Watermelon juice: a promising feedstock supplement, diluent, and nitrogen supplement for ethanol biofuel production
© The Article is a work of the United States Government; licensee BioMed Central Ltd. 2009
Received: 12 March 2009
Accepted: 26 August 2009
Published: 26 August 2009
Two economic factors make watermelon worthy of consideration as a feedstock for ethanol biofuel production. First, about 20% of each annual watermelon crop is left in the field because of surface blemishes or because they are misshapen; currently these are lost to growers as a source of revenue. Second, the neutraceutical value of lycopene and L-citrulline obtained from watermelon is at a threshold whereby watermelon could serve as starting material to extract and manufacture these products. Processing of watermelons to produce lycopene and L-citrulline, yields a waste stream of watermelon juice at the rate of over 500 L/t of watermelons. Since watermelon juice contains 7 to 10% (w/v) directly fermentable sugars and 15 to 35 μmol/ml of free amino acids, its potential as feedstock, diluent, and nitrogen supplement was investigated in fermentations to produce bioethanol.
Complete watermelon juice and that which did not contain the chromoplasts (lycopene), but did contain free amino acids, were readily fermentable as the sole feedstock or as diluent, feedstock supplement, and nitrogen supplement to granulated sugar or molasses. A minimum level of ~400 mg N/L (~15 μmol/ml amino nitrogen) in watermelon juice was required to achieve maximal fermentation rates when it was employed as the sole nitrogen source for the fermentation. Fermentation at pH 5 produced the highest rate of fermentation for the yeast system that was employed. Utilizing watermelon juice as diluent, supplemental feedstock, and nitrogen source for fermentation of processed sugar or molasses allowed complete fermentation of up to 25% (w/v) sugar concentration at pH 3 (0.41 to 0.46 g ethanol per g sugar) or up to 35% (w/v) sugar concentration at pH 5 with a conversion to 0.36 to 0.41 g ethanol per g sugar.
Although watermelon juice would have to be concentrated 2.5- to 3-fold to serve as the sole feedstock for ethanol biofuel production, the results of this investigation indicate that watermelon juice, either as whole juice fermented on-site or as a waste stream from neutraceutical production, could easily integrate with other more concentrated feedstocks where it could serve as diluent, supplemental feedstock, and nitrogen supplement.
Ethanol produced by fermentation of plant biomass is considered to be an environmentally friendly alternative to fossil fuels [1, 2]. The search for renewable biomass sources that do not serve as human food and animal fodder have focused primarily on plant biomass that possesses mainly cellulose and lignocellulosic materials . Although it is generally agreed that this type of biomass will ultimately be the primary source of ethanol biofuel in the future , there exist sources of simple hexoses that can fill a special niche in biofuel production but that have not been investigated for such an application. One such source is watermelon. Two factors make watermelon worthy of consideration as a possible feedstock for ethanol biofuel production. First, about 20% of each annual watermelon crop is rejected for fresh fruit marketing because of surface blemishes or because fruit are misshapen; these 'culls', although internally sound, are left in the field. For the 2007 growing season, this would have amounted to around 360,000 t lost to US growers as a source of revenue . Conversion on the premises of all or part of these cull watermelons to ethanol could, at the very least, provide supplemental on-farm fuel to the grower, or, in some cases, be sold on the ethanol biofuel market. Second, the neutraceutical value of compounds obtained from watermelon is at a threshold whereby a portion of the watermelon crop and all the culls could be employed as starting material to extract and manufacture these products. Lycopene, the powerful antioxidant carotenoid that imparts the red color to watermelon, has been shown to be important in prostate health . L-citrulline is a naturally occurring amino acid involved in the detoxification of catabolic ammonia and also serves as a precursor for L-arginine, the amino acid centrally involved in the production of the circulatory vasodilator, nitric oxide . Production of these two neutraceuticals from watermelon yields a waste stream that contains ~10% (w/v) sugars that can be directly fermented to ethanol.
