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Improved ethanol production from cheesewhey, whey powder, and sugar beet molasses by“Vitreoscilla hemoglobin expressing” Escherichia coliMeltem Yesilcimen Akbasa, Taner Sar & Busra Ozcelika

a Department of Molecular Biology and Genetics, Gebze Institute of Technology, Gebze,TurkeyPublished online: 15 May 2014.

To cite this article: Meltem Yesilcimen Akbas, Taner Sar & Busra Ozcelik (2014) Improved ethanol production from cheesewhey, whey powder, and sugar beet molasses by “Vitreoscilla hemoglobin expressing” Escherichia coli, Bioscience,Biotechnology, and Biochemistry, 78:4, 687-694, DOI: 10.1080/09168451.2014.896734

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Improved ethanol production from cheese whey, whey powder, and sugar beetmolasses by “Vitreoscilla hemoglobin expressing” Escherichia coli

Meltem Yesilcimen Akbas*, Taner Sar and Busra Ozcelik

Department of Molecular Biology and Genetics, Gebze Institute of Technology, Gebze, Turkey

Received October 17, 2013; accepted December 17, 2013

http://dx.doi.org/10.1080/09168451.2014.896734

This work investigated the improvement of etha-nol production by engineered ethanologenic Esche-richia coli to express the hemoglobin from thebacterium Vitreoscilla (VHb). Ethanologenic E. colistrain FBR5 and FBR5 transformed with the VHbgene in two constructs (strains TS3 and TS4) weregrown in cheese whey (CW) medium at small andlarge scales, at both high and low aeration, or withwhey powder (WP) or sugar beet molasses hydroly-sate (SBMH) media at large scale and low aeration.Culture pH, cell growth, VHb levels, and ethanolproduction were evaluated after 48 h. VHb expres-sion in TS3 and TS4 enhanced their ethanol produc-tion in CW (21–419%), in WP (17–362%), or inSBMH (48–118%) media. This work extends thefindings that “VHb technology” may be useful forimproving the production of ethanol from waste andbyproducts of various sources.

Key words: bioethanol; Escherichia coli; sugar beetmolasses; Vitreoscilla hemoglobin; whey

The production of sustainable energy alternatives tofossil fuels is a topic of great concern worldwide. Bio-ethanol is the most commonly used alternative liquidfuel, and can be produced from various kinds of rawmaterials by microbial fermentation. Of particular inter-est is fermentation of materials that are traditionallyconsidered waste products or byproducts of other pro-cesses.1–3) Among products with such potential arethose produced as by products in the food processingindustry.4,5)

Cheese whey (CW) is a byproduct of dairy industriesand, because it is produced in large amounts, its dis-posal represents a significant environmental problem.Generally to make 1 kg of cheese about 9 L of whey isgenerated.6) Although it is considered a waste product,whey is nutrient-rich, containing 5–6% lactose, 1% pro-tein, 0.06% fat, and 0.1–0.8% (w/v) lactic acid.7) Lac-tose is the sugar mainly responsible for whey’spolluting load. Similarly, raw juice from sugar beet pro-cessing can be used directly for sugar or ethanol pro-duction,8,9) or can be concentrated in an evaporator and

stored for several months as molasses.10,11) Sugar beetmolasses is a byproduct of sucrose production, and isthe noncrystallizable residue after most of the sucrosehas been crystallized. Because of its ready availabilityand low cost, molasses can be used as animal feed, inbaker’s yeast, bakery goods and pharmaceuticals, andfor ethanol production.12)

Brazil is the largest grower of sugarcane in the worldand the second largest ethanol producer behind theUnited States.13) Global production of ethanol was22.36 billion US gallons (84.6 billion liters) in 2011with production from US dominating (accounting for87.1% of world production).13) Ethanol productionfrom CW has been investigated by many research-ers14,15) as a way to convert a waste product into avaluable energy source. The amount of lactose in wheyavailable for ethanol production may be as high as 4million tons per year and this could yield 2.3 millionm3 of ethanol. This is about 3.5% of the total worldproduction of ethanol in 2008.16)

Several organisms have been investigated for wheyfermentation but ethanol production has been low17,18);this has led to efforts to increase this production.19–21)

Fermentation of whey is problematic, in part, becauseits fermentable sugar is lactose. Saccharomyces cerevi-siae, one of the most traditional ethanol producers, isunable to utilize pentose sugars22) or lactose for ethanolproduction. Kluyveromyces sp. ferments lactose but it isinhibited by moderate concentrations of sugar and saltand has low ethanol tolerance compared to S. cerevisi-ae.23) Fermentation of molasses to ethanol has alsobeen well-studied.10,24,25) In European countries sugarbeet molasses is the preferred starting material for bio-ethanol production26); in Serbia about 90% of ethanolproduction comes from molasses.10) Other sugar beetprocessing intermediates are also potential sources forbioethanol production.27)

E. coli has many advantages for ethanol production,including the ability to ferment a wide spectrum ofsugars and no requirements for complex growth factors.However, it uses mixed acid fermentation and normallyconverts only a small fraction of fermentable sugars toethanol. E. coli strains have been genetically engi-neered, however, to ferment with ethanol as the only

