Bioconversion of ferulate into vanillin by Escherichia coli strain JM109/pBB1 in an immobilized-cell...

Annals of Microbiology, 54 (4), 517-527 (2004)

Bioconversion of ferulate into vanillin by Escherichiacoli strain JM109/pBB1 in an immobilized-cell reactor

P. TORRE1, D. DE FAVERI1, P. PEREGO1, M. RUZZI2*, P. BARGHINI2, R. GANDOLFI3, A. CONVERTI1

1Department of Chemical and Process Engineering “G.B. Bonino”, University of Genoa, Via Opera Pia 15, 16145 Genoa, Italy;

2Department of Agrobiology and Agrochemistry, Tuscia University, Via C. de Lellis, snc, 01100 Viterbo, Italy; 3Institut of Organic Chemistry “A. Marchesini”, University of Milan, Via Venezian 21, 20133 Milan, Italy

Abstract - The present work deals with a novel bioconversion of ferulate into vanillinusing resting cells of Escherichia coli strain JM109/pBB1 as a biocatalyst. Biomass recy-cling from four successive bioconversion steps demonstrated the possibility of employingthe proposed resting cell system for the continuous production of vanillin. Among the test-ed immobilization supports (polyurethane, synthetic sponge and porous glass) the syn-thetic sponge proved to be the best material in terms of both vanillin formation (Cv = 0.080g l-1) and productivity (Qv = 0.019 g l-1 h-1) at the end of entrapment tests. Thus, it wasused in preliminary continuous tests using a fixed-bed column with immobilized E. coliJM109/pBB1 cells. The highest vanillin yield (YP/S = 0.851 mol mol-1) was obtained at adilution rate of 0.022 h-1.

Key words: Escherichia coli JM109/pBB1, immobilization, continuous production, feru-late, vanillin, bioconversion.

INTRODUCTION

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is an important flavor and aromacompound, which is mainly extracted from the beans or pod of the tropicalVanilla orchid. However less than 1% of worldwide vanillin production comesfrom natural vanilla, because its cost is too high (2000 - 4000 $ kg-1) with re-spect to that (< 15 $ kg-1) of vanillin synthesized via chemical processes (Lo-mascolo et al., 1999; Muheim and Lerch, 1999). The high and variable cost of“natural” vanillin is due to various causes, among which scarce availability ofvanilla pods, climate-associated fluctuations of the harvest yields, economicaland political decisions, labor-intensive cultivation, pollination, harvesting andcuring of pods.

* Corresponding Author. Fax: +39-0761357242; E-mail: [email protected]

517

Vanillin is used either in food or non-food applications, in fragrances and inpharmaceutical preparations. Most of synthetic vanillin is used as an intermedi-ate in the productions of herbicides, antifoaming agents or drugs (Hocking,1997), and household products, such as air-fresheners and floor polishes. Be-cause of its antimicrobial and antioxidant properties (Burri et al., 1989; David-son and Naidu, 2000) vanillin could be used as food preservative, but this fieldof potential use is hindered by its strong flavor at the minimal inhibitory concen-trations required (Gould, 1996).

The large difference between the prices of natural and synthetic vanillin, theincreasing customer-led demand for “natural” and “healthy” flavors, and theserving of “bio” marketing claims have been leading to a growing interest of theflavor industry to produce natural vanillin from other natural sources by biocon-versions. This common trend in the production of flavors and fragrances wasextensively reviewed (Cheetham, 1993, 1997; Hagedorn and Kaphammer,1994; Rosazza et al., 1995; Häusler and Münch, 1997; Krings and Berger,1998).

To be economically viable, a precursor, chemically close to the vanillin,cheap and readily available, has to be found. Ferulate, a well-known productfrom lignin degradation by white-rot fungi (Kirk, 1971) and bacteria (Betts andDart, 1988), was thoroughly studied as raw material (Rosazza et al., 1995),being a naturally occurring phenolic compound. Free ferulate can be releasedfrom common agricultural residues, such as cereal bran and sugar-beet pulp,by a combination of physical and enzymatic treatments (Williamson et al.,1998). Various bacteria belonging to different genera are able to metabolize fer-ulate as the sole carbon source leading to the production of vanillin, vanillateand protocatechuate as catabolic intermediates (Sutherland et al., 1983; Guru-jeyalakshmi and Mahadevan, 1987). Pseudomonas fluorescens was shown toproduce vanillate from ferulate (Andreoni et al., 1995; Barghini et al., 1998),with formation of vanillin as an intermediate (Narbad and Gasson, 1998).

