Bioconversion of solid food wastes to ethanol

Transcription

1 Analyst, March 1998, Vol. 123 ( ) 497 Bioconversion of solid food wastes to ethanol Jothi V. Kumar *a, Abolghasem Shahbazi b and Rose Mathew a a Department of Chemistry, North Carolina A&T State University, Greensboro, NC 27411, USA b Department of Natural Resources and Environmental Design, North Carolina A&T State University, Greensboro, NC 27411, USA Solid food processing wastes and by-products are cofermented with cheese whey to produce ethanol. The experimental procedure involves the use of an enzymatic process to convert starch and lactose into fermentable sugars. These reducing sugars are then fermented to alcohol by distiller s dried yeast and a high-ethanol tolerant yeast, Saccharomyces cerevisiae. Cheese whey is used as a wetting agent and provides macro- and micronutrients for the microorganisms. Cofermenting food processing wastes with cheese whey, in the presence of high- and low-temperature enzymes, induces a 33-36% increase in alcohol yield. This procedure also significantly reduces the fermentation time from 60 to 12 h. Keywords: Bioconversion; cofermentation; cheese whey; ethanol; solid wastes; gas chromatography Energy and environmental issues take turns driving the development and use of alternative fuels for motor vehicles. As the availability of petroleum-derived fuels and industrial feedstocks decreases owing to depletion and also economic and political developments, renewable sources of organic compounds are tested for their suitability as alternatives to petroleum-based substances. Recent environmental concerns such as ozone non-attainment, solid waste management and control of toxic air pollutants have been other reasons for finding clean-burning alternative fuels. Ethanol production from agricultural products has been in practice for the past 80 years. Ethanol can be produced from many kinds of raw material that contains starch, sugar or cellulose. Wastes from food processing industries represent a severe pollution problem and need better waste management techniques. Utilization of food processing wastes to produce fuel alcohol with an increased efficiency has been under investigation in our laboratory for the past few years. We were able to develop a novel and highly efficient cofermentation system for food wastes containing starch and lactose. Fermentation is an anaerobic, energy-releasing transformation of carbohydrates by living organisms. Yeast can ferment a wide variety of sugars and oligosaccharides other than glucose. The d-hexoses and oligosaccharides fermented most often by yeast are glucose, mannose, fructose, galactose, maltose, lactose, melibiose, trehalose and raffinose. The yeast in most widespread use for alcoholic fermentation is Saccharomyces cerevisiae. Several studies on ethanol production via fermentation and the effects of different factors on the fermentation have been published 1 7 in the past decade. Utilization of cheese whey as the liquid portion of a fermenting corn mash has been investigated by Whalen et al. 8 Their work involved the fermentation of lactose/corn mash by the use of a dual yeast inoculum (Kluyveromyces marxianus and distillerís yeast). This lactose/glucose cofermentation process took h for completion. We investigated the use of whey with bakery products and other starchy waste products by the application of lactose hydrolysis in conjunction with a single yeast inoculum to reduce the fermentation time and an increase in alcohol yield. The objectives of this work were to study the effect of lowand high-temperature enzymes on hydrolysis of food wastes, to compare the fermentation of bakery products with mixed waste products and to study the cofermentation of cheese whey and starchy food wastes. Experimental Materials Feedstocks Raw materials for ethanol production include various types of starchy waste products from bakeries and food processors and cheese whey from dairy processing industries. The types of waste samples used were bread, biscuits, buns, cakes, donuts, potato chips and flour. The bakery products (2 3 weeks old) were collected from a local bakery and were stored below 5 C. Cheese whey was obtained from a local cheese plant and was refrigerated until used. Samples were ground to