*Corresponding author. Email: akbasm@gyte.edu.tr

Bioscience, Biotechnology, and Biochemistry, 2014Vol. 78, No. 4, 687–694

© 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry

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product.28–30) One of these strains, FBR530) has beenfurther engineered to express the hemoglobin (VHb)from the bacterium Vitreoscilla.31,32)

The gene encoding VHb (vgb) has been cloned andexpressed in many bacteria to improve growth andmetabolite production.33–35) VHb-expressing FBR5strains were shown to have increased ethanol produc-tion using either corn fiber hydrolysate31,32) or potatowastewater hydrolysate as primary carbon sources.36)

In the present work, E. coli strain FBR5, and twoFBR5 transformants expressing vgb (TS3 and TS4)31),were utilized to investigate the possible enhancementof ethanol from food processing byproducts, in thiscase CW and sugar beet molasses. This extends ourwork on VHb-related enhancement of ethanol produc-tion from potato wastewater36) to two additionalbyproducts of food processing, and suggests that theVHb strategy may be generally useful in enhancingethanol production from a wide variety of inexpensivesugar sources.

Materials and methodsBacterial strains, vectors and strain maintenance.

Strain FBR5, constructed by Dien et al.30), harborsplasmid pLOI29719) contains the pyruvate decorboxy-lase (pdc) and alcohol dehydrogenase (adhB) genesfrom Zymomonas mobilis, which encode the enzymesthat catalyze ethanol production from pyruvate. StrainsTS3 and TS4 were previously developed by transfor-mation of FBR5 with plasmids pTS3 and pTS4, respec-tively, both of which are compatible with pLOI297 andboth of which contain the Vitreoscilla hemoglobin gene(vgb)31); thus, each of strains TS3 and TS4 harbor twoplasmids. Strain TS4 produces generally about fivetimes as much VHb as strain TS3. To maintain plasmidstability during routine strain maintenance on 8% (w/v)xylose-containing LB plates and in certain experiments,antibiotics were added from filter sterilized stock solu-tions to media as follows: 100 µg/mL ampicillin (forFBR5; ampicillin resistance encoded by pLOI297); or100 µg/mL ampicillin and 50 µg/mL streptomycin (forTS3; ampicillin resistance encoded by pLOI297, strep-tomycin resistance encoded by pTS3); or 100 µg/mLampicillin and 5 µg/mL gentamicin (for TS4; ampicillinresistance encoded by pLOI297, gentamicin resistanceencoded by pTS4).

Preparation of CW medium. Sweet CW wasobtained from a regional Dairy industry (Ispey Milkproducts, Kutayha, Turkey). Its pH was 4.4 and it con-tained approximately 4.0% (w/v) lactose. For prepara-tion of CW solution, modified from Leite et al.21), thepH of whey was adjusted to 7.0 followed by autoclav-ing. The mixture was then filtered to remove denaturat-ed proteins and 5 g of yeast extract was added to a literof the solution, autoclaved again and then centrifuged(sterilely) at 3000 g (Heraeus Instruments, Labofuge400R, Osterode, Germany, with swinging bucket rotor)at 4 °C for 15 min. The supernatant (CW solution) wascollected sterilely and stored at 4 °C until use.

For preparation of CW medium (pH 7.0), 1 mL ofthiamine (0.1%, w/v) and 5 mL of trace elements

solution were added to a liter of CW solution as wasdescribed previously20). For preparation of control med-ium (CM1, pH 7.0) 5 g of yeast extract was added to aliter of lactose solution (4%, w/v lactose) and auto-claved. About 1 mL of thiamine (0.1%, w/v) and 5 mLof trace elements solution were then added.

Preparation of whey powder medium. Whey pow-der (WP) was donated by Maybi (Malkara Birlik Milkand Milk products C.A., Istanbul, Turkey). It containedapproximately 75% lactose, 10% protein, and 1% fatson a dry weight basis. WP was dissolved in distilledwater to 33% (w/v), adjusted to pH 7.0, and auto-claved. The mixture was then centrifuged at 3000 g(Heraeus Instruments, Labofuge 400R, Osterode,Germany, with swinging bucket rotor) at 4 °C for 15min. The supernatant was collected and stored at 4 °Cuntil use.For preparation of WP medium (pH 7.0), 500 mL

2X-LB (contains 10 g peptone and 5 g yeast extractwith no NaCl; pH 7.0), 3 g MgSO4, 1 g NH4HPO4, 1 gCH3COONa, 89 ml distilled water, 1 mL of thiamine(0.1% (w/v)), and 200 mL 0.5M sodium phosphatebuffer (pH 7.0) were added to 200 mL of WP solution,prepared as described above. Control medium for theseexperiments (CM2, pH 7.0, and 5.5% lactose (w/v))contained 500 mL 2X-LB (contains 10 g peptone and5 g yeast extract with no NaCl; pH 7.0), 3 g MgSO4,1 g NH4HPO4, 1 g CH3COONa, 89 mL distilled water,1 mL of thiamine (0.1% (w/v)), 200 mL 0.5 M sodiumphosphate buffer (pH 7.0), and 200 mL of lactose(27.5% (w/v)).

