Low pH process for fermentation of sugars from carbohydrates for the production of organic acids and biodegradable deicers

Abstract

This disclosure provides methods for fermentation of sugar substrates by acid-tolerant bacteria for producing acetic and lactic acids, and methods for further processing of these products to obtain acetate and propionate biodegradable deicers. Methods are also disclosed on the use of acid tolerant bacteria to produce acetate and propylene glycol deicing compounds.

Claims

1. A process for producing organic deicers comprising: (a) incubating an acid-tolerant Lactobacillus buchneri in a nutrient medium containing xylose at an incubation pH of 4.3 or less, and at a temperature of at least 30° C. to produce a solution containing at least 30 g/L of acetic acid and 50 g/L of lactic acid, (b) incubating, Propionibacterium acidipropionici, in sequential fermentation using a spent medium from (a) at an incubation pH of 5.0 to 5.5, and at temperature of at least 35° C. to produce a solution of at least 20 g/L of propionic acid and 10 g/L acetic acid; (c) recovering free acetic and propionic acids; and (d) reacting the acetic acid or a combination of acetic and propionic acids with, calcium, magnesium, potassium salts or a combination thereof to produce organic deicers.

2. The process of claim 1 wherein the incubation is conducted using free cells suspended in batch, fed-batch, or continuous bioreactors.

3. The process of claim 2 wherein the incubation is conducted using cells immobilized on alumina fibers or fibrous cloth material in the batch, fed-batch or continuous reactors.

4. The process of claim 1 or claim 2 wherein the incubation step (b) is conducted using co-culture of Lactobacillus diolivorans and Propionibacterium acdipropionici.

5. The process of claim 1 wherein the xylose fermentation is at least 99% complete, and the volumetric productivity is at least 1.35 g/Lh for lactic acid, and at least 0.85 g/Lh for acetic acid.

6. The process of claim 1 or 2 wherein the nutrient medium contains at least 50 g/L carbohydrate.

7. A process for producing deicers comprising: (a) incubating an acid-tolerant Lactobacillus buchneri in a nutrient medium containing glucose at an incubation pH of 4.3 or less, and at a temperature of at least 30° C. to produce a solution containing at least 50 g/L of lactic acid, 5 g/L of acetic acid, 5 g/L of propylene glycol and 20 g/L of ethanol; (b) incubating, Propionibacterium acidipropionici, in sequential fermentation using a spent medium from (a) to utilize the lactate at an incubation pH of 5.0 to 5.5, and at temperature of at least 35° C. to produce a solution of at least 20 g/L of propionic acid and 10 g/L acetic acid; (c) incubating Acetobacter aceti, in a spent medium from (b) at an incubation pH of 4.5 or less to convert the ethanol to acetic acid; (d) recovering the propylene glycol; (f) recovering the acetic and propionic acids; (h) reacting acetic acid or a combination of acetic and propionic acids with, calcium, magnesium, potassium salts or a combination thereof to produce organic deicers.

8. The process of claim 7 wherein the incubation is conducted using free cells suspended in batch, fed-batch, or continuous bioreactors.

9. The process of claim 8 wherein the incubation is conducted using cells immobilized on alumina fibers or fibrous cloth material in the batch, fed-batch or continuous reactors.

10. The process of claim 7 or claim 8 wherein the incubation step (b) is conducted using co-culture of Lactobacillus diolivorans and Propionibacterium acdipropionici.

11. The process of claim 7 or 8 wherein the nutrient medium contains at least 50 g/L carbohydrate.

12. A process for producing organic deicers comprising: (a) incubating an acid-tolerant Lactobacillus buchneri in a nutrient medium containing lactose at an incubation pH of 4.3 or less, and at a temperature of at least 30° C. to produce a solution containing at least 25 g/L of acetic acid and 30 g/L of propylene; (b) incubating an acid tolerant Lactobacillus diolivorans, in sequential fermentation using a spent medium from (a) or as a coculture in the medium of step (a) to produce a solution of at least 10 g/L of each propionic acid and propanol; (c) recovering the propionic acids; and (d) reacting the propionic acids with, calcium, magnesium, potassium salts or a combination thereof to produce organic deicers.

13. The process of claim 12 wherein the incubation is conducted using free cells suspended in batch, fed-batch, or continuous bioreactors.