a fine powder using an electrical blender. Enzymes Fungamyl is a purified fungal a-amylase produced from a selected strain of Aspergillus oryzae. This enzyme hydrolyzes 1,4-a-glucosidic linkages in amylose and amylopectin. Termamyl is an a-amylase isolated from a soil bacterium, Bacillus licheniformis. This enzyme hydrolyzes 1,4-a-glucosidic linkages in starch and possesses a high degree of heat stability. Amyloglucosidase Novo (AMG) is an exo-1,4-a-d-glucosidase (glucoamylase) obtained from a selected strain of Aspergillus niger by submerged fermentation. This enzyme hydrolyzes both1,4- and 1,6-a-linkages in starch. Lactozym (Novo Industries, Bagsvaerd, Denmark) is a b-galactosidase (lactase) preparation produced by submerged fermentation of a selected strain of the yeast Kluyveromyces fragillis. When Lactozym reacts with lactose, a mixture of glucose and galactose is formed. High T is Alltech s brand name for its improved bacterial a-amylase. High T is a carefully selected blend of enzymes derived mainly from Bacillus subtilis. Allcoholase II is a carefully selected blend of enzymes derived from Aspergillus niger and Rhizopus niveus. Allcoholase II is designed to be a complex blend of many enzyme activities including a- amylases, amyloglucosidase, protease and cellulase. In addition, it contains many vitamins and yeast growth factors. Since Allcoholase II works at the same temperature range as yeast, it can be added with the yeast. All the enzymes were bought from Novo Nordisk (Baltimore, MD, USA). Organism The yeast we used for fermentation experiments was Saccharomyces cerevisiae. Two types of yeast strains were used, distiller s yeast and Alltech Enriched Yeast. Distiller s yeast was obtained from the Redstar Division of Universal Foods (Milwaukee, WI, USA). Alltech Enriched Yeast (AEY) is a high-ethanol tolerant yeast obtained from Alltech (Lexington, KY, USA).

2 498 Analyst, March 1998, Vol. 123 Instrumentation YSI Model 27 industrial analyzer The YSI Model 27 industrial analyzer (Yellow Springs Instrument, Yellow Springs, OH, USA) is used to measure the concentration of certain carbohydrates, alcohols, enzymes and metabolites in food and beverages, agricultural products and fermentation processes and products. The precision and specificity of the Model 27 are comparable to those of the more timeconsuming and rigorous systems. The advantages of the Model 27 system include its speed, convenience and sensitivity. Reagents included immobilized enzyme membranes, buffer concentrates, calibrator, control solutions and other auxillary chemicals. The sensor system of the YSI Model 27 consists of three electrodes and a temperature-compensating thermistor embedded in the probe. The platinum working electrode measures hydrogen peroxide amperometrically, according to the following reaction: H 2 O 2? 2H + + O 2 + 2e 2 Current flow in the platinum anode circuit is linearly proportional to the local concentration of hydrogen peroxide, and is very nearly zero in the plain buffer solution without peroxide. By means of a signal conditioning circuit which functions as a three-electrode potentiostat, the platinum working electrode is maintained at an electrical potential of 0.70 V positive with respect to a silver/silver chloride reference electrode. The potential of the silver reference electrode is determined from the reaction AgCl + e 2? Ag + Cl 2 At constant chloride ion concentration and low reference electrode current, this reaction provides a very stable reference potential. The presence of silver chloride is assured by atmospheric oxidation of the silver electrode surface. In order that the reference electrode current may be kept very small, even when the working current is much larger, a third electrode is used to complete the working electrode circuit. An operational amplifier measures the potential difference between the working and the reference electrodes without drawing any significant current from the reference electrode. The amplifier forces significant current through the third electrode to meet the needs of the working electrode and hold its potential at the desired V. The membrane which fits over the enzyme probe face is a three layer laminate, with the middle layer containing one or more covalently immobilized