A watermelon is nominally 60% flesh, and about 90% of the flesh is juice that contains 7 to 10% (w/v) sugars. Thus, over 50% of a watermelon is readily fermentable liquid. A logical use for a watermelon juice processing waste stream would be integration into bioethanol production. Since it is water-based, watermelon juice could serve as a diluent for concentrated sources of fermentable sugars such as molasses that require an approximate dilution to ~25% (w/v) sugars before fermentation. In addition, the 7 to 10% (w/v) ready-to-ferment sugars in watermelon (glucose, fructose, and sucrose) would supplement the primary feedstock and reduce primary feedstock requirements proportional to the volume of watermelon juice used to dilute the concentrated feedstock. Third, the free amino acids at 15 to 35 μmol/ml in watermelon juice could serve as a nitrogen supplement for yeast in those feedstocks such as cane sugar and molasses that possess inadequate available nitrogen levels to maintain maximal yeast growth and ethanol production.
Little has been published on investigations for optimizing the parameters for fermentation of watermelon juice. Kim et al.  reported on parameters affecting the fermentation of watermelon juice for the ultimate manufacture of vinegar, but the optimization for maximal rates of ethanol production was not one of their objectives. The purpose of this investigation was to examine watermelon juice, either whole or as a waste stream from neutraceuticals production, as a diluent, feedstock supplement, and nitrogen supplement in ethanol biofuel production systems.
Watermelons employed in this study came from several sources covering two crop years. In 2007, watermelons came from a commercial field in Hinton, OK, USA. They were purposely selected because they had been graded 'culls' as the result of an anthracnose infection on their outer rinds. Other sources of watermelons included those from the 2007 crop in Terral, OK, USA, some from the 2008 crop in Louisiana, USA, and others from the 2008 crop raised at the South Central Agricultural Research Center, Lane, OK, USA. Lycopene-free juice was a waste stream from processing watermelon flesh to produce lycopene-containing chromoplasts by a procedure previously described (Fish, US Patent Appl. 60/752.279, 2005). Amino acid-free juice was prepared from the lycopene-free watermelon juice by procedures developed in this laboratory (Fish, unpublished). Watermelon juices at the various stages of processing were frozen and stored at -20°C until used. Granulated sugar and molasses employed for fermentations were purchased at a local supermarket.
Fermentations were conducted in a BF-110 benchtop modular fermentor system (New Brunswick Scientific Co., Inc., Edison, NJ, USA). The system included a 7.5 l thermostatted glass vessel, a pH/DO controller, a four-pump reagent addition module, an exhaust condenser, a DO probe, and a pH electrode. The fermentor was sanitized between fermentations by washing with detergent, rinsing with water, treating with 5% (v/v) Oxonia Active™ (Ecolab Inc., St Paul, MN, USA) for 6 h at room temperature, and rinsing thoroughly with water. Most of the experimental fermentations were conducted on a volume of 2 to 3 L of feedstock. Temperature was controlled at 32°C, and the medium stirred at 100 rpm to keep the yeast, substrate, and products evenly distributed. Media were routinely inoculated with hydrated and conditioned dried yeast to provide an initial yeast population of ~107 viable cells/ml in the fermentation medium. The pH of nearly all fermentations not pH-controlled quickly dropped to ~pH 2.8 and remained there regardless of the starting value. For those fermentations conducted at a pH other than ~3, the fermentor controller was programmed to add 1 M NaOH when needed to maintain the pH of the medium at the level desired. The starting pH of the processed watermelon juice used to prepare feedstocks was generally pH 3 so that the pH of the medium initially had to be adjusted to the value desired for the fermentation run. Fermentation runs under a specific set of conditions were repeated at least once, and in most cases, three replicate fermentations were performed. Data presented in the Figures and the text are the means of the replicates, and their standard deviations are represented by error bars in the Figures.
