Riboflavin (B2) Same 75% (nonrenewables) 50% 66% 50%
7-Aminocephalosporic acid — — 90% 33% –90% (environment-related)
Cephalexin Electricity þ,
steam — 80% 80% Considerable reduction
Amino acids Same — — 43%
Acrylamide –80% — Down Down Down
Acrylic acid — Down Down Down 54% (raw materials)
Enzyme-catalyzed synthesis of polyesters — — Down Down Down
Vegetable oil degumming enzyme 70% — — 80% 40%
enzymatic pulp bleaching 30–40% Down — Down
On-site production of xylanase — 35% (Cl2),65% (ClO2) — Down
Gypsum-free zinc refinery — Down (recycle) — Down
Source:ref. 3.
217
adapted from the report and contains a summary of some of the environmental and cost benefits identified for some of the case studies.
In the following paragraphs we discuss briefly some of the current industrial applications of bioprocesses.
8.4.1 Pharmaceuticals
The classic example of small-molecule pharmaceuticals produced through bioprocessing is found in antibiotics such as penicillins or cephalosporins. Of equal importance is the production of larger or more chemically complex molecules such as insulin or taxol, or the recent development and use of monoclonal antibody therapies. The success in producing these products has led to a dramatic increase in the life expectancy of many while contributing to a generally higher quality of life for those living with chronic diseases.
Henderson et al.4have recently reported a comparison of a chemical synthetic route with a route that employs biocatalysis to a cephalosporin precursor known as 7-aminocephalos- poric acid (7-ACA).
Example 8.1 Compare the chemical and biocatalytic routes to 7-ACA. Which route is greener, and why? Explain your answer.
Chemical route description. A four-step process is used to convert the potassium salt of cephalosporin C to 7-ACA, as shown in Figure 8.2(a). In the first step, a common protection strategy is used to convert the acid to an anhydride and the amine to an amide using chloracetyl chloride in the presence of the base dimethyl aniline. Next, phosphorous pentachloride is added to the mixed anhydride, which is held at37C to form the imodyl chloride, which is followed sequentially by the addition of methanol to form the transient imodyl ether and then water to form 7-ACA. 7-ACA is precipitated by using ammonia to change the pH to the isoelectric point and the 7-ACA is recovered methanol wet and then dried under vacuum.
Biocatalytic route. A three-step process is used to convert the potassium salt of cephalosporin C to 7-ACA, as shown in Figure 8.2(b). A solution of cephalosporin C is stirred with the immobilized biocatalyst D-amino acid oxidase (DAO), while air is bubbled through the solution to supply the required oxygen. The by-product of the bioconversion, hydrogen peroxide, reacts spontaneously with the keto intermediate to give glutaryl 7-ACA. The reaction is carried out at a constant temperature (18C) and elevated pressure (5 bar) under controlled pH (starting at pH 7.3 and rising to 7.7 at completion) to ensure the desired conversion. Additional hydrogen peroxide may be added to promote greater conversion to glutaryl 7-ACA if desired. Upon completion, the solution containing glutaryl 7-ACA is separated from DAO, and immobilized glutaryl 7-ACA acyclase (GAC) is added at a pH of 8.4 and a temperature of 14C to obtain the desired 7-ACA. Dilution may be required to control the concentration, but upon completion of the reaction, the 7- ACA is separated from GAC and isolated. In both cases, the enzymes may be recovered and reused.
Solution Table 8.7 compares the chemical and biocatalytic processes from the work of Henderson et al.