14. The process of claim 13 wherein the incubation is conducted using cells immobilized on alumina fibers or fibrous cloth material in the batch, fed-batch or continuous reactors.

15. The process of claim 12 or 13 wherein the nutrient medium contains whey permeate with at least 40 g/L of lactose.

16. The process of claim 12 wherein the nutrient medium of step (a) contains lactate from 1st stage fermentation of sugars by Lactobacillus plantarum or other lactate producing bacteria.

17. The process of claim 12 or 13 wherein the nutrient medium contains at least 50 g/L carbohydrate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a process flowsheet that displays the various operations on the raw biomass materials that will be conducted at low pH to produce acetic and lactic acids as products, and the separation processes to obtain separate streams of acetic acid and lactic acid as products. A variety of biomass wastes including whey permeate containing lactose, hydrolysates of lignocellulosic materials containing sugars such as glucose, xylose, or other sugars can be used as substrates for the production of the organic acids.

(2) FIG. 2a is a process schematic for the conversion of xylose to lactate and acetate as products. Lactic and acetic acids can be separated as indicated in FIG. 1, or lactate can be further converted to acetate and propionate as end products. Acetate and propionate are desirable as end products to produce biodegradable deicer products CMA, CMP, KA, and KP.

(3) FIG. 2b is a process schematic for the conversion of glucose as substrate to lactate, acetate, ethanol, and propylene glycol, and further conversion of these products to acetic and propionic acids.

(4) FIG. 3 is a process flowsheet for the conversion of glucose and xylose to lactate, acetate, ethanol, and propylene glycol (PG) as products, and further fermentation of the PG and ethanol products to propionic and acetic acids. These acid products are desirable to produce CMA, CMP, KA, and KP deicing compounds. The hydrolysis of woody biomass produces glucose and xylose as the predominant sugars in the hydrolysate, and this flowsheet illustrates the combined utilization of theses sugars as substrate.

(5) FIG. 4 is a process flowsheet for the conversion of the lactose or whey containing lactose to lactate, acetate, ethanol, and propylene glycol (PG) as products, and further fermentation of the lactate, PG and ethanol products to propionic and acetic acids. These acid products are desirable to produce CMA, CMP, KA, and KP deicing compounds.

(6) FIG. 5. This graph shows fermentation process data for the conversion of xylose to lactate and acetate at pH 4.2.

(7) FIG. 6. This graph shows the conversion of glucose as substrate to lactate and acetate and small amounts of propylene glycol and ethanol with a final fermentation pH of 3.75.

(8) FIG. 7. This graph shows the conversion of a combination of glucose and xylose as substrate to give lactate, acetate as major products, and propylene glycol and ethanol as minor products. The final pH of this fermentation was less than 4.

(9) FIG. 8. This graph shows conversion of lactose to lactate, acetate, and propylene glycol as major products and ethanol as a minor product. The final pH of this fermentation was less than 4.

(10) FIG. 9. This graph shows the low pH conversion of propylene glycol from fermentation in FIG. 2 to propionate as product.

(11) FIG. 10. This graph shows conversion of lactate from FIG. 1, 2, 3 or 4 to propionate and acetate at pH ˜5.5.

(12) FIG. 11. This graph shows the conversion of xylose to propionate at pH close to 5.

(13) FIG. 12. This graph shows pertains to immobilized cell fermentation using alumina fibers at pH 4.2. The graph shows lactic acid and acetic acid productivities, and xylose conversion rates as a function of the dilution rate.

(14) FIG. 13. This figure pertains to immobilized cell fermentation using cheese cloth as the support medium for L. buchneri immobilization. The graph show fed-batch fermentation of xylose at pH ˜4.2.

(15) FIG. 14. This graphs shows the conversion of whey permeate powder by L. buchneri to propylene glycol and acetic acid as major products, and lactic acid and ethanol as minor products at pH<4.0.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(16) The present process allows the efficient production of lactic and acetic acids at high concentrations via incubation of acid-tolerant heterofermentative bacteria in a suitable nutrient medium. The bacterial species utilized, Lactobacillus buchneri, can be isolated from a variety of sources including silages, tomato pomace, cucumber pickling, ethanol production, etcetera. L. buchneri is chosen as the preferred organism due to its ability degrade a variety of sugar substrates including waste byproducts and lignocellulosic hydrolysates to acetic and lactic acids, and propylene glycol as major products depending on the sugar utilized.

