Pathway Design for Industrial Fermentation E-Book

Walter Koch

• Herausgeber: Wiley-VCH GmbH
• Kategorie: Fachliteratur
• Sprache: Englisch

Seitenzahl: 1004

Veröffentlichungsjahr: 2023

Beschreibung

Pathway Design for Industrial Fermentation

Explore the industrial fermentation processes of chemical intermediates

In Pathway Design for Industrial Fermentation, distinguished researcher Dr. Walter Koch delivers an expert overview on industrial fermentation production technology as compared with natural extraction, organic chemistry, and biocatalysis. The book offers key insights for professionals designing and monitoring fermentation processes.

The author explores the applications, alternative production, biochemical pathways, metabolic engineering strategy, and downstream processing of various products—including C1 to C6 products—with a focus on low-value products with market prices below 4€ per kilogram. Products will include methane, ethane, acetate, lactic acid, alanine, and others.

With specific commentary and insightful perspectives on the cost drivers and technological aspects critical to commercially successful applications, the book also includes:

• Thorough introductions to methane, ethanol, acetate, lactic acid, alanine, and 3-Hydroxypropionic acid
• Comprehensive explorations of 1,3-Propanediol, butanol, isobutanol, and isobutene
• Practical discussions of 1,4-butanediol, succinic acid, itaconic acid, and glutamic acid
• Fulsome treatments of isoprene, pentamethylenediamine, lysine, citric acid, and adipic acid

Perfect for process engineers, biotechnologists, and chemical engineers, Pathway Design for Industrial Fermentation will also benefit biochemists and professionals working in the chemical and food industries.