Sugar beet molasses hydrolysis and preparation ofsugar beet molasses hydrolysate medium. Sugar beetmolasses was donated by Pakmaya Inc., Izmit, Koca-eli, Turkey; its initial sucrose concentration was deter-mined to be about 50% (w/v). Molasses was dilutedwith distilled water to 20% (v/v), adjusted to pH 3.0with 2M H2SO4 and hydrolyzed at room temperatureovernight to convert sucrose to glucose and fructose,followed by autoclaving. The pH was adjusted to 7.0with NaOH followed by centrifugation at 3000 g(Heraeus Instruments, Labofuge 400R, Osterode,Germany, with swinging bucket rotor) at 4 °C for15 min. The supernatant was filter sterilized and storedat 4 °C until used.Sugar beet molasses hydrolysate (SBMH) medium

(pH 7.0) contained 500 mL 2X-LB (contains 10 gpeptone and 5 g yeast extract with no NaCl; pH 7.0),3 g MgSO4, 1 g NH4HPO4, 10 mL CH3COONa (10%(w/v)), 89 mL distilled water, 1 mL of thiamine (0.1%(w/v)), 200 mL 0.5 M sodium phosphate (pH 7.0), and200 mL 20% (v/v) hydrolysate molasses, prepared asdescribed above. The medium was filter sterilized bypassage through a 0.22 µm membrane (Nalgene). Thefructose and glucose concentrations in SBMH mediumprepared in this way were both found to be about 1.2%(w/v). Control medium for these experiments (CM3,pH 7.0) contained 500 mL 2X-LB (with no NaCl; pH7.0), 3 g MgSO4, 1 g NH4HPO4, 1 g CH3COONa,89 mL distilled water, 1 mL thiamine (0.1% (w/v)),

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200 mL 0.5 M sodium phosphate buffer (pH 7.0), and200 mL of sugar mixture (6% glucose and 6% fructose(w/v)).

Preparation of precultures. Precultures werestarted from single colonies from 8% xylose (w/v) con-taining LB plates in 5 mL of CW, WP or SBMH, ortheir respective control media (CM1, CM2, or CM3)containing antibiotics appropriate for each strain asdescribed above; cultures were incubated at 37 °C at200 rpm overnight (18–20 h).

Growth conditions. Experiments were run with CWmedium or CM1 (containing appropriate antibiotics) atboth large and small scales under both high and lowaeration conditions. Because the ethanol levels werefound higher for large cultures at low aeration conditionsfor the fermentation with WP and SBMH media (withand without antibiotics), only large-scale cultures at lowaeration conditions were conducted.

For small-scale high aeration conditions, cultures werestarted with inocula of approximately 15 μL of stationaryphase preculture into 20 mL of fresh CW medium orCM1. Volumes of precultures were adjusted using pre-culture optical density to equalize the biomass of allinocula across all cultures (with the same initial opticaldensity of 0.02 OD600 nm units. An OD600 nm of 0.02was equivalent to about 106 CFU/mL). Each culture con-tained 20 mL of CW medium or CM1 in a 100 mLErlenmeyer flask. Incubation was at 37 °C and 180 rpm.

Small-scale cultures at low aeration were inoculatedin the same way, with approximately 300 μL of precul-ture into 80 mL of fresh CW medium or CM1 in125 mL Erlenmeyer flasks (with the same initial opticaldensity of 0.02 OD600 nm units). Flasks were cappedwith a rubber stopper pierced with a 22-gauge needlefor CO2 exhaust. Incubation was at 37 °C and 180 rpm.

For large-scale high aeration conditions, cultures weregrown in 200mL of CW medium or CM1 in 1000mLErlenmeyer flasks at 37 °C and 180 rpm. For low aeration,the cultures were grown in 800mL of CW, WP or SBMH,or their respective control media in 1000mL Erlenmeyerflasks at 37 °C and 180 rpm. As described above, inocula-tion volumes were adjusted to give initial optical densitiesof 0.02 OD600 nm units across all cultures.

Some growth experiments were run with antibiotics(as described above) in order to force plasmid stability.For comparison, some experiments were run withoutantibiotics. Stabilities of plasmid pTS3 (vgb+) in strainTS3 and plasmid pTS4 (vgb+) in strain TS4 were moni-tored after 48 h of growth in WP and SBMH medialacking antibiotics as was described by Urgun-Demirtaset al.37) using LB agar and the appropriate antibioticsand concentrations used for liquid media. The stabilityof plasmid pLOI297 (contains pyruvate decorboxylase,pdc and alcohol dehydrogenase, adhB genes) in TS3and TS4 was determined separately, and was found tobe about 100% in both strains after 48 h of growth inampicillin-free WP or SBMH.