(17) FIG. 1 provides a general schematic wherein a number of alternatives are provided for the feed substrate. One of the alternatives is the use of whey lactose from cheese processing or yoghurt production operations which can be pre-concentrated and fermented in the bioreactor at a pH ˜4 and a temperature of ˜34° C. Sugars such as glucose and xylose can be fed to the fermenter without pretreatment. Lignocelluosic biomass and wastes will undergo pretreatment including acid or enzymatic hydrolysis prior to being fed to the bioreactor. A cell separator is used to separate the cells from the fermentation broth, and the cells are recycled to the fermenter to maintain a high cell concentration in the reactor. The unused sugar in the fermentation broth is separated from the product and recycled. The lactic and acetic acids are separated in extractors, and the products can be concentrated to obtain lactic acid and acetic acid as products.

(18) The cell separator in FIG. 1 can be chosen from a variety of solid-liquid separation devices including a settler, centrifuge, ceramic or polymeric membrane filter, and a variety of mechanical filters including, but not limited to, belt filter, vacuum filter, and filter press.

(19) FIG. 2a is a schematic for the fermentation of xylose to lactate and acetate at pH ˜4.2 to lactic and acetic acids, separation of lactate and acetate, and the further conversion of lactate to propionate and acetate by Propionibacterium acidipropionici at pH ˜5.5. In this two-stage process, approximately 60 g of acetate and 40 g of propionate can be obtained from 100 g of xylose for use in the production of acetate and propionate deicing salts. The acetate and lactate produced in the first stage can also be separated and sold as products without further conversion by P. acidipropionici. FIG. 5 shows typical results obtained from the fermentation of xylose by L. buchneri at pH<4. The fermentation of glucose or xylose to lactate (FIG. 7) and further conversion of product lactate to propionate and acetate by P. acidipropionici is shown in FIG. 10, and also is the subject of U.S. Pat. No. 5,324,442.

(20) FIG. 2b is a schematic for the conversion of glucose to acetate and propionate by a group of bacterial species. The products of bioconversion in the first stage using L. buchneri at pH ˜4 include lactate, acetate, ethanol and propylene glycol. After separation of the lactate from the broth, the ethanol is converted using Acetobacter to acetate at pH ˜4.5. The lactate can also be further fermented using P. acidipropionici to obtain propionate and acetate. In this case, 100 g of glucose will yield approximately 50 g of acetate and 40 g of propionate. FIG. 6 shows typical results of glucose the conversion lactate, ethanol and acetate with small amounts of propylene glycol and a final fermentation pH of 3.6.

(21) FIG. 3 shows a general schematic for glucose and xylose, the predominant sugars from the hydrolysis of lignocellulosic materials, as the feedstock. In the bioconversion of mixed sugars by L. buchneri at pH ˜4, lactic acid (50%) and acetic acid (25%) are the primary products, but there is also 10% of propylene glycol, and 15% of ethanol in the product. The propylene glycol is further converted to propionate and propanol at pH ˜4 by Lactobacillus diolivorans (see FIG. 9). The ethanol is further converted to acetate by Acetobacter at pH ˜4.5. The lactate is converted to acetate and propionate by P. acidipropionici at pH ˜5.5. This process will produce 55 g of acetate and 30 g of propionate from 100 g of mixed sugar feed.

(22) FIG. 4 shows schematic conversion of lactose and whey containing lactose to lactate, acetate and propylene glycol as major products. After separation of acetate, the lactate can be further converted to propionate and acetate by P. acidipropionici at pH ˜5.5, while propylene glycol is further oxidized to propionate and propanol at pH ˜4 by Lactobacillus diolivorans (see FIG. 9). This process would yield about 39 g of acetate and 16 g of propionate from 100 g of lactose. A typical fermentation of lactose and whey is shown in FIGS. 8 and 14.

(23) The steps identified in FIGS. 2 to 4 are to maximize the yield of propionate and acetate that can be reacted with calcium, magnesium, or potassium salts to produce acetate and propionate deicing chemicals. However, if the goal is to produce lactic and acetic acids, the subsequent fermentation steps using P. acidipropionici, Acetobacter, or L. diolivorans may be omitted.