It is only recently that interesting vanillin concentrations were obtained fromferulate using the gram-positive bacteria Amycolatopsis sp. (Rabenhost andHopp, 1997) and Streptomyces setonii (Müller et al., 1998). However, since theoptimization of the process appears to be very difficult, the construction of newrecombinant strains able to specifically produce vanillin is attractive.

The effects of temperature and starting substrate concentration (Torre et al.,2003) as well as the use of different co-substrates and aeration conditions(Torre et al., 2004) have recently been investigated on the ferulate-to-vanillinbioconversion by a vanillin overproducing strain of Escherichia coli JM109, ex-pressing two catabolic genes from P. fluorescens BF13 encoding key enzymesof the ferulate catabolic pathway: feruloyl-SCoA synthetase and feruloyl-SCoAhydratase/aldolase.

Recycling of biomass in successive bioconversion steps has firstly been in-vestigated in this work to verify the long-term stability of such a resting cell sys-tem for the continuous production of vanillin. The most appropriate support forcell immobilization (synthetic sponge) was then selected by the Response Sur-face Methodology applied to data of entrapment tests, using the final vanillinconcentration (Cv) and productivity (Qv) as responses and the contact time be-tween biomass and support (tc) and the immobilized biomass incubation time(ti) as independent variables. Preliminary tests of continuous vanillin production

518 P. TORRE et al.

were finally carried out using the above flexible support for E. coli JM109/pBB1cells entrapment.

MATERIALS AND METHODS

Microorganism growth and bioconversion conditions. Cells of Escherichiacoli JM109/pBB1, expressing both feruloyl-SCoA synthetase and feruloyl-SCoAhydratase/aldolase from Pseudomonas fluorescens BF13, were selected asbiocatalyst in this work. Recombinant plasmid pBB1 was generated cloning a5,000-bp fragment, including the first three genes of the ferulate upper pathwayand the entire promoter region from ferulate-degrading P. fluorescens strainBF13, into the low-copy vector pJB3Tc19 (Blatny et al., 1997). Donor fragmentcontained an engineered mutation in vanillin dehydrogenase (vdh) gene, whichprevented oxidation of vanillin to vanillate in bioconversion experiments with fer-ulate as substrate. The DNA sequence of the entire ferulate operon from P. fluo-rescens BF13 is available from the EMBL nucleotide sequence database underAccession No. AJ536325.

Cells were transferred into 250-ml Erlenmeyer flasks (EFs) containing LBmedium (25 ml) and ampicillin (0.1 g l-1) and then cultivated over-night on a ro-tary shaker at 37 °C (200 rpm). Fresh LB medium (30 ml) plus 0.1 g l-1 ampi-cillin was seeded with 0.6 ml of the above pre-culture in 100-ml EFs. Cells werecollected by sterile centrifugation (6000 rpm, 10 min) during the late log phase,washed with M9 saline phosphate buffer (6 g l-1 Na2HPO4, 3 g l-1 KH2PO4, 1 g l-1 NH4Cl and 0.5 g l-1 NaCl), and inoculated into the bioconversion medium(M9 buffer amended with 0.200 g l-1 ferulate and 0.050 g l-1 yeast extract).

The long–term viability of E. coli JM109/pBB1 was checked through a set offour successive 20 h-long bioconversions at a starting ferulate concentration (So)of 0.940 g l-1, to perform which the cells from the preceding test were centrifuged(6000 rpm, 10 min), washed and reused in the subsequent one. All batch runswere carried out in EFs at the same starting biomass concentration (Xo).

Continuous tests were carried out in a fixed bed reactor, consisting of a 75cm-high column with inner diameter of 4 cm and provided with an external jack-et for external water recirculation. The column was thermostated at 30 °C andfilled up to 50 cm with synthetic sponge pieces (1 x 0.7 cm) containing en-trapped cells and glass beads (diameter of 1 cm), in a ratio of 0.20 g spongeper g glass, in order to avoid the occurrence of too high head losses. The work-ing volume was 450 ml, and the ferulate concentration in the feed (So) wasmaintained at 0.200 g l-1.