Leseprobe

Table of Contents

Cover

Table of Contents

Title Page

Copyright

Preface

Introduction

Fermentation as Production Technology

Rising Interest in Fermentation as Production Technology

Technical Comparison of Fermentation Versus Organic Chemistry

References

1 Methane

1.1 Application

1.2 Conventional Production of Methane

1.3 Carbon Dioxide as Feedstock

1.4 Conversion of Carbon Dioxide into Methane

1.5 Biochemical Pathway Design

1.6 Integration of Hydrogen Production and the Biochemical Methanation

1.7 Process Development for the “Biochemical Sabatier” without Integrated Water Electrolysis

1.8 Commercial Application of Fermentative Methane Production

References

2 Ethanol Ex Glucose

2.1 Application

2.2 Production of Ethanol

2.3 Pathway Design

2.4 Process Development

2.5 Alternative Raw Material Source

2.6 Industrial Production and Capacity

References

3 Acetate and Ethanol Ex CO/H

2

3.1 The Wood–Ljungdahl Pathway

3.2 Formation of Acetate in

A. woodii

Based on Carbon Dioxide and Hydrogen

3.3 Formation of Acetate in

A. woodii

Based on Carbon Monoxide

3.4 Formation of Ethanol in

A. woodii

Based on Carbon Dioxide and Hydrogen without AOR

3.5 Formation of Ethanol in

A. woodii

Based on Carbon Dioxide and Hydrogen with AOR

3.6 Formation of Ethanol in

C. woodii

Based on Carbon Monoxide

3.7 Formation of Acetate in

C. autoethanogenum

Based on Carbon Dioxide and Hydrogen

3.8 Formation of Ethanol in

C. autoethanogenum

Based on Carbon Dioxide and Hydrogen

3.9 Industrial Fermentation and Capacity

References

4 Lactic Acid

4.1 Application

4.2 Chemical Synthesis of Lactic Acid

4.3 Pathway Design

4.4 Process Development

4.5 Evaluation of Alternative Feedstocks

4.6 Production Cost and Market Price

4.7 Industrial Application and Capacity

References

5 Alanine

5.1 Application

5.2 Chemical Production of L‐alanine

5.3 Pathway Design

5.4 Metabolic Engineering

5.5 Industrial Production and Application

References

6 3‐Hydroxypropionic Acid

6.1 Application

6.2 Chemical Synthesis

6.3 Pathway Design

6.4 Industrial Application

References

7 1,3‐Propanediol

7.1 Application

7.2 Alternative Production of 1,3‐Propanediol

7.3 Pathway Design Toward 1,3‐Propanediol

7.4 Metabolic Engineering

7.5 Process Development

7.6 Industrial Application and Capacity

References

8 Butanol

8.1 Application

8.2 Conventional Production of Butanol

8.3 Pathway Design Based on Glucose

8.4 Pathway Design Based on Carbon Dioxide, Carbon Monoxide and Hydrogen

8.5 Process Development for Fermentative Butanol

8.6 Alternative Raw Material Sources

8.7 Industrial Application

References

9 Isobutanol

9.1 Application

9.2 Conventional Synthesis of Isobutanol

9.3 Metabolic Engineering

9.4 Process Development

9.5 Industrial Application

References

10 Isobutene

10.1 Application

10.2 Conventional Synthesis

10.3 Pathway Design Toward Isobutene

10.4 Carbon Yield and Carbon Footprint

10.5 Industrial Fermentation and Capacity

References

11 1,4‐Butanediol

11.1 Application

11.2 Conventional Synthesis of 1,4‐Butanediol

11.3 Pathway Design

11.4 Process Design for Fermentative 1,4‐Butanediol Based on Glucose

11.5 1,4‐Butanediol Derived by Chemical Hydrogenation of Succinic Acid

11.6 Alternative Carbon and Energy Source for Fermentation

11.7 Industrial Application and Capacity

References

12 Succinic Acid

12.1 Application

12.2 Conventional Synthesis of Succinic Acid

12.3 Pathway Design and Metabolic Engineering

12.4 Production Host

12.5 Reactor Concepts

12.6 Downstream Processing

12.7 Industrial Capacity and Performance

References

13 Itaconic Acid

13.1 Application

13.2 Metabolic Engineering

13.3 Process Design

13.4 Industrial Application and Capacity

References

14 Glutamic Acid

14.1 Application

14.2 Native Biochemical Pathway

14.3 Metabolic Engineering

14.4 Process Development and Industrial Application

References

15 Isoprene

15.1 Application

15.2 Chemical Synthesis

15.3 Pathway Design

15.4 Metabolic Engineering Toward Isoprene

15.5 Metabolic Engineering Toward Mevalonate

15.6 Downstream Processing

15.7 Industrial Application and Capacity

References

16 Pentamethylenediamine

16.1 Application

16.2 Chemical Synthesis

16.3 Pathway Design

16.4 Metabolic Engineering

16.5 Downstream Processing

16.6 Industrial Application

References

17 Lysine

17.1 Application

17.2 Chemical Production

17.3 Metabolic Pathway via DAP and Metabolic Engineering

17.4 Metabolic Pathway via α‐Aminoadipate in Fungi

17.5 Secretion of Lysine

17.6 Process Development

17.7 Industrial Application

References

18 Citric Acid

18.1 Application

18.2 Chemical Production and Natural Extraction

18.3 Biochemical Pathway

18.4 Process Development

18.5 Industrial Production

References

19 Adipic Acid

19.1 Application

19.2 Chemical Production of Adipic Acid

19.3 Metabolic Engineering for Fermentation

19.4 Digression: Metabolic Engineering for C6+ Diacids

19.5 Process Development

19.6 Industrial Application and Capacity

References

20 Hexamethylenediamine

20.1 Application

20.2 Chemical Production of HMD

20.3 Metabolic Engineering for Fermentation Technology

20.4 Biocatalytic Routes Towards HMD

20.5 Process Design

20.6 Commercial Application

References

21 Caprolactam and 6‐Aminocaproic Acid

21.1 Application

21.2 Chemical Production of CPL

21.3 Metabolic Engineering for Fermentation Technology via Adipyl‐CoA

21.4 Industrial Application

References

22 Anthranilic Acid and Aniline

22.1 Application

22.2 Pathway Design

22.3 Metabolic Engineering for Anthranilate as Fermentation Product

22.4 Derivatives of Anthranilate as Fermentation Product

22.5 Alternative Fermentation Precursors for Aniline

22.6 Process Development with Focus on Product Isolation

22.7 Industrial Fermentation

References

23 Farnesene

23.1 Application

23.2 Chemical Production

23.3 Biochemical Pathway

23.4 Metabolic Engineering

23.5 Process Design with Second Liquid Phase

23.6 Industrial Application

References

Index

End User License Agreement

List of Tables

Introduction

Table 1 Comparison of production technologies for chemical molecules.