(24) The pH of the broth during fermentation can be expressed in several different ways, e.g., in terms of the average fermentation pH or the final pH. The present process is capable of producing high levels of the primary products, lactic acid and acetic acid, at an average pH no greater than 4.3. The final pH in the uncontrolled pH fermentation is a function of the initial pH and the sugar concentration in the broth. A final pH as low as 3.1 was tolerated by L. buchneri with a starting pH of 4.5 and initial mixed glucose and xylose concentration of 20 g/L. The present process allows the production of lactic and acetic acids at a pH no more than 4.3, and preferably no more than 4.0 for the complete conversion sugar substrates. The further conversion of lactate to propionate and acetate by P. acidipropionici, is accomplished at a pH no greater than 5.5. The conversion of propylene glycol to propionate, if desired, can be accomplished using L. diolivorans at pH no greater than 4.0. The conversion of ethanol in the products, if desired, can be conducted at a pH no greater than 4.5.

(25) The fermentation temperature for the acid-tolerant bacterium L. buchneri can range from 30° C. to 40° C. A temperature of incubation of 37° C. is preferred as the optimal temperature.

(26) The present fermentation process can be conducted in batch mode, fed-batch by mode by providing feed at periodic intervals, or in a continuous mode with continuous feed and withdrawal of product. In all types of fermentation operations the bacteria can be freely suspended in liquid or in immobilized form where the bacteria are attached to carriers. Immobilization can be accomplished in a variety of ways with carriers including, but not limited to, porous or nonporous surfaces including fibers and beads, calcium alginate beads, or fibrous cloth material.

(27) L. buchneri can utilize a number of carbon and energy sources for the growth and the generation of products. These include, but are not limited to, glucose, xylose, and lactose, or a combination of sugars, or sugars from the hydrolysis of starch and lignocellulosic materials. The nutrient medium required typically contains effective amounts of sources of carbon, nitrogen, phosphorus, minerals, and trace elements. Trace elements are elements needed in minute quantities for cell growth and product formation. Composition of the nutrient medium used is shown in Table 1. The nitrogen source in the medium can be replaced with other inexpensive nitrogen sources such as corn steep liquor and soy peptone. Previous studies using corn steep liquor with Lactobacillus sp., has been reported (Vadlani et al, 2008). In addition, with whey as substrate, the 6% to 13% protein present in whey can be used as a source of nitrogen.

(28) TABLE-US-00001 TABLE 1 Incubation nutrient medium Compound Concentration (g L.sup.−1) Yeast extract 5.0 Peptone 3.0 Casamino acids 2.0 Lab-Lemco Powder (oxoid) 2.0 Dipotassi um hydrogen phosphate 1.0 Ammonium dihydrogen phosphate 2.0 Sodium acetate trihydrate 1.0 Magnesium sulfate heptahydrate 0.2 Manganese(II) Sulfate Tetrahydrate 0.05 Copper sulphate hexahydrate 0.02 Tween 80 0.5 Note: The carbon source would vary according to end product requirement.

EXAMPLE 1

Batch Fermentation of Xylose by L. buchneri

(29) The fermentation studies were initially conducted with L. buchneri ATCC 4005 in batch serum bottles with a working volume of 0.05 L. The composition used was adopted from previous batch studies on L. buchneri reported in the literature (Elferink et al, 2001; Liu et al, 2008). The serum bottles were purged for 1-3 minutes with nitrogen gas of 99.95% purity, and capped using aluminum crimp caps with silicone rubber septa. The bottles were autoclaved for 15 minutes at 120° C., prior to inoculation. The inoculum size used was 3% throughout all the experiments. The serum bottles were incubated at 36° C. in an orbital shaker at 150 rpm for a period of 32-40 h.

