Production of catechols : microbiology and technology

L. Krab-Hüsken

Research output: Thesisinternal PhD, WU

Abstract

<FONT FACE="Arial" SIZE=3><p>Catechols play an important role in the fine-chemical and flavour industry, as well as in photography, dyeing fur, rubber and plastic production. Many of these compounds cannot easily be synthesised chemically, but some micro-organisms are capable of producing catechols from an aromatic compound in only two steps. In this thesis, microbial 3-methylcatechol production from toluene was considered a model system for simulating the production of 3-substituted catechols in general.</p><p>3-Methylcatechol production was studied in various process configurations. <em>Pseudomonas putida</em> MC2 was a newly constructed strain that was capable of producing 3-methylcatechol from toluene in two steps, in the presence of the inducer salicylate. This trait was stably expressed in this strain and no antibiotics were required to maintain this stability. Growing cells produced the desired product at very high specific production rates.</p><p>In aqueous medium, 10 mM of 3-methylcatechol accumulated at one-litre scale in twelve hours. Toxicity of the product prevented further production. In addition, the stability of the product in aqueous medium at physiological pH was poor and downstream processing of the culture medium containing the product was complicated. Therefore, other process set ups were studied as well.</p><em><p>P. putida</em> MC2 was tolerant to some - otherwise toxic - organic solvents. Octanol was a solvent in which substrate toluene and product 3-methylcatechol both dissolved very well, while the biocatalyst survived and was active in its presence. To obtain the product in octanol was advantageous for downstream processing in comparison to monophasic aqueous processes. Production of 3-methylcatechol in the presence of octanol resulted in improved volumetric production rates and higher product concentrations. Up to 65 mM of product accumulated in octanol, and a maximum overall product concentration (over octanol and aqueous volume together) of 25 mM was obtained at one-litre scale, depending on the applied phase ratio of both liquid phases.</p><p>The two-phase process with octanol in direct contact with the culture medium started slowly. A lag time of 24 hours (depending on the phase ratio applied) before production started was not unusual. Molecular toxicity of dissolved octanol in the culture medium was negligible. We therefore assumed that the direct contact with octanol was very inhibitory to the bacterial cells (phase toxicity). To avoid toxic phase effects of the second phase, a membrane was introduced as a barrier between both phases. In this process configuration, no lag time was apparent and the total process was faster than without a membrane, maximum volumetric production rates rising about 40 %, albeit that the product concentration in octanol was not as high as in the two-phase process with direct phase contact. Mass transfer in the membrane process still needs attention.</p><p>Preliminary experiments on the downstream processing of 3-methylcatechol from octanol were carried out. The disodium salt of the product was extracted in a sodium hydroxide solution and was separated from the octanol phase, that still contained the toluene and that could be recycled to the process. The salt solution was acidified to reprotonate the product, which was now stably captured at high concentration in an aqueous acidic phase. A new organic solvent that dissolves the product well and that would be more volatile than octanol, for instance dichloromethane, can be used to obtain the purified product after phase separation and solvent evaporation.</p><p>To attain a rational design and to be able to further optimise this process, a process model was developed, both for one- and two-phase processes. The importance of different process parameters on bacterial growth, on product accumulation in both phases and on process time follow from the model. It seemed the process would be faster (14 - 27 % for one- and two-phase process, respectively) if more growth substrate would be available, although this hardly affected the final product concentrations.</p><p>The general discussion at the end of the thesis compares production rates and gives an overview of alternative 3-methylcatechol production processes. It furthermore discusses the industrial relevance and concluding remarks are presented.<font face="Arial"></font>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Wageningen University
Supervisors/Advisors
  • de Bont, J.A.M., Promotor
  • Tramper, J., Promotor, External person
  • Beeftink, H.H., Promotor, External person
  • Wery, J., Promotor, External person
Award date25 Oct 2002
Place of PublicationS.l.
Print ISBNs9789058086785
Publication statusPublished - 2002

Keywords

  • pyrocatechol
  • pseudomonas putida
  • industrial microbiology

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