Table 2 Maximum glucose uptake (

q

S

) rates for selected hosts.

Table 3 Very high productivity values achieved in lab‐scale.

Table 4 Process analysis for the fermentative production of ethanol and lact...

Table 5 Electron balance in glucose‐based fermentation based on the electron...

Table 6 Reduction potentials in biochemical systems. All values are defined ...

Table 7 Deduction of total energy equivalents in the aerobic metabolism of g...

Chapter 1

Table 1.1 Comparison of chemical and biological CO

2

methanation.

Table 1.2 Biochemical pathway for the generation of methane.

Table 1.3 Generation of methane coupled with the electrolysis of water.

Chapter 2

Table 2.1 G6P, Glucose‐6‐phosphate; F6P, fructose‐6‐phosphate; FBP, fructos...

Table 2.2 Biochemical conversion of glucose into ethanol.

Table 2.3 Reduction of acetaldehye to ethanol.

Table 2.4 Biochemical conversion of glucose into ethanol and formic acid.

Table 2.5 Biochemical conversion of ethanol into acetaldehyde and hydrogen....

Table 2.6 Global grain production and consumption.

Table 2.7 Technology for the digestion of lignocellulosic biomass.

Table 2.8 Conversion of D‐xylose into ethanol.

Table 2.9 Composition of lignocellulosic biomass and conversion into ethano...

Table 2.10 Feedstock, fermentable sugar, and ethanol G1/G2 production cost ...

Table 2.11 Consumption of ethanol 2020 and 2028.

Table 2.12 Planned capacity for plants using lignocellulosic biomass.

Table 2.13 Composition of wheat straw and conversion into ethanol.

Chapter 3

Table 3.1 Metabolic pathway toward acetate based on CO

2

and H

2.

Table 3.2 Metabolic pathway toward acetate based on CO

2

and H

2

in

A. woodii

Table 3.3 Hypothetical metabolic pathway toward ethanol based on CO

2

and H

2

Table 3.4 Potential metabolic pathway toward ethanol based on CO

2

and H

2

in...

Table 3.5 Metabolic pathway toward ethanol based on CO/CO

2

and H

2

in

A. woo

...

Table 3.6 Metabolic pathway toward acetate based on CO/CO

2

and H

2

in

C. aut

...

Table 3.7 Metabolic pathway toward ethanol based on CO/CO

2

and H

2

in

C. aut

...

Table 3.8 Metabolic pathway toward ethanol based on CO/CO

2

and H

2

in

C. aut

...

Chapter 4

Table 4.1 Biochemical conversion of glucose into lactic acid.

Table 4.2 Gibbs free energy for the hydrogenation of pyruvate.

Table 4.3 Biochemical conversion of glucose into lactic acid and ethanol.

Table 4.4 pH management with organic salt.

Chapter 5

Table 5.1 Alanine via direct amination.

Table 5.2 Kinetic data for the applied enzymes.

Chapter 6

Table 6.1 Synthesis pathway via malonyl‐CoA.

Table 6.2 Synthesis of 3‐hydroxypropionic acid via acetate and malonyl‐CoA....

Table 6.3 Synthesis via α‐alanine and β‐alanine.

Table 6.4 Synthesis of 3‐hydroxypropionic acid based on glycerol.

Chapter 7

Table 7.1 Chemical conversion of glucose into 1,3‐propanediol.

Table 7.2 Estimation of the molar yield for the syntheisis of 1,3‐propanedi...

