods and the increasing
interest of consumers in natural product (Sinha et al., 2008), reported in recent market surveys, have
led a great interest in the exploration of more “eco-friendly” procedures for production of natural
flavours.
Vanillin is the major compound responsible for vanilla aroma. It is one of the most commonly used
aromatic compounds in drugs and food industry. Since vanillin extracted from cured vanilla beans
is very expensive and its availability depends on plantations production, curing process duration and
labour costs. Synthetically produced vanillin is the most commonly used in vanilla flavoured
products, and covering 99% of the global market. According to the regulation No 1334/2008 of
the European Parliament and of the Council, vanillin produced by biotechnology from natural
feedstocks can be classified as ‘natural flavoring’, provided that the source is always specified. All
these factors make vanillin an important commercial target for biotechnological industry, and bases
and applied research.
Since vanillin is an intermediate product of the ferulic acid catabolism (a compound present in
significant quantities in the lignocellulosic material) it is possible to confer the ability to convert
ferulic acid to vanillin, through metabolic engineering, to strains unable to degrade ferulic acid.
Unfortunately these bioconversion processes are not economically competitive yet; the high
chemical activity and toxicity of both the product (vanillin) and the substrate (ferulic acid) cause
low yield in the bioconversion process.
With this research project, aiming at develop a competitive bioconversion process for vanillin
production, many factors influencing the system productivity and selectivity, have been optimized.
Bioconversion experiments carried out using resting cells of E. coli demonstrated that composition
and pH of bioconversion buffer affected the formation of vanillin and unwanted products such as
vanillyl alcohol. Using moderately alkaline bioconversion buffer (pH 9.0) it has been possible to
double the amount of final product, with respect to the systems until now described.
Using the statistical approach of the Response Surface Methodology (RSM) it has been possible to
evaluate the synergic effect of ferulic acid concentration and stirring speed on the productivity and
selectivity of the bioconversion process. Our results demonstrated that the highest vanillin title
obtained incubating cells at 135-165 rpm range and initial ferulic acid concentration of 12-18 mM.
Under optimized working conditions, vanillin yield increased from 8.51 ± 0.02 to 11.63 ± 0.1 mM,
while, ferulic acid concentration higher than 20 mM cause a drastic decrease in vanillin production.
Finally, to maintain low initial concentration of ferulic acid in the bioconversion buffer a two-phase
(solid-liquid) system for the controlled release of the substrate has been developed. Using agarose
gel cylinders containing ferulic acid, in conjunction with optimized buffer and nutrient amendments
(LB medium), we demonstrated that, compared to previous results (Barghini et al., 2007), it is
possible to increase vanillin final title (up 20%); to reduce the bioconversion time from 4 to 1 day;
to increase the final vanillin concentration in the liquid phase of 5-fold. The maximum amount of
accumulated vanillin in the liquid phase under optimized conditions was 20.57 ± 0.05 mM, the
highest found in the literature for recombinant E. coli strains.
In conclusion, results obtained demonstrated that vanillin production by resting cells of E. coli can
be increased significantly by acting on several parameters, including the bioconversion buffer
formulation and the way to modulate the substrate concentration.
Additional information
Dottorato di ricerca in Biotecnologia degli alimenti
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