A Comparative Study of Gluco

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BIOTECHNOLOGY LETTERSVobune 15 No.5 (May 1993) pp.499-504Received4th March

A COMPARATIVE STUDY OF GLUCOSE CONVERSION TO ETHANOLby Zymumonas mobilis and a RECOMBINANT Escherichia cdl

Hugh G. Lawford* and Joyce D. Rousseau

Department of BiochemistryUniversity of Toronto

Toronto, Ontario, Canada ~5s 1A8FAX (416) 978-8548

SUMMARY

Zymomonas mobilis and recombinant Escherichia coli B (pLOI297) were compared inside-by-side bat& fermentations using a synthetic cellulose hydrolysate (glucose/salts)medium with pH control at 6.0 and an inoculation cell density of 35-50 mg dry wt. cells/LAt a nominal glucose concentration of 696, both cultures achieved near maximal theoreticalethanol yields; however, the 2. mobilis fermentation was complete at 13h compared to 33hfor the E.coli fermentation. With approx.1295 glucose, the 2. mobilis fermentation wascomplete in 20h with a process yield of 0.49 g ethanol/g added glucose compared to the E.coli fermentation which remained 20% incomplete after 6 days resulting in a process yield ofonly 0.32 g/g. Nutrient supplementation (log tryptone/l) resulted in complete fermentationof 12% glucose (pH 6.3) by the recombinant E. coli in 4 days, with a yield of 0.48 g/g.

INTRODUCTION

The growing demand for fermentation ethanol as an renewable, environment friendly,alternative transportation fuel, means that the industry will soon be forced to expand beyondits present starch and yeast-based technology (Wyman et al., 1990). Various bacterialethanologens are being investigated in relation to the production of ethanol fromlignocellulosic biomass (Tolan & Finn, 1987a and 1987b; Ingram et aZ., 1990; Lawford &Rousseau, 1991a, 1992; Lacis & Lawford, 1989; Lynd, 1989). Zymomonas mobilis isgenerally recognized as being superior to brewers yeast with respect to both glucoseconversion efficiency and productivity (Rogers er al., 1982; Baratti Bt BuL.ock, 1986;Lawford, 1988), but both organisms are similarly disadvantaged with respect to thefermentation of lignocellulosic feedstocks because of their inability to utilize the pentosesugars that are present in hemicellulose, Whereas Escherichia coli is able to efficienctlymetabolize all the sugars present in lignocellulose, ethanol is only produced in very low yield(Gottschalk, 1985). The ethanol production pathway of Z. mobilis (pdc and udh genes) hasbeen cloned and E. coli recombinants have been genetically engineered which combine andexhibit the positive traits of both organisms such that the ethanol yield from both hexose andpentose sugars closely approaches the theoretical maximum (Ingram et al., 1987; Ingram &

Conway, 1988; Alterthum & Ingram, 1988; Ohta ez al., 1990)

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In a study comparing the fermentation performance of yeast (S. cerevisiae) and 2.mobilis, using acid-hydrolyzed softwood (pine) cellulose, the glucose-to-ethanol conversionefficiency was reported to be respectively 94% and 96% of theox%ical maximum (Par&h etal., 1989). Mart recently, in a study designed to promote the putative superior fermentationcharacteristics of a recombinant E. coli strain KOl 1, Barbosa et al. (1992) reported that theethanol yield based on monomer sugars in a nutrient-supplemented, CaOH-treated, pinecellulose acid hydrolysate exceeded 100% of the theoretical yield. Unfortunately, the vastlydiffenznt cell loadings used in these studies precludes any direct comparison with respect tokinetic parameters.

The purpose of the present study was to compare the fermentation performance of tworecog&& high-perfom~ance bacterial ethanologens, namely 2. mobilis and a recombinantE. coli l3, in side-by-side batch fermentations under identical environmental conditions withrespect to medium composition, pH and inoculation cell density. Since both these culture areknown to convert glucose to ethanol with very high efficiency, the technoeconomic parameterof particular interest is the comparative productivity displayed by each biocatalyst.

MATERIALS AND MEfHODS

Organisms Zymomona~ mobilis ATCC 29191 is a neotype strain and was obtained fromthe American Type Culture Collection (RocKlle, MD). Escherichia cofi B (ATCC 11303canying pLGI297) (Alter&m & Ingram, 1989) was a gift from L. 0. Ingram (Universityof Florida, Gain&lie, FL, USA).

Fermentation medium The synthetic glucose mineral salts medium contained 1.5 g/Lyeast extract (Difco) and ammonium chloride (1.6 g/L) as sources of assimilable nitxogen.Thiamine was added at O.OSmg/L. The composition with respect to all other inorganicelements and vitamins was as previously described (Lawford & Ruggiero, 1990). Certainexperiments +th E. coZi employed a complex LB medium, the composition of which was aspreviously described by Lawfoxd and Rousseau (1992).