Table 7.3 Estimation of the molar yield for the synthesis of 1,3‐propanedio...

Table 7.4 1,3‐Propanediol with oxygen as byproduct.

Table 7.5 Expressed genes and achieved molar yield.

Table 7.6 Selection of achieved titer and productivity for the fermentative...

Chapter 8

Table 8.1 Energy content and density of fuels.

Chapter 9

Table 9.1 Metabolic pathway toward isobutanol via 2‐oxoisovalerate.

Table 9.2 Enzyme portfolio for the generation of isobutanol (Noda et al. 20...

Table 9.3 ATP‐driven conversion of NADH into NADPH.

Table 9.4 Performance data with different strains.

Table 9.5 Biocatalytic pathway toward isobutanol without ATP as cofactor.

Chapter 10

Table 10.1 Metabolic pathway toward isobutene.

Table 10.2 Metabolic pathway toward isobutene via 2‐oxoisovalerate and isob...

Table 10.3 Metabolic pathway toward isobutene via 2‐oxoisovalerate and isov...

Table 10.4 Metabolic pathway toward isobutene via acetoacetyl‐CoA and 3‐met...

Table 10.5 Metabolic pathway toward isobutene via non‐oxidative glycolysis....

Table 10.6 Metabolic pathway toward isobutene via acetoacetyl‐CoA and 3‐hyd...

Table 10.7 Metabolic pathway toward isobutene via acetoacetyl‐CoA and aceto...

Table 10.8 Metabolic pathway toward isobutene via acetoacetyl‐CoA and non‐o...

Table 10.9 TMY and carbon yield of potential pathways toward isobutene.

Chapter 11

Table 11.1 Pathway toward butanediol with succinyl‐CoA as interlink of the ...

Table 11.2 Alignment of oxygen transfer rate and productivity.

Table 11.3 Carbon footprint of 1,4‐butanediol pending on synthesis route (h...

Table 11.4 Performance data for the use of alternative feedstocks.

Chapter 12

Table 12.1 Metabolic pathway toward succinic acid via PEP carboxylation.

Table 12.2 Pathway toward succinic acid via reductive citric acid cycle.

Table 12.3 Potential routes for the generation of the C4 backbone.

Table 12.4 Pathway toward succinate via reductive citric acid cycle and PPP...

Table 12.5 Metabolic pathway toward succinic acid via glycolysis and glyoxy...

Table 12.6 Overall reaction toward succinic acid via the glyoxylate cycle....

Table 12.7 Theoretical molar yield and carbon yield for different routes.

Table 12.8 Pathway toward succinic acid starting with glycerol.

Table 12.9 Pathway toward succinic acid starting with glycerol via pyruvate...

Table 12.10 Performance data for the four commercial technologies. These fi...

Chapter 13

Table 13.1 Metabolic pathway toward itaconic acid.

Table 13.2 Fermentation performance data for itaconic acid.

Chapter 14

Table 14.1 Biochemical pathway toward glutamic acid.

Table 14.2 Ammonia fixation via glutamine.

Chapter 15

Table 15.1 Metabolic pathway for the MVA pathway toward isoprene.

Table 15.2 Conversion of glucose into acetyl‐CoA via the pyruvate‐dehydroge...

Table 15.3 Conversion of glucose into acetyl‐CoA via the pyruvate‐dehydroge...

Table 15.4 DXP pathway toward isoprene.

Table 15.5 Coproduction of 1,3‐propanediol and isoprene.

Table 15.6 Adjusted MVA pathway via isoprenol toward isoprene.

Table 15.7 Simplified net reaction of the MVA and the DXP pathways toward i...

Table 15.8 Simplified net reaction with matching combination of MVA and DXP...

Table 15.9 Conversion of glucose into acetyl‐CoA via the pyruvate‐dehydroge...

Table 15.10 Selection of published performance data for the fermentative pr...

Chapter 16

Table 16.1 Comparison of methanol and glucose.

Table 16.2 Fixation of methanol into fructose‐6‐phosphate.

Table 16.3 Generation of ribulose‐5‐phosphate as receiving group for formal...