(30) A mid log phase culture of L. buchneri from serum bottle was used as a seed inoculum for bioreactor studies. A 1.3-L fermentor with a working volume of 1 L with automated pH and temperature controller (Bioflo 115, New Brunswick Scientific Inc.) was used for free cell fermentation. Batch fermentation was conducted using modified MRS medium consisting of 2.0 g L-1 of Casamino acids, 2.0 g L-1 of Lab-Lemco Powder (oxoid), 3.0 g L-1 of peptone, 5.0 g L-1 of yeast extract, 1.0 g L-1 of sodium acetate, 0.5 g L-1 of K2HPO4, 1.5 g L-1 of NH.sub.4H.sub.2PO4, 0.2 g L-1 of MgSO4.7H2O, 0.1 g L-1 of MnSO4.4H2O, 0.05 g L-1 of CuSO4.6H2O and 0.5 mL of Tween 80. Xylose was used as the carbon source. During fermentations, the temperature was maintained at 36° C., the agitation speed was set at 150 rpm and the pH was maintained at 4.22±0.02 with addition of 10 M NaOH. Filter sterilized industrial grade N2 was sparged at the rate of 0.2 L min-1 for a short duration (<20 min) through the vessel to obtain anoxic conditions. Normal operation was conducted without N2 sparging.

(31) FIG. 5 presents the results from the batch fermentation experiments. The data indicate that L. buchneri, is able to completely convert the xylose substrate within 35 h of fermentation time. The lactic acid and acetic acid concentrations reached up to 52 and 33 g L-1, respectively, while the biomass yield reached up to 0.1 g dry cells g-1 xylose. Under uncontrolled pH conditions, fermentations have been conducted with L. buchneri up to a pH of 3.42, though the conversion rate is somewhat decreased at the lower pH.

EXAMPLE 2

Continuous Fermentation of Xylose by L. buchneri Immobilized on Inorganic Fibers

(32) A known weight of sterile alumina fibers (2% w/v) coated with chitosan was introduced in to a batch grown culture fed with 48 g L.sup.−1 of xylose. Fed batch was accomplished with addition of 25 g L.sup.−1 of xylose. The reactor was shifted to continuous mode after xylose concertation was reduced to less than 0.5%. The feed medium used for continuous immobilized cell operation consisted of 2.0 g L.sup.−1 of peptone, 3.0 g L.sup.−1 of yeast extract, 0.5 g L.sup.−1 of sodium acetate, 0.2 g L.sup.−1 of K.sub.2HPO.sub.4, 0.2 g L.sup.−1 of NH.sub.4H.sub.2PO.sub.4 and 0.2 g L.sup.−1 of MgSO.sub.4.7H.sub.2O. The feed flow rate was varied according to the run and required dilution rate was obtained using the Masterflex peristaltic pump. A sterile filter membrane cloth tied with cheesecloth was used in the suction line to prevent the loss of alumina fibers. Intermittent purging of nitrogen was done to prevent clogging at membrane. A total reaction volume of 0.9 L and pH of 4.22±0.02 was maintained throughout the fermentation. Minimal stirring (approximately 70-120 RPM) was used to prevent excessive shear disruption of the attached bacterial matrix.

(33) FIG. 12 displays the results for acid productivities and xylose conversion at various dilution rates. from continuous fermentation with cells immobilized on inorganic fibers. The data indicate that a high lactic acid productivity of 2.8 g L.sup.−1 h.sup.−1 for lactic acid and 1.7 g L.sup.−1 h.sup.−1 for acetic acid with 96% xylose conversion can be obtained immobilized cell fermentation at a dilution rate of 0.12 h.sup.−1. High productivities up to 6 g L.sup.−1 h.sup.−1 of lactic acid and 4.5 g L-1 h-1 of acetic acid could be achieved using immobilized fermentation at a dilution rate of 0.5 h.sup.−1. Though the xylose utilization at this dilution rate is less at 60%, the unutilized sugar can be recycled after acid separation as shown in the process flow sheet in FIG. 1.

EXAMPLE 3

Fed-Batch Fermentation of Xylose by L. buchneri Immobilized on Cheese Cloth

(34) Approximately 1.1 m.sup.2 of cheese cloth was wound around the propeller blades, sparging tube, and the sampling tube of the 2 L bioreactor. The reactor was charged with sterile medium, and inoculated with L. buchneri cells. The total working volume was 1.8 L. The fermentation pH was 4.2. The bacteria completely immobilized the cloth matrix resulting in a more clear fermentation broth, and eliminating the need for expensive filtration/clarification steps downstream. The stirring rate was reduced to 70 RPM or less to minimize shear and cell detachment. The results shown in FIG. 13 indicate that L. buchneri cells immobilized on cheese cloth tolerate high lactic and acetic acid concentrations of ˜60 g/L and 40 g/L respectively at a pH of 4.2. These high concentrations will reduce downstream processing costs in the separation and concentration of the acids.