S N
CO2COCH2Cl O
CH3 O O
N H
O ClCH2CO2CO
NH O Cl
S N
CO2 O
CH3 O O
H3N
- S N
CO2COCH2Cl O
CH3 O O
N Cl ClCH2CO2CO
N Cl Cl
S N
CO2COCH2Cl O
CH3 O O
ClCH2CO2CO N
Cl N OMe
OMe
7-ACA chloroacetyl chloride dimethyl aniline DCM
dimethyl aniline PCl3 PCl5
methanol
water ammonia methanol
CO2 O
O -
D-amino acid oxidase
S N
CO2 O
CH3 O O
NH O O2C
- -
S N
CO2 O
CH3 O O
NH O O2C
O
- -
S N
CO2 O
CH3 O O
H3N
- O2C CO2
+O2,+H2O -NH3, -H2O2
spontaneously +H2O2
-CO2, -H2O
+
glutaryl 7-ACA acylase +H2O
- -
-
alpha-ketodiapoyl-7-ACA [keto intermediate]
glutaryl 7-ACA
7-ACA
(a) (b)
FIGURE 8.2 Chemical (a) and biocatalytical (b) routes for 7-ACA.
219
TABLE 8.7 Process Comparison
Attribute Chemical Process Biocatalytic Process
Renewability Cephalosporin C salt is derived from a fermentation. Cephalosporin C salt is derived from a fermentation.
Reagents and solvents are not renewable. Enzymes can be produced from renewable feedstocks.
Toxics Dichloromethane—suspect human carcinogen.
PCl3: Reacts violently with water, very toxic by inhalation.
Highly reactive.
PCl5: Reacts violently with water, very toxic by inhalation.
Highly reactive.
Dimethyl aniline: limited evidence of carcinogenic effect, toxic by inhalation, ecotoxic effects—aquatic.
Ammonia: corrosive, toxic by inhalation, explosive.
Chemoselectivity Protection/deprotection strategy required. Chemoselective.
Process safety PCl3and PCl5: require special handling. Hydrogen peroxide is produced and may be added, although there is a very low risk associated with this.
Mass efficiency Requires about 50% of the mass of the chemical synthesis.
Energy Chemical route requires considerable chilling (to37C) to control exotherms.
Requires about 80% of the process energy of the chemical synthesis.
Complexity Batch operation with greater number of steps. Batch operation largely aqueous based with simple mixing operations.
Reagent addition must be carried out with care to avoid worker exposures and process safety risks.
than the chemical route, so we could say that in this case the biocatalytic route is greener than the chemical route.
Additional Points to Ponder Given what you know to this point in the book, what can you say about the greenness of bioprocesses? What are the potential advantages? What are the potential drawbacks?
8.4.2 Biofuels
Given the recent volatility in petroleum availability and pricing, combined with the increased and steadily increasing concern about greenhouse gases leading to climate change, there has been considerable interest in the development of biofuels. Bioprocesses for the production of ethanol, butanol, biodiesel, and related products have been extensively reported in the literature, and bioderived ethanol is now a major business in the United States with an annual capacity exceeding 50 million gallons. In Brazil, ethanol derived from sugarcane is a very significant product. There is considerable controversy surrounding biofuel production and its impact on the environment and its societal impact, especially at the interface for its perceived competition with arable land and food production.5,6There are also major concerns about the use of marginal land and the continued influx of fertilizers, herbicides, and pesticides into the environment. This is discussed in more detail in Chapter 23.
8.4.3 Plastics
There has been considerable effort in recent years to develop polymers whose monomers are obtained from renewable resources. The following example discusses the production of poly(lactic acid) (PLA) from corn-derived glucose.6
Example 8.2 PLA is not a new polymer and was originally worked on by the pioneering DuPont polymer chemist Carothers in the 1930s.7Petrochemically derived lactic acid is generally undesirable because it is produced as a racemic mixture of theD- andL-isomers, which leads to an amorphous PLA. A simplified diagram of the petrochemical process for lactic acid is shown in Figure 8.3. However, by the 1980s, fermentation of corn-derived glucose was seen as a possible source of obtaining almost pureL-lactic acid and ultimately was first utilized in a joint venture between Dow and Cargill to provide high-molecular- weight PLAs. Cargill uses a solvent-free process that begins by taking lactic acid from its fermentation process followed by polymerization to a low-molecular-weight polymer. The
Petrochemical Feedstock
Ethylene
Production Acetaldehyde
Lactonitrile Racemic
Di-lactic acid Amorphous
Polylactic acid
oxidation
HCN
FIGURE 8.3 Petrochemical process for polylactic acid.