Table 16.4 Generation of GAP ex MeOH according to the RuMP pathway (transal...

Table 16.5 Ratio of reducing equivalents per building block.

Table 16.6 Selected performance data for the fermentation of glucose toward...

Table 16.7 Performance data for the biocatalytic decarboxylation of lysine ...

Table 16.8 Carbon footprint of polyamides.

Chapter 17

Table 17.1 First section of the metabolic pathway toward lysine until aspar...

Table 17.2 Biochemical pathway toward lysine (section 2; Wittmann 2010)....

Table 17.3 Technology development of lysine via fermentation. The maximum c...

Table 17.4 Technology development of lysine via fermentation. The performan...

Table 17.5 Global lysine capacity based on fermentation, selection of major...

Chapter 18

Table 18.1 Biochemical pathway toward citric acid.

Chapter 19

Table 19.1 Biochemical steps for the conversion of glucose into adipate.

Table 19.2 Thermodynamics for the conversion of succinyl‐CoA and acetyl‐CoA...

Table 19.3 Performance data for the conversion of fats into DCAs.

Table 19.4 Estimation of the pH given an adipic acid titer of 100 g/l witho...

Chapter 20

Table 20.1 Biochemical conversion of glucose into HMD.

Table 20.2 Biochemical conversion of glucose into HMD via 6‐hydroxyhexanoat...

Chapter 21

Table 21.1 Biochemical pathway for the conversion of glucose into CPL.

Chapter 22

Table 22.1 Biochemical pathway to anthranilate.

Table 22.2 Titer and productivity for biochemical precursors for aniline.

Chapter 23

Table 23.1 Metabolic pathway for the MVA pathway toward IPP.

Table 23.2 Simplified metabolic pathway of non‐oxidative glycolysis (Bogora...

Table 23.3 Metabolic pathway for the MVA pathway toward IPP.

Table 23.4 Metabolic pathway for the DXP pathway toward IPP.

Table 23.5 Summary of maximum theoretical molar yield (TMY) for several pat...

Table 23.6 Selection of published fermentation performance data on β‐farnes...

List of Illustrations

Introduction

Figure 1 Pathway steps with acid to aldehyde reduction.

Chapter 1

Figure 1.1 Potential role of biological methanation in the generation of met...

Figure 1.2 Biochemical pathway for the conversion of carbon dioxide into met...

Figure 1.3 Integrated bioelectrochemical system consisting of a water electr...

Figure 1.4 Flow diagram for the conversion of carbon dioxide with hydrogen i...

Chapter 2

Figure 2.1 Glycolysis with the conversion of glucose into ethanol in

S. cere

...

Figure 2.2 Glycolysis toward ethanol in

S. cerevisiae

. The alternative ED pa...

Figure 2.3 Process flow of the dry milling process for fermentative ethanol....

Figure 2.4 Artificial metabolic pathways for the conversion of D‐xylose and ...

Chapter 3

Figure 3.1 The Wood–Ljungdahl pathway for the conversion of carbon dioxide, ...

Figure 3.2 Pathway reconstruction for the generation of acetate in

A. woodii

Figure 3.3 The Wood–Ljungdahl pathway for the conversion of carbon dioxide, ...

Figure 3.4 Pathway reconstruction for the generation of ethanol in

C. autoet

...

Figure 3.5 Pathway reconstruction for the generation of ethanol in

C. autoet

...

Chapter 4

Figure 4.1 Pathway of homofermentative Lactobacillus toward lactate.

Figure 4.2 Pathway of heterofermentative Lactobacillus.

Figure 4.3 Metabolic engineering of

E. coli

for the production of D‐lactate....

Figure 4.4 Process steps for the fermentative production of lactic acid.

Chapter 5

Figure 5.1 Glycolysis with subsequent conversion of pyruvate into L‐alanine ...

Figure 5.2 Biocatalytic reaction cascade for the synthesis of L‐alanine base...

Chapter 6

Figure 6.1 Recombinant biochemical pathway toward 3‐hydroxypropionic acid. T...