Analytical measurements. Measurement of pH,biomass (OD600 nm), ethanol, VHb; lactose levels and

residual lactose levels for CW or WP, or their respec-tive control media, and glucose and fructose levels, aswell as residual glucose and fructose levels, for SBMHor its respective control media, were performed after48 h in culture. pH measurements were performed witha Hanna Instruments, pH 211 Microprocessor, pHmeter (Ann Arbor, Michigan, USA). Optical densitieswere measured at 600 nm using a Bio-Rad, SmartSpec3000 spectrophotometer (cuvette path length of1.0 cm); All OD600 nm readings were kept below 0.6 bydilution, if necessary, with the corresponding mediumand read against blanks of the corresponding medium.Ethanol levels were measured using an ethanol assaykit (ethanol-UV-test, Boehringer-Mannheim, R-Biop-harm, Germany) according to the instructions of thesupplier. VHb levels were determined by dithionitetreated-minus-untreated difference spectra (Δε435–405 =34M−1 cm−1; cuvette pathlength of 1 cm) as previouslydescribed38) and normalized to g wet weight of cells.The lactose, glucose, and fructose levels as well as

residual sugar levels in CW, WP, and SBMH mediawere determined after 48 h of growth by thin layerchromatography using comparisons with correspondingsugar samples of known concentrations as describedpreviously.39) TLC was conducted using two ascents ofacetonitrile/water (85:15 (v/v)) on Whatman K5 silicaplates. Carbohydrates were visualized by dipping theplates into 5% (v/v) H2SO4 in ethanol containing 0.5%(w/v) α-naphthol, followed by heating at 110 °C for 10min. Intensities of spots were quantified by TLC-imag-ing densitometry using a Bio-Rad GS-670 densitometer(Philadelphia, PA, USA).

Statistical analysis. Student’s t–tests were per-formed using Excel 2007. P values were calculatedwith one-tailed t-tests, corresponding to the hypothesisthat VHb would enhance ethanol production comparedto the matched strain without VHb (i.e. TS3 vs. FBR5and TS4 vs. FBR5). Results were considered significantfor P values less than or equal to 0.05.

Results and discussionCW medium and its control medium (CM1)

experimentsCW medium was a much better growth medium than

CM1, as reflected in higher biomass and ethanol levelsand greater metabolism of lactose for comparisonsacross all combinations of strain and aeration conditions.It is presumed that the additional nutritional compo-nents, such as proteins or minerals, provided to CWmedium by the whey are responsible. The ethanol levelsin CM1 were found to be much lower than those in CWmedium across all conditions. The highest ethanol levelswere obtained with strains TS3 and TS4, producing 174and 39% more ethanol than FBR5, respectively, atlarge-scale and low aeration. The highest enhancementof ethanol with VHb expression was achieved in large-scale fermentations at high aeration conditions, withTS3 producing 419% more and TS4 producing 48%more ethanol than FBR5 (Tables 1 and 2). Notunexpectedly, in general, low aeration conditionsresulted in better ethanol yields and higher ethanol/

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biomass than high aeration conditions. As expected,growth was greater at high aeration than low aeration forall pairwise comparisons in CW medium. But this wasnot true of CM1; the reasons for this are not yetunderstood. For otherwise identical conditions, thelarger-scale cultures produced much more ethanol thanthe smaller-scale cultures; the reasons for this are alsonot yet known.

There was no clear correlation between growth(OD600 nm) and ethanol production for all strains(Table 1) across all growth conditions. pH values werefound to be lower after 48 h fermentation in all cultureconditions (Tables 1 and 2). The decrease in pH valuesare presumably because of the production of CO2 inthe ethanol pathway.30) However, a correlation betweenpH values and ethanol production was not observed.

E. coli strains (FBR5, TS3 and TS4) produced nativebeta galactosidase for utilization of lactose in whey.The lactose (4% w/v) was consumed to a greater

extent by TS4 than by FBR5 in CW medium in allconditions in the 48 h fermentation period. Strain TS3,however, consumed all of the lactose after 48 h. In gen-eral, then, a 48 h fermentation duration was adequatefor effective and economical fermentation. In CM1,corresponding to the much lower ethanol production,lactose consumption was much lower than for the samecombination of strains and conditions in CW medium.In both media for all four combinations of aeration

conditions and scale, the production of ethanol wasenhanced by VHb expression for both TS3 and TS4(compared to FBR5). A very close correlation betweenthe increase in ethanol produced and the increase in

Table 1. Growth parameters for strains FBR5, TS3, and TS4 grown in CW medium and control medium (4% lactose containing) (CM1) underhigh aeration conditions as described in Materials and methods.

Strains VHb (nmol/ g) pH OD (600 nm) EtOH (g/100 mL) p EtOH/OD600 nm p Residual lactose (%)