enzymes. These enzymes convert the substances to be measured into hydrogen peroxide. The hydrogen peroxide is sensed by the platinum anode, and the result is a signal current proportional to the concentration of the substance to be measured. The circuitry of the Model 27 converts this signal according to the zero and calibrate control settings, and displays the assay value on the digital meter. The Model 27 can be set up to perform a variety of analyses using the appropriate enzymatic membrane systems and accessories as outlined in the YSI Model 27 Instuction Manual. We used this instrument mainly to monitor the glucose concentration. Hewlett Packard Model 5890 gas chromatograph This instrument, purchased from Hewlett-Packard (Rockville, MD, USA), was used to measure the alcohol content in the fermentation product. The column used for the analysis was a 0.10% SP 1000 high performance capillary column from Supelco (Bellefonte, PA, USA). The experimental conditions for the GC were as follows: sample size, 1 2 ml; Carrier gas flow rate, 21 ml min 21, initial temperature of the column, 50 C; final temperature, 150 C; programming rate of 30 C min 21 ; injection port temperature, 250 C; and detector (flame ionization) temperature, 260 C. The concentration of ethanol from the fermented samples were calculated from a calibration curve constructed using ethanol standards. Atomic absorption spectrometer A Perkin Elmer (Norwalk, CT, USA) Model 560 atomic absorption spectrometer with appropriate hollow cathode lamps (Fisher Scientific, Norcross, GA, USA) was used to measure the concentration of the metal ions in cheese whey. A calibration curve prepared using standard solutions was used to determine the metal ion concentration in cheese whey. Benchtop fermenter A 14 l Microferm Benchtop Fermenter equipped with an automatic stirrer and temperature control units was obtained from New Brunswick Scientific (Edison, NJ, USA). It was also assembled with a sample outlet tube and a carbon dioxide exhale tube. Distillation column A Kontes distillation unit from Fisher Scientific with a flask of 14 l capacity was used to distil off the alcohol from the fermented mixture. Procedure Starch is a polymeric carbohydrate produced by plants as a food reserve which can be broken down into soluble sugars. Starch consists of two components, amylose and amylopectin, that are both polymers of glucose. Amylose, which normally makes up 20 25% of the starch mass, is a linear polymer of glucose units joined by 1,4-a-linkages. The number of glucose units contained in one amylose molecule typically varies between 500 and 1500, corresponding to molecular masses of Amylopectin makes up 75 80% of most starch types. It is a branched polymer in which the glucose units are joined by 1,4-a linkages in the linear molecular section and by 1,6-a-linkages at the branching points, which typically occur every glucose units. Molecules of amylopectin can be very large, consisting of glucose units, corresponding to molecular masses from around up to several million. In its native state, starch consists of microscopic, partly crystalline granules in which the amylose and amylopectin molecules are arranged in a complex folded and stratified manner. To produce ethanol from starch, it must be first converted into sugar, which is then fermented to alcohol. To facilitate the starch treatment processes, the raw material was ground to a fine powder using an electric blender. At ambient temperature the starch granules are virtually insoluble in water and not very susceptible to enzymatic hydrolysis. However, when treated with hot water, the starch granules swell and gradually rupture, the amylose and amylopectin molecules unfolding and dispersing into solution. This process is referred to as gelatinization of starch. Enzymatic hydrolysis Liquefaction. With a-amylase (endo-amylase), the 1,4-alinkages in both amylose and amylopectin units can be hydrolyzed almost at random. This results in the breakdown of starch to dextrins and oligosaccharides. Saccharification. When treated with glucoamylase (exo-amylase), liquefied starch is broken down into glucose. The 1,4- and 1,6-a-linkages of amylose and amylopectin are hydrolyzed in a stepwise manner from the non-reducing end of the molecule.