Chapter 7

Figure 7.1 Recombinant pathway for the fermentative production of 1,3‐propan...

Figure 7.2 Reductive and oxidative branch in the fermentative production of ...

Figure 7.3 Recombinant metabolic pathway of Genencor/Dupont for the fermenta...

Figure 7.4 Recombinant metabolic pathway toward 1,3‐propanediol with glycero...

Figure 7.5 Theoretical molar yield in the conversion of glycerol to 1,3‐prop...

Chapter 8

Figure 8.1 Metabolic pathway for the fermentative production of butanol.

Figure 8.2 Biochemical pathway for the conversion of glucose into butanol wi...

Figure 8.3 Biochemical pathway toward butanol via the pyruvate dehydrogenase...

Figure 8.4 Biochemical pathway for the conversion of glucose into butanol (L...

Figure 8.5 Simplified process flow for the butanol & hexanol technology of E...

Figure 8.6 Simplified process flow in conventional ABE fermentation.

Figure 8.7 Simplified process flow of a sugarcane refinery in Brazil includi...

Chapter 9

Figure 9.1 PET value chain and potential entry points via fermentation produ...

Figure 9.2 Metabolic pathway toward isobutanol in

E. coli

with overexpressio...

Figure 9.3 Recombinant pathway in

E. coli

for the synthesis of isobutanol. F...

Figure 9.4 Recombinant pathway toward isobutanol based on glycine and butyry...

Figure 9.5 Recombinant pathway toward isobutanol based on carbon monoxide an...

Figure 9.6 Fermentative production of isobutene as operated by Gevo in Luver...

Figure 9.7 Biocatalytic conversion of glucose into isobutanol via the modifi...

Chapter 10

Figure 10.1 Proposed biochemical pathways toward isobutene. KICD: α‐ketoisoc...

Figure 10.2 Biochemical pathway for the conversion of pyruvate into isobuten...

Figure 10.3 Biochemical pathway for the conversion of pyruvate into isobuten...

Figure 10.4 Biochemical pathway for the conversion of pyruvate into isobuten...

Figure 10.5 Biochemical pathway for the conversion of pyruvate into isobuten...

Chapter 11

Figure 11.1 Biochemical pathway design toward 1,4‐butanediol starting with s...

Figure 11.2 Biochemical pathway design toward 1,4‐butanediol with α‐ketoglut...

Figure 11.3 Alternative feedstock approach to derive α‐ketoglutarate from li...

Figure 11.4 Alternative pathway toward 1,4‐butanediol based on D‐xylose (Liu...

Chapter 12

Figure 12.1 Metabolic pathway toward succinic acid with glucose as feed. The...

Figure 12.2 Glyoxylate cycle for the synthesis of succinate with recycling o...

Figure 12.3 Combination of glycolysis and pentose phosphate pathways for the...

Chapter 13

Figure 13.1 Metabolic pathway toward itaconic acid in

A. terreus

(Cruz et al...

Figure 13.2 Conversion of

cis

‐aconitate into itaconic acid (Geiser et al. 20...

Figure 13.3 Pathway for the conversion of p‐coumaric acid into itaconic acid...

Figure 13.4 Downstream process for the purification of itaconic acid.

Chapter 14

Figure 14.1 Biochemical pathway toward glutamic acid. The formation of oxala...

Figure 14.2 Redox balance in the joint synthesis of glutamate and 1,3‐propan...

Chapter 15

Figure 15.1 Routes for the chemical production of isoprene.

Figure 15.2 Simplified MVA and DXP pathways for the synthesis of IPP and DMA...

Figure 15.3 MVA pathway toward isopentenyl‐PP and dimethylallylpyrophosphate...

Figure 15.4 Schematic representation of the pyruvate‐dehydrogenase bypass to...

Figure 15.5 Classical MVA pathway compared with modified MVA pathway I (

red

...

Figure 15.6 Advanced versions of the MVA pathway toward prenol, isoprenol, a...