Immobilization procedure and optimization. Porous glass, reticulatedpolyurethane and synthetic sponge were tested as immobilization supports. Inorder to evaluate the capacity of entrapping biomass of each material, a sus-pension of cells was inoculated in 250 ml-EFs each containing 50 ml of M9saline solution and 0.6 g of flexible materials or 10 g of porous glass. The con-tact time between cells and each material was varied according to the statisticaldesign mentioned forward. Immobilized cells were collected by sterile filtration,incubated for variable time in fresh LB medium (50 ml) plus 0.1 g l-1 ampicillin,recovered by sterile filtration, and finally used in the bioconversion process.

Ann. Microbiol., 54 (4), 517-527 (2004) 519

To select the most suitable support for the process, batch bioconversionswere performed according to a 22 factorial central composite design repeatedfor each of the three materials and the results were evaluated by the ResponseSurface Methodology (RSM) (Montgomery, 2001). Vanillin concentration andproductivity were selected as the response variables of the statistical study. Thetested ranges of the independent variables, i.e. the times of contact (tc) and in-cubation (ti), were 24-72 h and 6-42 h, respectively. The experimental resultswere fitted by the equation:

y = bo + b1x1 + b2x2 + b12x1x2

where bo is the interception coefficient, b1 and b2 the linear terms, b12 the inter-action term, and x1 and x2 the coded values of the above independent vari-ables, tc and ti, respectively.

Analytical determinations. Biomass concentration was determined by opticaldensity (OD) measurements at 600 nm using an OD versus biomass dry weightcalibration curve. Vanillate, vanillin and ferulate concentrations were deter-mined by an HPLC 1100 HP provided with a Vydac 201TP54 C18 reverse-phase column and UV/Vis detector (254 nm, 35 °C). The mobile phase (0.5 mlmin-1) was 40% methanol plus 60% of a 6.8 g l-1 KH2PO4 water solution, whosepH was adjusted at 2.5 with H3PO4.

RESULTS AND DISCUSSION

Effect of biomass recycling in successive bioconversion stepsIn order to check the suitability of employing the proposed resting cell systemfor the continuous production of vanillin, its long-term viability was firstlychecked through four successive steps of biomass recovery and reuse. The re-sults of these tests (Fig. 1) did not evidence any appreciable loss of cell capa-bility to convert ferulate into vanillin within the first 6 h, even after 3 successiverecycling operations.

Such a long-term stability of the biosystem confirms the significance ofyeast extract as essential co-substrate to sustain the basal metabolism of rest-ing cells in bioconversions, like the one under consideration, that are not selfsustained from the bioenergetic viewpoint (Torre et al., 2004). On the contrary,vanillin concentration detected at the end of bioconversion (about 20 h) exhib-ited a remarkable and progressive decrease (up to 35%) in favor of vanillate for-mation. As previously proposed (Torre et al., 2003), this observation can be as-cribed to the progressive importance of a non-specific oxidase activity, whichwould allow the microorganism to generate reduced cofactors to be used forATP synthesis by oxidative phosphorylation.

These results suggest that resting cells of E. coli JM109/pBB1 could be use-fully employed for the production of vanillin by continuous or sequencing-batchoperations.

520 P. TORRE et al.

Cell immobilization testsTo select the most appropriate support for continuous vanillin production infixed-bed column, three different porous materials, specifically porous glass,polyurethane and synthetic sponge, were selected among those reported in theliterature as good cell supports (Huysman et al., 1983; Converti, 1994) and thentested for their ability in immobilizing E. coli JM109/pBB1. The experimentaldata of these immobilization tests are listed in Table 1.

Although porous glass ensured the highest yield of cell entrapment (EY =

Ann. Microbiol., 54 (4), 517-527 (2004) 521

FIG. 1 – Influence of successive biomass recycling operations on the capability of Es-cherichia coli JM109/pBB1 cells to produce vanillin (A) from ferulate (B).

, I cycle, ■, II cycle, ■, III cycle, ■, IV cycle. Bioconversion medium: M9 salt solu-tion supplemented with yeast extract. Xo = 4.0 g l-1; So = 0.94 g l-1; Tempera-ture = 30 °C.