3 Analyst, March 1998, Vol Conversion of lactose to glucose and galactose. Lactose is a disaccharide consisting of one molecule each of glucose and galactose connected by 1,4-b-linkages. By the action of a b- linkage-breaking enzyme (lactase), lactose can be converted into glucose and galactose, which are fermentable. Fermentation. The sugars formed can be converted into ethanol and carbon dioxide by the action of yeast. The conversion of glucose into ethanol involves a complex sequence of chemical reactions which can be summarized as follows: C 6 H 12 O 6? 2 C 2 H 5 OH + 2 CO 2 + heat During fermentation, approximately 5% of the fermentable sugar was lost in yeast and formation of byproducts, 9 including approximately 3% as glycerol, 0.4% as succinic acid and 0.8% as yeast biomass. Cheese whey analysis (Table 1) Cheese whey obtained from a local cheese plant was acid whey with a ph of It was tested for its lactose content using the YSI Model 27 industrial analyzer. The whey contained 4.85% of lactose. The whey was further tested for its protein content (0.47% m/v) by the Lowry protein determination method. 10 The metal content was determined using an atomic absorption spectrometer. Conversion and fermentation of cheese whey (Table 2) For the conversion of lactose in whey into fermentable sugars, the enzyme Lactozym was used. Whey (2 l) was put in the fermenter and its ph was adjusted to 6.50 with NaOH solution. The temperature was kept at 37 C and 1.5 ml of Lactozym was added. The whey was stirred and samples were removed at 30 min intervals. The glucose formed was measured by injecting the samples into the YSI Model 27 instrument. The conversion was almost complete in 3 h. The temperature of the whey was then brought to 32 C and 1 g of distiller s yeast was added. The decrease in glucose concentration was measured with the YSI Model 27 and the increase in ethanol concentration was determined using a gas chromatograph at 30 min intervals. Fermentation was continued until the maximum ethanol concentration was obtained. Table 1 Analysis of cheese whey (ph 4.30) Component Composition (%) Lactose Protein Fat Potassium Calcium Sodium Magnesium Table 2 Fermentation of cheese whey in the presence of Lactozym and yeast Time/h Glucose/g l 21 Ethanol/g l 21 Conversion (%) Measurement of starch The starch content of the feedstock (food processing wastes) was determined indirectly by an enzymatic procedure. 11 The starch contents of mixed bakery wastes and mixed waste products were measured to be 516 and 597 g kg 21 respectively. This procedure required the use of a-amylase and amyloglucosidase to convert starch into glucose. Fermentation of bakery products The waste feedstock was first tested for its starch content, then the starch was hydrolyzed to glucose using three different enzymes, as described below. Hydrolysis and fermentation in the presence of fungamyl and AMG. A 500 g amount of finely ground bakery products was mixed with 2 l of water in the fermenter. The ph and the temperature of the mash were adjusted to 4.70 and 37 C, respectively. A 2 ml volume of the enzyme Fungamyl was added and the mixture was stirred for 2 h. Both the initial and final glucose concentrations were measured. After the liquefaction was complete, the ph of the mash was brought to 4.30, the temperature was adjusted to 25 C and 2 ml of AMG were added. Glucose formation was monitored with the YSI Model 27. The saccharification process was continued until a constant concentration of glucose was obtained. The ph and the temperature of the hydrolyzed mash were adjusted to 5.0 and 30 C, respectively. A 2 g amount of Distiller s yeast was added to the mash with stirring and the fermentation was monitored by measuring the decrease in glucose concentration (YSI) and the increase in ethanol content (GC) with sampling every hour. Fermentation was continued to obtain the maximum ethanol yield. Hydrolysis and fermentation in the presence of termamyl and AMG. The temperature of the finely ground bakery waste, 500 g in 2 l of water, was adjusted to 70 C and its ph to 5.0, then 2 ml of Termamyl were added and the mixture was stirred for 2 h. The mash was then mixed with 2 ml of AMG at ph 4.50 and 55 C. The hydrolysis was continued to obtain the maximum glucose concentration. The ph and the temperature of the hydrolyzed mash were adjusted to 5.0 and 30 C, respectively, and 2 g of Distiller s yeast were added to the mash with stirring. The fermentation was monitored by measuring the decrease in glucose concentration and the increase in ethanol content with hourly sampling. Fermentation was continued to obtain the maximum ethanol yield. Hydrolysis and fermentation in the presence of High T and alcoholase II. The mash containing 500 g of bakery products and 2 l water was heated to 90 C and 1.5 ml of the enzyme High T was added at ph 6.0. The mash was stirred at this temperature for h. When the liquefaction was complete, the mash was cooled to 32 C to prepare it for saccharification and fermentation. Allcoholase II at the rate of 0.03 g l 21, Alltech Enriched Yeast at 0.13 g l 21, and Allpen at g l 21 were added to the mash in the fermenter. The samples were tested for alcohol concentration at 1 h intervals and fermentation was continued until the maximum alcohol yield was obtained. Fermentation of mixed waste products. The fermentation experiments were repeated with a mixture of waste products such as different types of bakery products, donuts, potato chips and flour. The mixture was hydrolyzed with Termamyl/AMG and fermented with Distiller s yeast. Cofermentation of bakery and mixed waste products. In the cofermentation process, 1 kg of waste products was mixed with 4 l of cheese whey. The experiments included an additional step for the conversion of lactose in whey into fermentable sugars. Initially, the substrate whey mixture (250 g l 21 ) was treated with Lactozym at ph 6.5 and 37 C. After the lactose conversion, the mixture was liquefied and fermented as described in the previous experiments.