polymer undergoes a controlled depolymerization to produce a cyclic dimer known as the lactide, which is maintained as a liquid and distilled to increase its purity.8Catalytic ring opening of the lactide will produce a range of PLAs with controlled molecular weights. The entire process from the raw lactic acid to the production of the high-molecular-weight PLAs is continuous, with no need to separate the intermediate lactide. Figure 8.4 is a block diagram of PLA production.
Briefly describe the benefits of the PLA process developed by Cargill from a green chemistry and engineering perspective.
Solution The range of benefits of the Cargill PLA process are summarized in Table 8.8.
Additional Point to Ponder What other environmental impacts would the fermentation process for PLA have?
8.4.4 Biocatalysts
We learned in Chapter 6 that biocatalysts are beginning to be used extensively in a number of applications in chemical, pharmaceutical, and other parts of the chemical manufacturing enterprise. Table 8.9 is similar to Tables 8.3 and 8.4, but is specific to biocatalyst production, and Table 8.10 presents some advantages and disadvantages of biocatalysts. Biocatalysts are an extremely important part of the evolution toward a more sustainable chemical enterprise. In the first place, they are usually extremely chemo- selective and especially useful for performing specific stereochemical or regiochemical syntheses. Second, they may be derived from renewable feedstocks, which at this point may or may not also be sustainable. Third, they hold the greatest promise for getting away from certain transition metal and platinum group metal catalysts that are both toxic and in limited supply. From a green chemistry viewpoint, in general biocatalysts have the advantages of operating in aqueous media, carrying out reactions at moderate temperatures, and having very good regio-, chemo-, and stereoselectivity. On the other
Lactic acid
Low-m.w. PLA
Lactide
High-m.w. PLA Polymerization
Depolymerization
Catalytic polymerization
Separation by continuous distillation OH
CH3 H O H
O O
O O
CH3
C H3
O
* O
* H CH3
O n
H2O
Lactic acid Lactide Poly(3,6-dimethyl-1,4-dioxan-2,5-dione)
FIGURE 8.4 Polylactic acid production.
hand, they have challenges on their operational stability and normally a very low volumetric productivity.
Example 8.3 Pfizer’s Lyrica (Pregabalin) The initial chemical synthesis of Pfizer’s Lyrica (pregabalin) is as follows:
CHO NH2
CO2H
NH2 CO2H (S)-mandelic acid
29% overall
>99% enantiomeric excess (e.e.) 3 steps
The original chemical synthesis1began with isovaleraldehyde and diethyl malonate and in three steps arrived at the desired intermediate, (þ)-3-(aminomethyl)-5-methylhexanoic acid. A classic resolution requiring stoichiometric addition of (S)-mandelic acid was used to obtain the desired (S)-3-(aminomethyl)-5-methylhexanoic acid, but the undesired enantiomer was difficult to recycle and over 75% of the input material became waste.
The overall yield was low, as was the throughput, and the high cost to make this was undesirable.
Because of the undesirable aspects of the chemical synthesis, the development of a biotransformation route was undertaken.2Racemic 2-carbethoxy-3-cyano-5-methylhexa- noic acid, ethyl ester (CNDE), an intermediate in the isovaleraldehyde process, was mixed with a lipase to obtain the desired isomer, the (3S)-3-cyano-5-methyl hexanoic
Attribute Petrochemical Process Cargill Process
Renewability Fossil fuel (petroleum): No Glucose (corn-based but could be derived from other plants, e.g., sugarcane)
Toxics Ethylene oxide: human carcinogen Acetaldehyde: Potential human
mutagen and carcinogen; possible sensitization; severe eye irritant HCN: acutely toxic
Chemoselectivity Racemic mixture: loss of unwanted isomer or production of inferior amorphous plastic
L-Lactic acid
Process safety Ethylene: extremely flammable Ethylene oxide: explosive HCN: extremely flammable Energy Range of 79–140 MJ/kg energy for a
range of petrochemically derived plastics such as nylon 66, PET, PP, HDPE, and PE
First-generation PLA used about 57 MJ/kg energy
Complexity Multiple unit operations of higher hazard, storage of toxics, etc.