Equipment and Fermentation Conditions Batch fermentations were conductedanaerobically in 2L stirred-tank biorectors (MultiGen= F2000 from New BrunswickScientific Co., Edison, NJ) containing about ISL medium. Agitation was minimal at about150 RPM. The temperature was 30C and the pH was controlled automatically at thespecified value by addition of 2N KOH. Inoculation was made from an overnight static flaskcultme. The iuitial cell density was in the range 35-50 mg dry wt. cells per litnz (equivalent toanODat55OnmofaboutO.l too.15

Analytical Procedures Growth was measured turbidometrically at 55Onm (lcm lightpath)and culture dry weight was measured by microfrltration (0.45~), washing and drying thefilter to constant weight under an i&a-red heatlamp. Compositional analyses of fermentationmedia and cell-free spent media were determined using an HPLC equipped with anHPX-87H column and a RI monitor with computer-interfaced controller/integrator (Bio-RadLabs) as described previously (Lawford & Rousseau, 1992).

Calculation of Operational Parameters The average volumetric productivity ( ) wasdetermined by dividing the foal ethanol concentration by the time (h) required to9 hievecomplete glucose utilization. The maximum volumetric productivity (Q -) was estimated asthe maximum slope in plots of ethanol concentration versus elaspsed f&mentation time. Thepmcessethano1 yield (Y ) was determined as the mass of ethanol produced divided by themass of glucose added% the medium. In determining glucose-to-ethanol conversionefficiency (I theoretical max.), the theoretical maximum value for Ytis is assumed o be 0.51

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Glucose Utilization (pH 6)

0 20 40 60 60 100 120 140

7Ethanol Production

7d recombinant /

0 20 40 60 80 100 120 140Time thl

FIGURE 1 Fermentation Profiles for 2. mobilis and Recombinant E. coli:

[A] Glucose. Symbols:A and A, 2. mobilis and E. coli (p~OI297) as per lines 4 and3 of Table 1 respectively; W, E. coli in LB-supplemented medium as per line 1 in Table 2.

[B] Bwoduction. Symbols: 0 and 0, E. coli(pLOI297) and E. coli (KOl 1) as per lines 2and 3 of Table 2 respectively; other symbols as in (A).

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TABLE 1Comparative Fermentation Parameters for Z mobilis and

Recombinant E. co/i (pLOI297) In a Synthetic Ceilulose Hydrolysate(Glucose/salts) Medium at pH 6.0

PRODUCTS PRODUCTIVITY YIELD

Organism[Glucose] [Biomass] [EtOH] av.QP QPmax Yp, Conver.

an- gDwh sn gPhnp de Effi. (s)

E. co/i 6 (p~o1297) 66.7 2.4 30.7 0.93 1.10 0.46 90

z. Il7ObiiiS (29191) 61.5 2.1 30.8 2.40 4.78 0.50 98

E. co/i B(pumn) 134.0 43.1 0.30 0.50 0.41' 80

z. mObiliS (29191) 123.5 2.5 60.5 3.03 4.80 0.49 96

l Sii ghmse utilii was incompkIe aftfz lSOh, the process Yield (based on sugar added to the medium)is 0.32 g &OH/g gl~se.

TABLE 2Comparative Fermentation Parameters for Recombinant E. co/i

in Nutrient-supplemented Glucose Media

Organism

PRODUCTS PRODUCTIVITY YIELD

[Glucose] [Biomass] (EtOH] av.Q cl ma

t&-P

YP/s

Conver.

pn gDwh tin- BIB Effic. (Q)

Lurla broth wE. co/i B(pmmn)This work [expt.B13 ] 6.3 123.0 2.7 59.0 0.61 0.92 0.48 94

E. co/i B (PLDIZW)~7.0 120.0 3.6 58.0 0.48 1.40' 0.48 94

E. co/i B (K011)2 6.0 100.0 3.8 53.0 0.74 1.40 0.53 104

E. co/i B (~011)pinewood cellulose 6.6 63.1 (-IRS) 34.0 0.71 1.33acid hydrolysate3

0.61f 119

Note: The hostculture is E. coli B (ATCC 11303) - in strain pLOI297. the PET operon is plasmid encoded,whereas for strain KOll. it is chromosomally integrated (ref. Ohta ti al.. 1991a)

1. Altmthum and Ingram (1989) [data taken from Fii. 1A]2 Guimaaea u al. (1992) [data taken from Fig. 2A]3. Barbosa et ul. (1992) [Fig. lC] @yptone-supplanenti. CaOH-treated. pinewood cellulose hydrolysate;

fTRS was 82.4% glucose. whae TRS = total reducing sugarshigh yield may result from catabolii of cellobiose and/or complex exogenous nutrients (Barbosa et al.. 1992)

* In all these fermentation trials ( refs, 1-3). the inoculation cell density was approx. 10 times (330 mg dry wet. ceb/f.,)that used in the present investigation