4 500 Analyst, March 1998, Vol. 123 Results and discussion The substrates for our fermentation experiments included cheese whey, mixed bakery products and mixtures of different waste products. The enzymes employed were Fungamyl, Termamyl, AMG, High T and Allcoholase II. Fermentation was brought about by two different types of yeast strains: Distiller s Yeast and Alltech Enriched Yeast. Table 3 presents the glucose and ethanol yields from the fermentation of bakery wastes with the low-temperature enzyme Fungamyl, AMG and yeast. The saccharification process took 8 h and the maximum glucose yield was 540 g kg 21. The fermentation process took 12 h and the maximum ethanol yield was 221 g kg 21. The conversion of glucose to ethanol was 80.15%. Table 4 gives the results of cofermentation of bakery products with cheese whey using the same enzyme and yeast. The increase in ethanol yield due to the addition of cheese whey as the liquid portion of the fermenting mash was 36.5% and conversion was 89%. The saccharification time was shortened by 2 h. Table 5 gives the data for the bioconversion of bakery products in the presence of high-temperature enzyme, Termamyl, AMG and yeast after the saccharification for 4 h. The maximum ethanol yield was 249 g kg 21 with 89% conversion. The results of cofermentation of the bakery products with cheese whey in the presence of Termamyl, AMG and yeast are presented in Table 6. An increase of 30% in ethanol yield was observed with 93% conversion after only 2 h of saccharification. Table 5 Fermentation of bakery products in the presence of Termamyl, AMG and yeast Average final yield * * RSD = 0.85%. Table 3 Fermentation of bakery products in the presence of Fungamyl, AMG and yeast Average final yield * * RSD = 0.96%. Table 6 Fermentation of bakery products and cheese whey in the presence of Lactozym, Termamyl, AMG and yeast Average final yield * * RSD = 1.10%. Table 4 Fermentation of bakery products and cheese whey in the presence of Lactozym, Fungamyl, AMG and yeast Average final yield * * RSD = 0.95%. Table 7 Fermentation of bakery products in the presence of High T, Allcoholase II and yeast Time/h Ethanol/g kg Average final yield * * RSD = 1.16%.

5 Analyst, March 1998, Vol The fermentation of bakery products with High T, Allcoholase II, Altech Enriched Yeast and Allpen yielded 231 g kg 21 of the substrate (Table 7) and an increase of 36% in ethanol yield with cofermentation (Table 8). The process time decreased from 24 to 22 h. When 1 kg of a mixture of food wastes including bakery products, potato chips and grain flour was hydrolyzed using Termamyl and AMG and fermented by distiller s yeast, it produced 245 g kg 21 of ethanol (Table 9). Cofermentation of the same mixture with cheese whey increased the alcohol yield by 33% (Table 10) with 95% conversion and a decrease in Table 8 Fermentation of bakery products and cheese whey in the presence of Lactozym, High T, Allcoholase II and yeast Time/h Ethanol/g kg Average final yield * * RSD = 0.97%. Table 9 Fermentation of mixed waste products in the presence of Termamyl, AMG and yeast Time/h Glucose/g kg 21 Ethanol/g kg Average final yield * * RSD = 1.03%. process time. The time for saccharification also decreased from 4 to 2 h. The increase in efficiency of the saccharification and fermentation process by the addition of whey can be attributed to the fact that the protein and minerals present in the whey act as stabilizing agents and nutrients for the enzyme and the yeast cells during their growth phase. It was observed that the ethanol concentration increased steadily with increase in metal ion concentration up to a certain point, after which it decreased with any further addition of metal ion. The increase in ethanol yield can be explained by the fact that the enzyme and yeast cells use the metal ions as stabilizing agents and nutrients during the growth phase. The metal ions permeate the cell and increase the reaction rate. There is an initial rapid uptake of metal ions due to surface adsorption on the cell walls and a subsequent slow uptake due to the membrane transport