AFBR5 0 4.97 (0.04) 4.23 (0.32) 0.45 (0.01) 0.11 (0.01) 1.91 (0.23)TS3 166 (7) 7.71 (0.88) 6.76 (0.51) 1.50 (0.11) 0.03 0.22 (0.04) 0.06 0.00 (0.00)TS4 271 (61) 5.99 (1.33) 3.05 (1.48) 0.14 (0.04) 0.02 0.05 (0.01) 0.00 0.12 (0.12)BFBR5 0 5.22(0.12) 2.77 (0.52) 0.42 (0.01) 0.15 (0.03) 1.49 (0.15)TS3 174 (42) 6.43 (0.43) 4.98 (0.25) 2.18 (0.06) 0.01 0.39 (0.04) 0.06 0.00 (0.00)TS4 622 (11) 5.21 (0.03) 3.29 (0.76) 0.62 (0.08) 0.07 0.19 (0.02) 0.05 1.30 (0.04)CFBR5 0 6.14 (0.11) 1.02 (0.03) 0.06 (0.01) 0.06 (0.01) 3.41 (0.01)TS3 16 (5) 6.19 (0.54) 1.33 (0.17) 0.13 (0.01) 0.05 0.09 (0.01) 0.05 3.08 (0.04)TS4 80 (10) 5.94 (0.08) 1.74 (0.06) 0.16 (0.01) 0.03 0.09 (0.01) 0.10 2.88 (0.04)DFBR5 0 6.16 (0.37) 1.45 (0.07) 0.16 (0.01) 0.11 (0.01) 3.00 (0.13)TS3 18 (6) 5.24 (0.62) 1.50 (0.01) 0.25 (0.01) 0.02 0.16 (0.00) 0.03 2.53 (0.06)TS4 29 (3) 5.29 (0.60) 1.08 (0.11) 0.20 (0.01) 0.05 0.18 (0.01) 0.06 2.79 (0.01)

Notes: A, small-scale cultures in CW medium with antibiotics; B, large-scale cultures in CW medium with antibiotics; C, small-scale cultures cultures grown in CM1with antibiotics; D, large-scale cultures cultures grown in CM1 with antibiotics. VHb concentrations are nmol/ g wet weight of cells. Values are averages of two tothree independent measurements after 48 h of growth; standard deviations are in parantheses. Columns (labeled p) to the right of the EtOH and EtOH/OD600 nm

columns are p values for t-tests of differences between TS3 or TS4 and FBR5 for the EtOH and EtOH/OD600 nm values, respectively.

Table 2. Growth parameters for strains FBR5, TS3, and TS4 grown in CW medium and control medium (4% lactose containing) (CM1) underlow aeration conditions as described in Materials and methods.

Strains VHb (nmol/g) pH OD (600 nm) EtOH (g/100 mL) p EtOH/OD600 nm p Residual lactose (%)

AFBR5 0 5.22 (0.29) 2.08 (0.04) 0.38 (0.03) 0.18 (0.01) 2.09 (0.16)TS3 201 (24) 6.13 (0.04) 4.53 (0.46) 1.11 (0.11) 0.04 0.24 (0.00) 0.05 0.00 (0.00)TS4 433 (88) 5.51 (0.12) 2.26 (0.06) 0.46 (0.02) 0.02 0.20 (0.01) 0.00 0.54 (0.11)BFBR5 0 5.42 (0.06) 2.00 (0.14) 0.81 (0.01) 0.41 (0.02) 1.14 (0.14)TS3 227 (63) 5.64 (0.03) 4.04 (0.06) 2.22 (0.10) 0.02 0.55 (0.04) 0.02 0.00 (0.00)TS4 508 (8) 5.33 (0.18) 1.98 (0.04) 1.13 (0.06) 0.02 0.57 (0.01) 0.01 0.65 (0.18)CFBR5 0 5.31 (0.81) 1.30 (0.28) 0.03 (0.01) 0.02 (0.01) 3.57 (0.04)TS3 75 (17) 5.19 (0.83) 1.26 (0.11) 0.05 (0.00) 0.06 0.04 (0.01) 0.00 3.27 (0.04)TS4 136 (13) 5.14 (0.80) 1.01 (0.01) 0.04 (0.01) 0.06 0.04 (0.01) 0.00 3.66 (0.06)DFBR5 0 4.85 (0.08) 1.17 (0.11) 0.23 (0.02) 0.19 (0.00) 2.71 (0.04)TS3 86 (42) 5.51 (0.01) 1.41 (0.05) 0.34 (0.01) 0.03 0.24 (0.01) 0.04 2.71 (0.30)TS4 334 (87) 5.38 (0.11) 1.26 (0.06) 0.42 (0.01) 0.02 0.33 (0.02) 0.04 2.52 (0.06)

Notes: A, small-scale cultures grown in CW medium with antibiotics; B, large-scale cultures grown in CW medium with antibiotics; C, small-scale cultures grown inCM1 with antibiotics; D, large-scale cultures grown in CM1 with antibiotics. VHb concentrations are nmol/g wet weight of cells. Values are averages of two to threeindependent measurements after 48 h of growth; standard deviations are in parantheses. Columns (labeled p) to the right of the EtOH and EtOH/OD600 nm columnsare p values for t-tests of differences between TS3 or TS4 and FBR5 for the EtOH and EtOH/OD600 nm values, respectively.

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ethanol per unit of cell mass was also observed in bothmedia and at both aeration conditions (Tables 1 and 2).In almost all cases, the increase in ethanol/cell masscompared to FBR5 was statistically significant (Tables1 and 2).

A number of studies have indicated that respirationand ATP production are increased due to VHb expres-sion.40) In the work reported here, such a VHb-relatedeffect may have provided additional ATP for enhancedgrowth and enzyme levels, especially those of betagalactosidase and the ethanol pathway. Therefore, mea-surement of these enzyme levels may add to the under-standing of how VHb is correlated with increasedethanol production.