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A pH of 6.0 is near optimal for growth and ethanol production by genetically engineeredEscherichia coli B (Beall et al., 1991; Ohta et al., 199la). For Zymomonas mobilis, pH 6.0is near optimal for growth (Lawford et al., 1988); however, because maintenancemetabolism is increased at lower PI-I, pH 6.0 is not optimal with respect to the rate of ethanolproduction (Lawford & Ruggiero, 1990) and a pH value of 5.0 is most often used inZymomonas fermentations (Rogers et al., 1982). Therefore, by choosing to control the pH at

6.0, the recombinant E. coli culture receives an advantage n terms of productivity.Using a 6% (W/V) glucose defined salts medium with pH controlled at 6.0, Z. mobilis(ATCC 29191) and recombinant E. coli B (pLOI297) (Alterthum & Ingram. 1989) producedethanol at a conversion efficiency which was 98% and 90% (max. theoretical) respectively(Table 1). However, the volumetric pmductivity of the Z. mobilis fermentation was 2.6 timesthat of the recombinant E. coli fermentation with times for complete fermentation being 13hand 33h respectively (Iable 1).

When the glucose concentration was doubled (range 12-13s w/v), there was a morepronounced difference in fermentation performance displayed by these two bacterialethanologens (Fig. 1, Table 1). Whereas the Zymomonas culture maintained highperformance characteristics with respect to both product yield and productivity, therecombinant E. coli displayed a markedly lower fermentation performance (Table 1). At thehigher glucose concentration, about 20% of the sugar remained unfermented by the E. coliculture after 6 days (Fig. 1), resulting in a process yield (based on sugar added) of only 0.32g/g (equivalent to a conversion efficiency of 63%) (Table 1). Nutrient supplementation(log/L tryptone) resulted in an improved performance by the recombinant E. coli withcomplete fermentation in 4 days at a conversion efficiency of 94% (max. theoretical) (Fig. 1,Table 2). For comparison purposes, data taken from Alterthum and Ingram (1989) relating tothe production of ethanol by E. coli B (pLOI297) using a nutrient-rich medium (Luria broth),containing 12% glucose, is plotted in Figure 1B and presented in Table 2. Also included, forcomparison purposes, is data taken from Guimaraes et al. (1992) in connection with theirinvestigation of a closely related genetic construct, namely strainKOl1 (Ohta et at., 1991b),in which, for purposes of culture stability, thepdc and adh genes from Zymomonas werechromosomally integrated into E. coli B (Fig. 1B and Table 2). Figure 1B illustrates that thefermentation performance characteristics of recombinant E. coli B (pLO1297) and strain

KOll, in a nutrient-rich medium, are very similar. The faster rates of ethanol production(Fig. 2) observed in the experiments of Alterthun & Ingram (1989) and Guimaraes et al.(1992), are due to the 10 times inoculation cell density employed (see Table 2). Recently,Barbosa et al. (1992) proposed using recombinant E. coli strain KOll for fuel ethanolproduction from softwood (pine) acid hydrolysate. The results of their investigation relatingspecifically to the fermentation of the acid hydrolyzed cellulose fraction (detoxified usingCaOH) are presented in Table 2. The fact that the observed product yield (0.61 g EtOH/gglucose) exceeded the theortical maximum may be due to the catabolism of cellobiose and/orthe tryptone used as a nutritional supplement (Barbosa et al., 1992). The inoculation celldensity used by Barbosa et al. (1992) was also approx. 10 times that used in the presentinvestigation.

Using a similar fermentation feedstock (acid hydrolyzed pinewood cellulose), Parekhetal. (1989) compared the fermentation performance of S. cerevisiae and Z. mobilis and foundthat both organisms exhibited high conversion efficiencies; however, the productivity of thebacterial fermentation was 2.6 times higher than the yeast fermentation. Unfortunately,because of the vastly different inoculation densities employed by Pa&h et al. (1989). theresults of these investigations using different ethanologens cannot be compared directly interms of productivity.

The newly developed and patented ethanologenic recombinants of Esherichia coli haverecently been the focus of considerable attention in terms of their potential for the productionof fuel ethanol from biomass and wastes (Ingram et al., 1991; Lawford & Rousseau, 199 1and 1993; Beall & Ingram, 1992). Nevertheless, the results of the present study, together


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witb observations reported in the literature, suggest that the fermentation performancecharacteristks of Zymovnonusare not inferior to those of the recombinant E. coli cults andthat, in f&t, further R&D with 2. mobilis seems ustified particularly in terms of the potentialfa implwzd productivity.

AcknowledgementsThis research was supported by an aperating grant from the Natural Sciences and Engineering Research Councilof Canada. We are grateful to Professor Lonnie Ingram (University of Florida) for the patented recombinant

Escherichia coli B (pLOI297)

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A Comparative Study of Gluco