of the metals into the cytoplasm of the cells. The cell walls of the microorganism consists mainly of polysaccharides, proteins and lipids and therefore they offer a number of functional groups which are capable of binding to the metal ions. These functional groups, such as amino, carboxylic, phosphate and thiol groups, differ in their affinity and specificity for metal binding. The amount of a metal species bound on the cell surface is therefore determined by the relative affinities of the sites for that particular metal. The surfacebound metal ions are then diffused through the cell membrane into the cytoplasm. The cheese whey also contains some amount of lactose which breaks down to fermentable sugar during hydrolysis, thus increasing the amount of sugar that is fermented. It was also observed that there was an increase in ethanol yield when high-temperature enzymes were used. The high temperature helps the saccharification process to produce Table 10 Fermentation of mixed waste products and cheese whey in the presence of Lactozym, Termamyl, AMG and yeast Time/h Glucose/g kg 21 Ethanol/g kg Average final yield * * RSD = 1.16%. Table 11 Ethanol yields from various waste products with different enzymes Substrate * Enzyme Ethanol/g kg 21 s t-test * MBP Fungamyl and AMG MBP and whey Lactozym, Fungamyl and AMG MBP Termamyl and AMG MBP and whey Lactozym, Termamyl and AMG MBP High T and Allcoholase II MBP and whey Lactozym, High T and Allcoholase MWP Termamyl and AMG MWP and whey Lactozym, Termamyl and AMG * MBP = mixed bakery products; MWP = mixed waste products. 95% CI.

6 502 Analyst, March 1998, Vol. 123 more glucose, which in turn results in an increased ethanol yield. Hence it was concluded that the bioconversion of food processing wastes is favored in the presence of higher temperature enzymes and cofermenting with cheese whey. Some of the results of the statistical evaluation of our data, the average ethanol yields and relative standard deviations are given in various tables and the t-test values presented in Table 11. Statistical evaluation of our data by the t-test to determine the effect of cheese whey in fermenting mixed bakery and waste products indicates that the calculated t-values at the 95% confidence level are higher than the reference value of Therefore, we rejected the null hypothesis and concluded that cheese whey additives play a significant role in fermenting solid food processing wastes to ethanol. Also, the enzymes we tested perform differently from each other. Conclusion Bioprocessing of food wastes using high-temperature enzymes is more promising than using the low-temperature enzymes. Cofermentation with cheese whey increases the ethanol production (33 36%) and shortens the processing time from 60 to 12 h. A preliminary kinetic study shows the reactions are first order and the rate constants range from 18 to 22 h 21 for different enzymes. Details of the kinetic study will be presented in a future publication. There is no significant increase in ethanol yield between the two substrates, bakery products and mixed waste products, that we tested using the same enzyme. This project was supported by the USDA/CSRS Evans Allen Fund, Grant No.. NC X References 1 Chen, S. L., and Gutmanis, F., Process Biochem., 1982, 2. 2 Chen, S. L., Biotechnol. Bioeng., 1981, 23, Garcia, A. III., Fischer, J. R., and Ianotti, E. L., Energy Agric., 1984, 3, Marlatt, J. A., and Datta, R., Biotechnol. Prog., 1986, 2, Bajpai, P. and Bajpai, P. K., Biotechnol. Appl. Biochem., 1989, 11, Ramponi, G., Liguri, G., Stefani, M., Taddei, N., and Nassi, P., Biotechnol. Appl. Biochem., 1988, 10, Tchorbanov, B., and Lazarova, G., Biotechnol. Appl. Biochem., 1988, 10, Whalen, P. J., Shahani, K. M., and Lowry, S. R., Biotechnol. Bioeng. Symp., 1985, 15, Novo Enzymes in the Production of Ethanol from Starch Containing Crops, Novo Industries, Bagsvaerd, Lowery, D. H., Rosebrough, A. L. and Randall, R. J., J. Biol. Chem., 1951, 193, Aman, P., and Hesselman, K., Swed. J. Agric. Res., 1984, 14, 135. Paper 7/06088B Received August 19, 1997 Accepted November 14, 1997