Yeast conditioning and nutrition
The yeast (Ethanol Red™) employed for fermentations was a commercial dried yeast (Fermentis of Lesaffre, Milwaukee, WI, USA). Sufficient active dried yeast (0.5 g/L of fermentation medium) was rehydrated with the goal of providing a starting concentration of ~107 viable cells/ml in the fermentation medium. The yeasts were rehydrated/conditioned by first suspending into water an amount of Go-Ferm™ (Lallemand, Montreal, Canada) that equaled 1.25 parts by weight of that of the dried yeast that was to be used. The Go-Ferm™ was suspended into 20 times its weight of sterile H2O at 43°C. Once the temperature of the Go-Ferm suspension reached 39 to 40°C, the dried active yeast was added and gently stirred to evenly disperse the cells. The suspension was allowed to stand for 15 to 30 min during which time the yeast slurry cooled to ~32°C. The yeast slurry was then added to the feedstock (already at 32°C) in the fermentation vessel. Nitrogen supplementation was provided, when desired, by the addition of diammonium phosphate (DAP) (Sigma-Aldrich, St Louis, MO, USA) and/or yeast extract, HY-Yest 412 (10.2% total nitrogen; 5.2% amino nitrogen) (Sigma-Aldrich, St Louis, MO, USA). The yeast nutrient complex (Fermaid K™, Lallamand, Montreal, Canada), was routinely added at 0.25 g/L to the ongoing fermentation when the starting sugar levels were 1/3 depleted. For fermentations that utilized greater than 20% (w/v) sugars, yeast hulls were added at a level of 0.2 g/L to furnish additional sterols to the yeast and to adsorb part of the yeast autotoxins that are produced during fermentation.
Yeast and bacterial counts
The density of viable yeast cells was routinely estimated by determining the absorbance at 420 nm of a diluted sample of fermentation medium. This relationship had been determined earlier by sampling, diluting, plating, and counting colony-forming units from fermentations that were in the log phase of growth. The number of colony-forming yeast cells per ml of media was then correlated with the absorbance at 420 nm of a sample from the fermentation medium measured at the same time as the sample was removed for plating. Microbial counts were taken from media after 48 h of fermentation. Dilutions of the fermentation medium were plated onto nutrient agar that contained 10 μg/ml of cyclohexamide (Sigma, St Louis, MO, USA) to inhibit yeast growth.
Determination of ethanol and CO2 production rates
The rates of ethanol production at various pH values were estimated by plotting the ethanol levels versus fermentation times between 12 and 28 h and fitting the data with a linear least squares equation. The slope of the line was then used as a measure of the maximal rate of ethanol production. Although it is recognized that this type of direct plot is not truly linear, data in this region approach linearity (0.990 <R2 < 0.999), and the slope of a linear fit to the data approaches the slope of a tangent to a point in this region.
The rate of CO2 evolution from the fermentor was estimated by counting the rate of bubbles released into the water trap per unit time. The conversion from number of bubbles per min to liters of CO2 per h was possible after first determining the number of CO2 bubbles from the water trap that it took to yield a liter of CO2 gas by displacement of water from an inverted water-filled graduated cylinder.
High performance liquid chromatography (HPLC) was carried out on a Varian ProStar ternary solvent system equipped with an autosampler and diode array and RI detectors. Quantitative carbohydrate profiles of the feedstocks were obtained with a 250 mm × 4 mm 5 μm Luna™ amino column (Phenomenex, Torrance, CA, USA). The sugars, glucose, fructose, and sucrose, were eluted with an isocratic system of 80% acetonitrile/20% H2O at a flow rate of 1 ml/min and a column temperature of 35°C. Fermentation substrates, glucose and fructose, and fermentation products, including ethanol, acetate, glycerol, citrate, and lactate, were separated and quantified on an Aminex™ HPX-87H 300 mm × 7.8 mm column (BioRad, Hercules, CA, USA). Components were eluted with an isocratic system of 5 mM H2SO4 in H2O. The flow rate was 0.6 ml/min, and the column temperature was maintained at 50°C. Three standard solutions containing fructose, glucose, and ethanol at different concentration levels were run each time a fermentation analysis was performed in order to confirm the fidelity of the respective calibration curves. In order to quantify fermentable sugars with the Aminex™ column during fermentation runs, samples that contained sucrose had to be pre-treated with invertase before column chromatography. This was necessary because the acid conditions of the 5 mM H2SO4 eluting solvent catalyzed hydrolysis of the glucose-fructose glycosidic bond during time on the column and created