Trace metals and thiamine are important factors forefficient fermentation in E. coli KO1120,41), and sowere added as supplements to CW medium in the workreported here. The trace metals could be cofactors ofenzymes or play important roles in oxidation–reductionreactions. Guimeras et al.20) found that trace metalswere important for whey fermentation in E. coli KO11.Supplementing sweet whey with a trace metal mix andammonium sulfate reduced the required fermentationtime and increased final ethanol from 26 g ethanol/L in144 h to 28 g ethanol/L to 72 h. Leite et al.21) alsofound that trace metals such as Zn2+, Ca2+, and Mn2+,and 0.5% yeast extract improved ethanol yield by100% in E. coli KO11.

In the present work, the highest ethanol levelsattained (about 2.2% (w/v)) were with strain TS3 atboth low and high aeration at large scale. The reasonsfor these results are unknown, but suggest that scale-upto fermentor scale may be feasible without a loss inethanol productivity. It has been reported that direct fer-mentation of CW to ethanol yielded ethanol levels ofabout 2–3% from 5–6% lactose containing whey. Etha-nol separation from 2% ethanol containing media is noteconomically feasible42); exptrapolating our results(from 4% lactose containing whey) to the higher initiallactose concentration predicts about 3% or moreethanol, which is well above the 2% limit and thus

suggests that commercial scale-up of such a process ispossible.

WP medium and its control medium (CM2)experimentsSimilar to the results with CW medium, in WP med-

ium, or CM2 both strains TS3 and TS4 grew betterand produced significantly more ethanol than strainFBR5 for all conditions (with or without antibiotics)(Table 3). Although in WP medium and CM2, the etha-nol enhancement was achieved by both VHb express-ing strains, the lower VHb expressing strain, TS3,produced more ethanol across all conditions. Theincrease in ethanol production with TS3 and TS4 ran-ged from 17 to 362% (Table 3).The highest cell mass was achieved generally by

TS3, and the lowest by FBR5. With these media, thestability of the plasmids encoding both vgb and the eth-anol productivity were also assessed. The presence ofantibiotics did negatively affect the growth of the VHbexpressing strains. However, inclusion of antibioticswas necessary to ensure plasmid stability (Table 4) andthe maximum enhancement of ethanol production inWP medium (Table 3); inherent plasmid instability wasgreater for strain TS3 than for TS4. As was seen withCW medium and CM1, there was no correlationbetween the magnitude of the pH drop at 48 h and eth-anol production, but the pH at 48 h was generallyhigher with WP medium, likely owing to 0.5 M sodiumphosphate (pH 7.0) acting as a buffer.The lactose (5.5% w/v) was consumed completely

by all of the three strains in both WP medium andCM2 in all conditions in the 48 h fermentation period.Because typical raw CW contains 5–6% lactose, wedetermined to work with a final lactose concentrationof 5.5% (w/v) in the WP culture medium. This higherlactose concentration was correlated with greater etha-nol production (comparing the data in Table 3 withthose in Table 2). It is interesting, however, that con-centrated lactose or whey media, especially containing

Table 3. Growth parameters for strains FBR5, TS3, and TS4 grown in WP medium and control medium (contains 5.5% lactose, w/v) (CM2)under low aeration conditions, with and without antibiotics, as described in Materials and methods.

Strains VHb (nmol/g) pH OD (600 nm) EtOH (g/100 mL) p EtOH/OD600 nm p

AFBR5 0 6.35 (0.29) 1.83 (0.53) 0.85 (0.17) 0.46 (0.03)TS3 47 (32) 6.38 (0.04) 5.78 (2.16) 3.93 (0.64) 0.01 0.77 (0.15) 0.03TS4 119 (27) 6.24 (0.08) 3.70 (0.42) 1.19 (0.20) 0.02 0.32 (0.01) 0.03BFBR5 0 6.24 (0.20) 1.95 (0.57) 0.97 (0.08) 0.50 (0.08)TS3 22 (1) 6.35 (0.14) 4.80 (0.57) 2.74 (0.26) 0.05 0.57 (0.02) 0.05TS4 94 (6) 6.22 (0.04) 2.92 (0.46) 1.14 (0.03) 0.01 0.39 (0.14) 0.04CFBR5 0 6.11 (0.12) 2.30 (0.00) 0.81 (0.15) 0.35 (0.06)TS3 47 (1) 6.26 (0.16) 2.28 (0.11) 1.14 (0.11) 0.03 0.50 (0.02) 0.06TS4 724 (95) 6.35 (0.24) 2.23 (0.21) 0.95 (0.18) 0.05 0.42 (0.05) 0.02DFBR5 0 5.96 (0.27) 2.48 (0.04) 0.51 (0.16) 0.20 (0.06)TS3 47 (1) 6.01 (0.37) 2.43 (0.13) 1.04 (0.25) 0.04 0.43 (0.12) 0.06TS4 811 (27) 6.15 (0.07) 2.89 (0.01) 0.84 (0.21) 0.03 0.29 (0.04) 0.05

Notes: A, Cultures grown in WP medium with antibiotics; B, cultures grown in WP medium without antibiotics; C, cultures grown in CM2 with antibiotics; D,cultures grown in CM2 without antibiotics. VHb concentrations are nmol/g wet weight of cells. Values are averages of two to three independent measurements after48 h of growth; standard deviations are in parantheses. Columns (labeled p) to the right of the EtOH and EtOH/OD600 nm columns are p values for t-tests ofdifferences between TS3 or TS4 and FBR5 for the EtOH and EtOH/OD600 nm values, respectively.