Time (h)

Time (h)

Con

cent

ratio

n (g

/L)

Con

cent

ratio

n (g

/L)

A

B

65% biomass removal after tc = 72 h) as well as the highest vanillin concentra-tion (Cv = 0.126 g l-1), it showed, under the same entrapment conditions, lowermaximum productivity (Qv = 0.013 g l-1 h-1) at tc = 24 h and ti = 6 h thanpolyurethane (Qv = 0.020 g l-1 h-1) and synthetic sponge (Qv = 0.019 g l-1 h-1).Both flexible porous materials tested also exhibited interesting Cv values (0.091g l-1 with polyurethane and 0.080 g l-1 with synthetic sponge), which suggeststheir use for continuous operation in immobilized-cell reactor. The response sur-faces for Cv and Qv, drawn selecting the contact and incubation times as the in-dependent variables (Fig. 2), demonstrate that vanillin production can be opti-mized by simultaneously increasing tc and decreasing ti, while a reduction ofboth independent variables would allow for productivity maximization, regard-less of the immobilization support.

The general trends of the observed response surfaces show that an in-crease in tc caused the extent of cell entrapment to increase, the concentrationof vanillin to rise and the productivity to decrease, probably due to the occur-rence of mass transfer limitation in the bioconversion medium. As a conse-quence, the highest product concentration and productivity were obtained at tc= 72 and 24 h, respectively. Moreover, the statistical model demonstrated that adecrease in the incubation time simultaneously maximized both Cv and Qv. Theoptimal value of this independent variable for both responses (ti = 6 h) wasclose to that detected for maximum vanillin production by free cells (t = 4 h)

522 P. TORRE et al.

TABLE 1 – Experimental results of tests of Escherichia coli JM109/pBB1 cells immobilization according to a 22 factorial central composite design

Test tca ti

b Support EYc Cvd Qv

e

(h) (h) (%) (g l-1) (g l-1 h-1)

1 72 42 Reticulated polyurethane 17.6 0.065 0.003Synthetic sponge 34.0 0.069 0.008Porous glass 64.5 0.092 0.004





a Contact time between biomass and support.b Incubation time of immobilized cells.c Yield of cell entrapment.d Final vanillin concentration.e Vanillin productivity.

(Torre et al., 2003), thus pointing out the absence of any transport limitation inthe incubation medium.

On the basis of these observations, the synthetic sponge was chosen as thebest material for continuous tests, because it ensured the highest average Cvand Qv values under all tested conditions, in addition to its commercial avail-ability and long term stability (Converti et al., 1991, 1994). The values of the sig-

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FIG. 2 – Response surface plots of final vanillin concentration (Cv, g l-1) and produc-tivity (Qv, g l-1 h-1) as simultaneous functions of the times of incubation (ti, h)and contact (tc, h), during immobilization tests using as cell supports: A) retic-ulated polyurethane, B) synthetic sponge, and C) porous glass.

A Reticulated polyurethane

B Synthetic sponge

C Porous glass

nificance level (p) (Table 2) demonstrate that the mathematical models used forCv and Qv optimization were highly significant and described well the region ofthe independent variables under investigation.

Continuous testsPreliminary tests of continuous vanillin production were carried out in a fixed-bedcolumn with E. coli JM109/pBB1 cells entrapped in synthetic sponge. The inletferulate concentration (So) was kept at 0.200 g l-1 and the dilution rate (D) wasvaried between 0.011 and 0.033 h-1. The time variations of ferulate, vanillate andvanillin concentrations were plotted versus the bioconversion time (Fig. 3).

The steady-state conditions were reached at D = 0.022 h-1 after about 150

524 P. TORRE et al.

TABLE 2 – Linear statistical models employed for the evaluation of results of Escherichia coli JM109/pBB1 cell immobilization tests

Support Modela-d pe

Reticulated polyurethane y1 = 0.055 + 0.021x1 – 0.008x2 – 0.005x1x2 0.0377y2 = 0.008 - 0.004x1 – 0.005x2 + 0.003x1x2 0.0097

Synthetic sponge y1 = 0.065 + 0.009x1 – 0.002x2 – 0.003x1x2 0.0991y2 = 0.010 - 0.003x1 – 0.002x2 + 0.004x1x2 0.0253

Porous glass y1 = 0.052 + 0.045x1 – 0.018x2 0.0611y2 = 0.006 - 0.001x1 – 0.004x2 + 0.003x1x2 0.0069

a y1: Final vanillin concentration (Cv).b y2: Vanillin productivity (Qv).c x1: Coded values of contact time (tc).d x2: Coded values of incubation time (ti).e Significance level.