a reaction zone so that none of the three sugars could be quantified. Pre-column hydrolysis of sucrose with invertase was performed by incubating 100 μl of sample (up to 40% sucrose) with 100 μl of yeast invertase (Sigma, St Louis, MO, USA) at ~100 U/ml in 0.05 M sodium acetate buffer, pH 4.6, at 37°C for 30 min. By the end of the incubation, all sucrose had been hydrolyzed to glucose plus fructose, and these two hexose sugars could be separated and quantified by HPLC analysis on the Aminex™ column. Samples for analysis during fermentation were collected via the sterile sampler on the fermentor. Two 1 ml aliquots of the fermentation broth were centrifuged for 2 min at 10,000 × g on a microfuge (Eppendorf, Hamburg, Germany) to pellet all insoluble material. After invertase treatment and appropriate dilution (~50-fold), samples for HPLC analysis were filtered through 17 mm disposable nylon filters of 0.45 μm pore size before being placed in the autosampler. A sample loop of 100 μl was employed in the autosampler. Amino nitrogen was estimated in feedstocks by the ninhydrin method . The concentrations of L-citrulline in the respective watermelon juices were estimated by separating the juice amino acids by thin layer chromatography  and comparing the intensity of each juice's L-citrulline spot, after staining with ninhydrin, to those of authentic L-citrulline co-developed on the chromatogram at known concentrations. The concentration of total nitrogen in a watermelon juice was estimated by measuring its amino nitrogen and adjusting for the additional nitrogen furnished by its L-citrulline content. This then assumed that all other amino acids in the watermelon juice each contained only one nitrogen. So while the estimate for amino nitrogen is accurate, the estimated numbers for watermelon juice total nitrogen may be as much as 5 to 15% lower than the true value since there was no quantitative measure of those amino acids that contained more than one nitrogen.
Results and discussion
Feedstock watermelons employed and their respective juice compositions
2007 Hinton, OK, USA
2007 Terral, OK, USA
2008 Louisiana, USA
Summer Flavor 800
2008 Lane, OK, USA
2008 Lane, OK, USA
Respective contents of amino nitrogen and concomitant citrulline varied among the watermelon juices. It is not known whether these differences are due to cultivar inherent differences or the result of variation in fertility and growing conditions. This can be indirectly illustrated by comparing the citrulline results for two of the cultivars that were used in this study with those for the same two cultivars that were also part of a study which compared citrulline levels in several watermelon cultivars . The value for 'Sangria' flesh reported for the 2000 growing season was ~9.1 mmol/ml (1.6 mg/g flesh) while a citrulline content of 5.8 mmol/ml for 'Sangria' from the 2008 crop season was measured in the present study. Conversely, the value of ~6.8 mmol/ml (1.2 mg/g flesh) reported for citrulline in 'Summer Flavor 800' in 2000 is lower than 12.6 mmol/ml measured for citrulline in this cultivar in 2008.
Fermentation of whole watermelon juice and as a waste stream from various processes
Lycopene-containing chromoplasts from the complete watermelon juice could still be solublilized in sodium dodecyl sulfate detergent (SDS) after 24 h in the fermentor at 32°C and in the presence of a final concentration of 3+% (w/v) ethanol. This SDS-solubility indicated that the integrity of the chromoplasts' membranes was still intact  so that the chromoplasts were still viable entities that could be isolated from the fermentation broth, if desired. Subsequently, chromoplasts from a fermentation that produced 12% (w/v) ethanol were also determined to be 97% viable. These results suggest that this neutraceutical could be successfully harvested intact post-fermentation and before removal of the ethanol by distillation.
Watermelon juice as a nitrogen source for fermentation
The negative impact on fermentation rate by depletion of amino nitrogen from watermelon juice prompted an examination in more detail of the effect of nitrogen levels on fermentations involving this juice. Sucrose (table sugar) was employed as the primary feedstock. It was dissolved in chromoplast-free watermelon juice to a final sugar concentration of ~20% (w/v). This meant that the watermelon juice served as diluent, feedstock supplement (supplying ~70 g/L fermentable sugars to that of the total 200 g/L of sugars in the feedstock), and nitrogen source for the yeast. The level of free amino nitrogen in the fermentation media was varied by combining various proportions of amino acid-containing watermelon juice with amino acid-free watermelon juice. Nitrogen in these fermentations came almost exclusively from watermelon juice and varied from ~857 mg N/L down to ~46 mg N/L.