EXAMPLE 4

Batch Fermentation of Whey and Lactose by L. buchneri

(35) A concentrated seed culture of L. buchneri grown in 2% glucose, 2% xylose and 0.5% lactose was used as inoculum for batch fermentation of lactose. The lactose fermentation was carried out in a fermenter with a working volume of 1.0 L at an initial pH of 5.0 and the pH was allowed to drop gradually via fermentation to 4.25 and maintained at 4.25 thereafter by the addition of 10 M NaOH. Fermentation temperature was 36° C., and the impeller speed was 150 rpm. The nutrient media composition is similar to that outlined in Table 1, where lactose was used as a major carbon source supplemented with 0.3% of each of glucose and xylose. Filter-sterilized industrial grade N.sub.2 was sparged at the rate of 0.2 L min.sup.−1 for a short duration (≤20 min) through the vessel initially to obtain anoxic conditions. Further operation was conducted without N.sub.2 sparging. The fermentation with lactose of initial concentration 60 g L.sup.−, resulted in 20 g L.sup.−1 of propylene glycol, 18 g L.sup.−1 of acetic acid, 10 g L.sup.−1 of lactic acid and 2.5 g L.sup.−1 of ethanol (FIG. 8).

EXAMPLE 5

Batch Fermentation of Whey Permeate and Whey Permeate Powder by L. buchneri

(36) Fermentation of whey was carried out in 0.05 L serum bottles with initial pH at 5.0. The whey permeate obtained from Kansas State University dairy cheese processing operation was heat treated, centrifuged, and filtered prior to use. Whey permeate powder from Foremost Farms, USA was mixed with warm water to obtain lactose concentration equal to 5.3%. The constituents of whey permeate powder can be obtained at www.formostfarms.com. Nutrient media used for the study was as outlined in Table 1. In either case the nutrient constituents were dissolved in clarified whey permeate solution. The bottle headspace were purged with N.sub.2 for 1 to 3 minutes, and sealed using aluminum crimp cap and silicone rubber lined septa. The setup was sterilized at 121° C. prior to inoculation. A concentrated seed culture of L. buchneri grown in 2% glucose, 2% xylose and 0.5% lactose was used as the inoculum for batch fermentation of whey permeate. The fermentation was carried out at 36° C. and mixer speed at 150 rpm. The final pH of fermentation was between 3.5 and 4.0. Table 2 lists the product composition for whey permeate and whey permeate powder fermentation at 96 h. The data show that L. buchneri can utilize the lactose in whey permeate or whey permeate powder to produce lactate, acetate and propylene glycol.

(37) TABLE-US-00002 TABLE 2 Analysis of broth after 96 h fermentation of whey permeate and whey powder from by L. buchneri, ATCC 4055 Whey permeate from cheese Whey permeate powder production: 5.3% lactose; obtained from Foremost 0.25% lactate Farms*: 5.3% lactose Final media Concentration Final media Concentration composition g/L composition g/L Lactose 2.4 Lactose 5.3 Lactate 9.5 Lactate 9.1 Acetate 12.5 Acetate 12.6 PG 13.4 PG 14.2 EtOH 3.2 EtOH 0 *www.foremostfarms.com

(38) Batch fermentation of whey powder was carried out in a fermenter with a working volume of 1.0 L at an initial pH of 5.0. The pH was allowed to drop gradually via fermentation to 4.25 and maintained at 4.25 thereafter with addition of 10 M NaOH. Fermentation temperature was 36° C., and the impeller speed was 150 rpm. The nutrient media composition is similar to that outlined in Table 1, where whey powder containing lactose was used as a major carbon source supplemented with 0.3% of each glucose and xylose. Filter sterilized industrial grade N.sub.2 was sparged at the rate of 0.2 L min.sup.−1 for a short duration (≤20 min) through the vessel initially to obtain anoxic conditions. Further operation was conducted without N.sub.2 sparging. FIG. 14 shows that with whey powder, the product concentrations obtained from 50 g/L lactose feed concentration were 15.5 g L.sup.−1 propylene glycol, 13.5 g L.sup.−1 of acetate, and 2.7 g L.sup.−1 of ethanol.