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100 to 150 g/L lactose, is inhibitory to ethanol produc-tion43); the initial sugar concentration should be below75 g/L to obtain the highest yields of ethanol in theyeast Kluyveromyces marxianus and avoid substrateinhibition.44)

SBMH medium and its control medium (CM3)experiments

We extended the examination of whether VHbexpression enhances ethanol production from byprod-ucts to molasses using SBMH medium and CM3. Theglucose and fructose in SBHM medium and CM3 werecompletely exhausted in 48 h. These results indicatedthat the 48 h fermentation time was adequate for all ofthe sugar to be consumed for ethanol production inthese media.

In SBMH medium or CM3, TS3 generally grew thebest of the three strains, but TS4 produced more etha-nol than strain TS3 and FBR5 for all conditions, espe-cially with presence of antibiotics (Table 5). Similar toCW or WP media, SBMH medium supported greaterethanol production than CM3. It is presumed that theadditional nutritional components, such as vitamins orminerals, provided by SBMH are responsible. The

highest ethanol production was achieved with TS4, pro-ducing 90% more ethanol than FBR5; TS3 produced48% more ethanol than FBR5 (Table 5). In previousstudies, the lower level of VHb expression in TS3strain enhanced ethanol levels more than occurred inthe higher VHb expressing TS4 strain;31) these resultsare consistent with our results with CW or WP mediabut not with SBMH medium.Similar to the case for WP medium and CM2, the

presence of antibiotics in SBMH medium and CM3 didnot negatively affect the growth of the VHb expressingstrains; the stability of plasmid pTS4 in strain TS4 wasgreater than that of plasmid pTS3 in strain TS3 in bothSBMH medium and CM3 (Table 4).Concerning strain TS3 the current results are, for the

most part, qualitatively consistent with those of Sannyet al.31), Arnaldos et al.32) and Abanoz et al.36) Quanti-tatively, however, ethanol enhancement levels weremuch larger than those of Sanny et al.31) and Arnaldoset al.32) particularly for TS4 in SBMH medium andTS3 in WP medium. The differences between the pres-ent and previous studies could be due to the differencesin the medium supplements (sugar content; content ofother nutrients and minerals) used.

Comparisons VHb- producing vs. VHb-negativestrainsEngineering of a variety of heterologous hosts to

express VHb has been shown to increase cell growthand production of many and various recombinant prod-ucts33,35) or improve biodegration of aromatic com-pounds.37,45–48) The available data indicate that theVHb-associated enhancement may work in severalways: increasing respiratory ATP production viaincreased delivery of oxygen to the terminal respiratoryoxidase, delivery of oxygen to oxygenases, and modu-lation of activity of certain oxygen responsive tran-scription factors.40,49) In the case of enhancedproduction of ethanol and other metabolites, the mainVHb effect is likely mediated through the increased

Table 4. Plasmid stabilities measured are for the vgb-bearingplasmids (pTS3 for Strain TS3 and pTS4 for Strain TS4) for strainsTS3 and TS4 grown in WP or in control medium (CM2) and SBMHMedium or in control medium (CM3), under low aeration conditions,without antibiotics, as described in Materials and methods.

Medium Strains Plasmid stability (%)

WP TS3 41TS4 73

SBMH TS3 48TS4 100

CM2 TS3 56TS4 94

CM3 TS3 64TS4 100

Table 5. Growth parameters for strains FBR5, TS3, and TS4 grown in SBMH medium and control medium (contains 1.2% glucose and 1.2%fructose, w/v) (CM3) under low aeration conditions, with antibiotics, as described in Materials and methods.

Strains VHb (nmol/g) pH OD (600 nm) EtOH (g/100 mL) p EtOH/OD600 nm p

AFBR5 0 6.36 (0.17) 3.92 (0.33) 0.87 (0.54) 0.22 (0.07)TS3 354 (48) 6.46 (0.05) 4.45 (0.49) 1.29 (0.08) 0.04 0.29 (0.10) 0.08TS4 961 (72) 6.49 (0.06) 5.20 (0.14) 1.65 (0.03) 0.03 0.31 (0.09) 0.03BFBR5 0 6.20 (0.40) 3.75 (0.92) 0.67 (0.17) 0.17 (0.00)TS3 252 (93) 6.43 (0.04) 4.65 (1.06) 1.26 (0.32) 0.03 0.27 (0.02) 0.03TS4 784 (51) 6.42 (0.06) 3.23 (0.75) 1.46 (0.34) 0.04 0.45 (0.05) 0.04CFBR5 0 6.35 (0.22) 5.51 (1.49) 0.66 (0.06) 0.12 (0.01)TS3 169 (12) 6.42 (0.01) 5.85 (0.54) 0.97 (0.02) 0.03 0.16 (0.01) 0.08TS4 853 (127) 6.50 (0.04) 5.28 (0.76) 1.05 (0.01) 0.04 0.19 (0.02) 0.04DFBR5 0 6.61 (0.05) 4.77 (0.04) 0.67 (0.01) 0.14 (0.00)TS3 96 (26) 6.43 (0.04) 4.94 (0.01) 0.94 (0.11) 0.04 0.19 (0.02) 0.05TS4 495 (60) 6.41 (0.06) 5.43 (0.60) 1.07 (0.06) 0.05 0.19 (0.01) 0.02