Effect of pH on fermentation rates of feedstocks that include watermelon juice
Watermelon juice in the fermentation of cane sugar
Fermentation of processed sugar supplemented and diluted with watermelon juicea
Beginning (Fermentable sugars)
24 h (Yeast)
24 h Respiration rate
Final (Unfermented sugar)
Time to completion
6.8 × 107
7.8 × 107
5.1 × 107
3.7 × 107
11.0 × 107
9.0 × 107
7.9 × 107
Watermelon juice as diluent, sugar supplement, and nitrogen supplement in the fermentation of molasses
Applications of the technology
At a nominal total watermelon yield of 42 t/ha and 20% of the crop being culls, there would be about 8.4 t/ha of watermelons left in the field. A yield of 520 L juice/t containing 10% (w/v) fermentable sugars means the culls would produce 4370 L of juice possessing 435 kg fermentable sugars per ha. At a production ratio of ~0.4 g ethanol/g sugar as measured in this study, approximately 174 kg/ha or 220 L/ha of ethanol would be produced from these cull watermelons.
Two approaches exist for ultimately converting cull watermelons to ethanol. The most direct alternative would be to utilize the juice harvested from the culls to produce ethanol on-site at the production field with a mobile fermentation/distillation unit. Juice collected with a modified watermelon seed harvester would then only need to be transported from the rows of watermelon to the ethanol production system at the edge of the field. Using an ethanol production rate of 2 g/L/h of ethanol from watermelon juice as measured in this study (from Figures 1, 4, and Table 2), a required fermentation capacity can be calculated. The above approach would provide the grower with fuel for on-farm use and/or to sell on the ethanol fuel market.
In the second approach, watermelon juice in the form of a waste stream from neutraceuticals processing would be utilized as diluent and feedstock supplement in ethanol production. This scenario would likely involve a neutraceuticals production facility in physical proximity to an existing ethanol biofuel production facility. Watermelon production at a nominal rate of 42 t/ha has the potential for simultaneous production of 1.5 kg lycopene, 90 kg L-citrulline, and 2000 L ethanol per ha by utilizing both flesh and rinds. The removal of first lycopene-containing chromoplasts and then L-citrulline from watermelon has been developed into an integrated process that leaves a waste stream of sugar-containing juice (Fish, unpublished results). Both cull watermelons and those appropriate for the fresh food market are acceptable for this processing. Watermelon juice as a waste stream from neutraceutical production offers several advantages when integrated into a nearby bioethanol production facility. First, watermelon juice can be used in place of potable water as a diluent for the primary feedstock. Although wastewater treatment costs from post-fermentation waste would be little affected with either type of diluent, the use of a watermelon juice waste stream would save the cost of potable water at the front end of the process. Second, by using watermelon juice as diluent, the amount of primary feedstock required would be reduced by ~7 to 10% as a result of the readily fermentable sugars in the diluent juice. Based on the results using watermelon juice as a diluent for molasses fermentation, it is estimated that there would be an approximate 15% savings on molasses consumption and ~22,000 L savings of potable water per 40,000 L fermentation run. As well as serving as diluent and feedstock supplement, watermelon juice that retains its amino acids would also serve as a source of readily assimilable nitrogen for yeast growth and vitality and thus also save the costs associated with nitrogen supplementation.
The results of this investigation indicate that watermelon juice as a source of readily fermentable sugars represents a heretofore untapped feedstock for ethanol biofuel production. The 8.4 t/ha of unmarketable watermelons left in the field at harvest would produce about 220 L/ha of ethanol for on-farm use or as an additional revenue stream for the grower. Whole watermelons utilized for the production of the neutraceuticals, lycopene and L-citrulline, yield a waste stream of sugar-containing juice. This juice waste stream would easily integrate with other more concentrated feedstocks employed in ethanol biofuel production where the watermelon juice would serve as diluent, supplemental feedstock, and nitrogen supplement.
sodium dodecyl sulfate.
The authors thank Rick Houser, Diann Baze, and Kelli Baze for providing valuable technical support. The fermentor utilized in this investigation was generously provided by Abbott & Cobb Seed Company, Feasterville, PA, USA. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. All programs and services of the US Department of Agriculture are offered on a non-discriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap.
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