Fewer unit operations.
TABLE 8.9 Characteristics of Biocatalysts Biocatalyst
Production
Device Raw Material Time Scale Purification Examplesa
Enzymes Bioreactor Pure substrates Short Simple Cyclodextrin, acrylamide,L-dopa
Bacteria and yeasts Bioreactor Simple media Short Medium Lysine, vitamin B2, insulin
Fungi Bioreactor Simple media Medium Medium Citric acid, antibiotics
Mammalian cells Bioreactor Complex media Medium Medium Monoclonal antibodies, interferons
Plant cells Bioreactor Simple media Medium Medium Taxol, shikonin, methyldigoxin
Transgenic plants Bioreactor Fertilizer, CO2, various others
Long Complex Antibodies, antibody fragments, HSA, PHB Transgenic animals Whole plant Various plants and
animal materials
Long Complex a1-Antitrypsin, HSA, lactoferrin Extractive technology Whole animal Certain parts of plants,
animals, and humans
Long Complex Plasma components, taxol Source:ref. 1.
aHSA, human serum albumin; PHB, poly(3-hydroxybutyrate).
acid, and in an additional step, taken to the desired (3S)-3-cyano-5-methylhexanoic acid, ethyl ester [(S)-CNE]. Reduction of the cyano group was accomplished with hydrogen in the presence of a spongy nickel catalyst to obtain the desired product, pregabalin:
EtO2C CO2Et CN
H2O
EtO2C CO2Et CN
R-1, 85% ee
–O2C CN CO2Et
H2O CN
CO2Et H2, Ni
H2O CO2H
NH2 lipolase
+
racemic CNDE 1
NaOEt, 100%
>98 % ee @ 45% conversion Step 1
765 g/L of total volume
recycling of R-1
(S)-CNDE acid Step 2 (S)-CNE 85–90%
Pregabalin Step 3
90–95%
99% purity, >99.7% ee overall 40–45% yields after one recycling (3.25 kg/L of H2O)
This synthesis resulted in the elimination of 11 million gallons of solvent, better solvent profile use (see Figure 8.5), a reduction in CNDE use of more than 800 metric tons, and elimination of 1600 metric tons of (S)-madelic acid and 500 metric tons of nickel.
Additional Points to Ponder On the basis of Table 8.2 and Example 8.3, could you outline the disadvantages of the process described in Example 8.3 that are not mentioned? Do you think the process is green? How could you improve it?
Type of
Biocatalyst Advantages Disadvantages
Whole cells General No need for cofactor recycling
Low substrate concentration High biomass production Low specificity
Unstable at high temperatures and low pH
Fermented High activities High biomass production More by-products Fermented Easier to process Low activities Immobilized Ease of separation Low activities
Ease of purification
Isolated enzymes General Very high selectivities Requires cofactor recycling Ease of use
Simple processing High substrate
concentrations
Accesible for only few reactions
Instable at high temperaturas and low pH
Immobilized High selectivity Ease of separation Ease of purification
Loss of activity during immobilization
PROBLEMS
8.1 Describe several examples of the beneficial use of bioprocessing not mentioned in Section 8.1.
8.2 Look at the applications in Table 8.1. Imagine that you are a policymaker and want to write legislation that would spur on particular applications of bioprocessing. Which applications would you choose to focus on? Defend your answer.