Notes: A, cultures grown in SBMH medium with antibiotics; B, cultures grown in SBMH medium without antibiotics; C, cultures grown in CM3 with antibiotics; D,cultures grown in CM3 without antibiotics. VHb concentrations are nmol/ g wet weight of cells. Values are averages of two to three independent measurements after48 h of growth; standard deviations are in parantheses. Columns (labeled p) to the right of the EtOH and EtOH/OD600 nm columns are p values for t-tests ofdifferences between TS3 or TS4 and FBR5 for the EtOH and EtOH/OD600 nm values, respectively.

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ATP production, which may result in greater cell mass,etc.35,40) The enhancement of ethanol production byVHb was surprising, as ethanol fermentation is ananaerobic process, and VHb should increase oxygenlevels in the cell, but physical provision of limitedamounts of oxygen to ethanologenic E. coli cultureshas the same effect.50,51)

Enhancement of ethanol production by VHb expres-sion in E. coli strains FBR5, TS3, and TS4 has beenpreviously studied with a variety of media and mediasupplements, and in all cases substantial increases havebeen seen. This includes media supplemented with purexylose and glucose,31,32) but also with sugar sourcesthat might be of practical usefulness because they arewaste products of other processes (corn fiber hydroly-sate31,32); potato wastewater36)). The work reported hereextends the supplements of practical potential even fur-ther, adding two additional byproducts of food process-ing to the list of those in which VHb expression isassociated with increased ethanol production.

In the first such study, using glucose and xylose assupplements, only the strain (TS3) expressing lowerlevels of VHb had higher ethanol production than theVHb-free control strain (FBR5),31) and so TS3 waschosen for further work.32) It was postulated that themuch higher VHb levels in TS4 might make intracellu-lar conditions too aerobic to support high-level fermen-tation to ethanol. When TS3 and TS4 were againcompared, however, using potato wastewater as a sup-plement, TS4 was generally superior, even though itsVHb level was still higher than that of TS3 by aboutthe same relative amount as the previous studies.36) Inthe work reported here either TS3 was superior forwhey or TS4 was superior for molasses, like the bulkof the previous studies, although both were generallysubstantially better than FBR5. It is presumed thatthese variations are due to some as yet undefined dif-ferences provided by the various supplements. Thegreater advantage of TS3 seen here occurred even withits higher inherent plasmid instability compared to TS4,and even when plasmid stability was not forced byinclusion of antibiotics.

VHb expression levels were fairly consistent acrossall media, and were generally greater at low aerationthan high aeration for all pairwise comparisons in CWmedium or CM1. This is as expected given the knownmechanism of induction of vgb expression at low oxy-gen concentrations.49) Also as expected, when high andlow aeration conditions were compared, low aerationconditions generally gave greater levels of ethanol. Thisis consistent with previous work with this system.31,36)

Although depending on the growth conditions theVHb–associated ethanol increases seen previously varyover a fairly large range, the increases seen here coinci-dent with VHb expression are generally within therange seen previously.31,32,36)

In the previous studies with the FBR5, TS3, andTS4 system, there has been no overall consistent corre-lation between ethanol production and cell mass, andas a general conclusion the VHb-mediated ethanolincrease is due to a combination of greater growth andgreater ethanol produced per unit of biomass. In thework reported here is no clear correlation betweengrowth (OD600 nm) and ethanol production for FBR5,

TS3, and TS4 strains in any of the growth media, and,as in previous work, the VHb-related ethanol increaseis sometimes due to increased cell mass, sometimes toincreased ethanol/unit of cell mass and sometimes toboth effects.

Conclusion

Our results indicate that the vgb/VHb system maybenefit ethanol production using sugar beet molasses orWP as sources of fermentable sugars, as seems to be thecase for corn fiber hydrolysate and potato waste. Thisextends the possible utility of this strategy regardingbiofuel production from waste materials and byproducts.It may also be possible to use mixtures of such wasteproducts/byproducts (such as of WP and SBMH) in fer-mentation media. In this case, the catabolite repressionissue (due to the high glucose content of molasses)would need to be overcome by using mutant strains.

Acknowledgment

We would like to thank Dr. Benjamin C. Stark forvaluable help in the preparation of manuscript and inproviding the strains for this work. We would like tothank Ispey Milk products, Kutayha, Turkey for provid-ing cheese whey, Maybi (Malkara Birlik Milk and Milkproducts C.A., Istanbul, Turkey) for providing wheypowder, and Pakmaya Inc., Izmıt, Kocaeli, Turkey forproviding sugar beet molasses.

Funding

This work was supported by TUBITAK [grant number212T172] and by the Gebze Institute of Technology, Turkey.

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