FIG. 3 – Experimental data of continuous tests of ferulate-to-vanillin bioconversion in afixed-bed column with Escherichia coli JM109/pBB1 cells entrapped onto syn-thetic sponge. Transient responses at variable dilution rate. Concentrations (g l-1): (▲) ferulate; ( ) vanillin; ( ) vanillate.

Time (h)

Con

cent

ratio

n (g

l–1)

D = 0.022 h–1 D = 0.011 h–1 D = 0.018 h–1 D = 0.027 h–1 D = 0.033 h–1

h, yielding average vanillin concentration and volumetric productivity of Cv =0.050 g l-1 and PD = 1.10 mg l-1 h-1, respectively. Ferulate was only partiallyconsumed, more than 0.120 g l-1 being detected in the effluent stream. In orderto improve substrate consumption and vanillin production, D was then de-creased up to 0.011 h-1; although, ferulate consumption was improved (from0.084 to 0.164 g l-1), it was not preferentially addressed to vanillin accumula-tion, only 0.034 g l-1 of aldehyde having been produced under this condition, cor-responding to PD = 0.37 mg l-1 h-1. At intermediate dilution rate (D = 0.018 h-1),vanillin concentration and productivity were Cv = 0.051 g l-1 and PD = 0.93 mg l-1 h-1 (Fig. 4), whereas 0.108 g l-1 of ferulate were consumed.

From the results of the molar yield (YP/S) versus D under steady state-con-ditions (Fig. 4), it is evident that an increase in the residence time from 45 to 90h led to a remarkable decrease (from 0.851 to 0.261 mol mol-1) in the fraction offerulate addressed to vanillin production. It is likely that, under these conditions,the residence time was too long, so as to lead the system to a condition of co-substrate (yeast extract) shortage.

According to the above hypothesis, the resulting low level of acetyl-CoAcould have become insufficient to sustain the activity of feruloyl-CoA syn-thetase, the first enzyme involved in ferulate-to-vanillin bioconversion (Priefertet al., 2001), thereby becoming the limiting factor of the process from the bion-ergetic point of view. A material balance for the carbon source (results notshown) pointed out that a significant fraction of ferulate was completely oxidizedto carbon dioxide, which suggests that the microorganism could have activateda metabolic response to face such stress energy conditions. As a result, astrong oxidase activity, already proposed in previous work (Torre et al., 2003),

Ann. Microbiol., 54 (4), 517-527 (2004) 525

FIG. 4 – Continuous tests of vanillin production in a fixed-bed column with Escherichiacoli JM109/pBB1 cells entrapped onto synthetic sponge. Steady-state resultsof (O) ferulate-to-vanillin yield (YP/S) and (■) volumetric vanillin productivity(PD).

D (h–1)

YP

/S(m

ol m

ol–1

), P

D(m

g–1h–1

)

was likely to be expressed, leading to the oxidation of a significant portion ofvanillin to vanillate and subsequent consumption of this intermediate throughthe TCA cycle. The quick decrease in YP/S for D > 0.022 h-1 might be ascribed,in addition to the easy washout of resting cells, also to an excess substrate in-hibition of the activity of one of the key enzymes of the ferulate degradationpathway.

Although the yield of vanillin on consumed ferulate obtained in this work wassatisfactory at D = 0.022 h-1 (YP/S = 0.851 mol mol-1), the fraction of unconvert-ed substrate was always very high. These results suggest that significant per-formance enhancement could be ensured by improvements of the bioconver-sion technology; therefore, the next effort will be addressed to the set-up andoptimisation of a bi- or tri-phasic system to implement the reactor scheme forthe continuous product removal and unreacted substrate recycling.

AcknowledgmentsThe authors thank the Italian MIUR, prot. 2001075847, for the financial sup-

port of this work.

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