8.3 Look at the production volumes in Table 8.2. Which of these applications would you expect to grow? Would you expect any of these to decrease? What basic chemicals do you think might be useful targets for bioprocessing, and why?
8.4 Tables 8.4 and 8.5 list common carbon and nitrogen sources and cite a few general environmental issues associated with agriculture. Investigate and describe some of these potential impacts. How might these impacts be reduced through further improvements in bioprocessing?
8.5 Choose from the literature an application of an enzyme that is replacing a chemical synthesis. Evaluate its greenness. If you were a scientific director for a company, would you invest in developing the proposed replacement?
8.6 Adipic acid is a monomer produced in large quantities for the production of nylon 66 and polyurethane. We will look at three different routes to adipic acid.
Traditional chemical synthesis:
O OH
HO2C
CO2H
Ni — Al2O3 Co — O2 HNO3
+
370–800 psi 120–140 psi
benzene cyclohexane cyclohexanone cyclohexanol
adipic acid
Draths–Frost biotechnical synthesis:11
HO2C
CO2H O
O
O O O
O
CO2H
O OH
OH
HO2C
CO2H
Pt, H2
D-glucose 3-dehydroshikimate cis-cis-muconic acid adipic acid
E. coli E. coli
50 psi
Water IPA Toluene
Water THF MeOH EtOH IPA
(a) (b)
FIGURE 8.5 Solvent use of the chemical (a) and biocatalytic (b) routes to Pfizer’s Lyrica (pregabalin).
10 g Bacto tryptone 1 g NH4Cl Yieldẳ20.4 mmol 5 g Bacto yeast 10 g glucose (62 mmol) % Yieldẳ33%
10.5 g NaCl 0.12 g MgSO4 6 g Na2HPO4 1 mg thiamine 3 g KH2PO4
Synthesis by Kazuhiko et al.9:
HO2C
CO2H
+ 4H2O2
Na2WO4[CH3(n-C8H17)3N]HSO4
+ 4H2O
cyclohexene adipic acid
A typical procedure for the oxidation and reuse of the water phase proceeds as follows. In the first run, a 1-L round-bottomed flask equipped with a magnetic stirring bar and a reflux condenser was charged with 4.1 g (12.2 mmol) of Na2WO42H2O, 5.67 g (12.2 mmol) of [CH3(n-C8H17)3N]HSO4 (used as a phase-transfer catalyst), and 607 g (5.355 mol) of aqueous 30% H2O2. The mixture was stirred vigorously at room temperature for 10 min and 100 g (1.217 mol) of cyclohexene was added. The biphasic mixture was heated successively at 75C for 30 min, at 80C for 30 min, at 85C for 30 min, and at 90C for 6.5 h, with stirring at 1000 rpm. The homogeneous solution was allowed to stand at 0C for 12 h, and the resulting white precipitate was separated by filtration and washed with 20 mL of cold water. The product was dried in a vacuum to produce 138 g (78% yield) of adipic acid as a white solid (with a melting point of 151.0 to 152.0C). A satisfactory elemental analysis was obtained without further purification. Con- centration of the mother liquor produced 23 g of pure adipic acid; the yield determined by GC (OV-1 column, 0.25 mm50 m, GL Sciences, Tokyo) was 93%. The by-products identified were 1,2-cyclohexanediol (2% yield) and glutaric acid (4% yield). In the second run, a 2-l round-bottomed flask was charged with the water phase of the first run, which contained the W catalyst, 5.67 g (12.2 mmol) of [CH3(n-C8H17)3N]HSO4, and 552 g (4.868 mol) of aqueous 30% H2O2. After the mixture was stirred vigorously at room temperature for 10 min, 100 g (1.217 mol) of cyclohexene was added. This mixture was heated successively at 75C for 30 min, at 80C for 30 min, at 85C for 30 min, and at 90C for 46.5 h, with stirring at 1000 rpm; the homogeneous solution was allowed to stand at 0C for 12 h. The resulting white precipitate was separated, washed, and dried in a vacuum to produce 138 g (78% yield) of analytically pure adipic acid as a white solid.
(a) Which of the three syntheses would be the greenest? Explain your answer.
(b) Which synthesis is more mass efficient?
(c) Assess the recycle and reuse strategy in the synthesis by Kazuhiko et al. Do you think this would be commercially viable? Defend your answer.
(d) Which of these syntheses would have a greater environmental impact? Why?
8.7 A pharmaceutically important building block, pyruvic acid (2-oxopropanoic acid CAS No. 127-17-3), may be produced through a fermentation from glucose usingEscher- ichia coli.1,4This is an important route, as traditionally the process used to produce pyruvic acid is via pyrolysis of tartaric acid and possesses a number of economic and
environmental drawbacks. There are two alternatives for downstream processing: a solvent-based process, and recovery of product by electrodialysis. Tables P8.7A and P8.7B contain a mass balance and energy use for the downstream process.
(a) What would lead to the large differences in energy use between the two options?
(b) What would lead to a greater quantity of solvent and water being used for the extraction process?
(c) Based on these two tables, which downstream processing alternative is best, and why?
8.8 Citric acid is one of the few commodity chemicals produced by bioprocessing with a worldwide production on the order of 1.1 million tons. It has been produced for over 80 years, predominantly using the filamentous fungusAspergillus niger.1Biwer et al.
describe a process that uses starch as a feedstock instead of glucose, the starting material normally used.10,11 Starch is a mixture of two different polymers of glycopyranose (amylose and amylopectin), whose only building block is glucose (C6H12O6), linked by predominantly bya-1,4-glycosidic bonds. The basic process is depicted in Figure P8.8(a).
TABLE P8.7A Mass Balance
Input (kg/kg) Output (kg/kg)
Component Extraction Electrodialysis Extraction Electrodialysis
Acetic acid 0.09 0.09
Ammonium 0.03 0.03
Ammonium sulfate 0.19 0.19 0.18 0.18
Biomass <0.01 <0.01 0.09 0.09
Carbon dioxide — — 0.05 0.05
Glucose 1.19 1.18 0.10 0.10
Hydrogen chloride 0.62 <0.01
Solvent 1 0.31 — 0.31
Product loss — — 0.08 0.08
Other organic material — — 0.15 0.15
Oxygen 0.19 0.18
Salts
Inorganic — — 0.75 0.32
Mineral 0.14 0.14
Sodium hydroxide 0.37 0.38
Water 46.9 32.3 47.3 32.7
Mass Intensity 52.6 34.5 51.6 33.5
Without water 3.1 2.2 1.7 0.8
TABLE P8.7B Energy Use
Energy Use Extraction Electrodialysis
Process energy use (kWh/kg) 1.9 2.4
Steam (kg/kg) 50 15
Cooling (kg/kg) 265 50
Chilled water (m3/kg) 2.7 1.5
(a) What would be the advantage of using starch instead of glucose or molasses as a starting material?
(b) What other components should be added to the flow diagram to account more accurately for what is added and taken from the process?
(c) In the flow diagram in Figure P8.8(b), what might the missing unit operations be?
(d) How would you change the process to make it greener?
(e) The material balance for the citric acid process is as shown in Table P8.8. For every 100 kg of starch, the bioreaction yield is 84% (kg/kg) and the downstream processing yield is 94%. What is the overall yield?
(f) The authors report that 12,630 tons of citric acid monohydrate is produced over 330 days in 565 batches. What is the total CO2produced in a year from the bioreaction?What would be the composition of the aqueous waste?
(g) What would happen to the waste biomass? Outline some of the potential disposal options and the pros and cons of each.
Starch Dextrin Glucose
Glycolysis Pyruvate
Tricarboxylic Acid Cycle
Citric Acid
(a) Starch Hydrolysis
Ultrafiltration
Decolorization
Vacuum Filtration
(b) FIGURE P8.8