Figure 15.7 DXP pathway toward isoprene via IPP and DMAPP.

Figure 15.8 MVA pathway in

E. coli

with origin of heterologous genes.

Figure 15.9 MVA pathway in

E. coli

with origin of heterologous genes.

Figure 15.10 Mixotrophic pathway for the synthesis of isoprene in an acetoge...

Figure 15.11 Engineered pathway for the synthesis of mevalonate in

S. cerevi

...

Figure 15.12 Conversion of mevalonate into mevalonolactone and isoprene.

Chapter 16

Figure 16.1 Ribulose monophosphate pathway (SBPase variant) in

B. methanolic

...

Figure 16.2 Ribulose monophosphate pathway (aldolase variant) in

B. methanol

...

Figure 16.3 Metabolic flux via glycolysis toward succinate (red rectangle) i...

Figure 16.4 Metabolic flux via PPP toward pentamethylenediamine (red rectang...

Figure 16.5 Consortium of two

E. coli

strain to synthesize PMD.

Chapter 17

Figure 17.1 Biocatalytic synthesis of L‐lysine based on cyclohexene as start...

Figure 17.2 Pathway covering the section glucose to aspartate (section 1; Wi...

Figure 17.3 Pathway toward lysine covering the section aspartate to lysine (...

Figure 17.4 Simplified pathway model with focus on reduction equivalents....

Figure 17.5 Synthetic lysine pathway via aminoadipate in fungi.

Figure 17.6 Downstream process for the generation of pure lysine hydrochlori...

Chapter 18

Figure 18.1 Metabolic pathway toward citric acid.

Figure 18.2 Process flow for the fermentative production and isolation of ci...

Chapter 19

Figure 19.1 Chemical pathway for the synthesis of adipic acid ex benzene....

Figure 19.2 Natural degradation pathway for aromatic compounds.

Figure 19.3 Metabolic pathway toward adipic acid with succinyl‐CoA and acety...

Figure 19.4 Metabolic pathway toward

cis

,

cis

‐muconic acid based on glucose....

Figure 19.5 Metabolic pathway toward adipic acid based on glucose.

Figure 19.6 Allocation of pathway sections toward

cis, cis

‐muconic acid in a...

Figure 19.7 Conversion of lignin derivatives into

cis, cis

‐muconic acid.

Chapter 20

Figure 20.1 Reverse adipate pathway towards HMD based on the condensation of...

Figure 20.2 Biochemical pathway towards HMD via 6‐hydroxyhexanoate.

Figure 20.3 Biochemical pathway towards HMD based on 2‐amino‐6‐oxopimelate a...

Figure 20.4 Biochemical pathway towards HMD via α‐ketopimelic acid.

Figure 20.5 Biochemical pathway toward HMD based on 6‐amino‐2‐hydroxy hexano...

Figure 20.6 Biocatalytic routes to HMD based on ADA. The thioester intermedi...

Figure 20.7 Options for pH management in the fermentative production of HMD....

Chapter 21

Figure 21.1 Chemical synthesis of CPL starting with benzene.

Figure 21.2 Reverse adipate pathway toward caprolactam.

Figure 21.3 Chain elongation from α‐ketoglutarate to α‐ketopimelic acid. The...

Figure 21.4 α‐Ketopimelate pathwayα toward CPL.

Figure 21.5 Proposed metabolic engineering routes toward ADA, CPL, and HMD....

Chapter 22

Figure 22.1 MDI value chain based on petrochemistry with alternative ferment...

Figure 22.2 Initial pathway steps until 3‐dehydroshikimate for the fermentat...

Figure 22.3 Metabolic network for the synthesis of anthranilate (Hirayama et...

Figure 22.4 Mechanism of the anthranilate synthase with 2‐amino‐2‐deoxyisoch...

Figure 22.5 Biochemical conversion of chorismate into tryptophan including p...

Figure 22.6 Coculture of

E. coli

strains for the fermentative production of

Figure 22.7 Process scheme for the fermentative production and isolation of ...

Chapter 23

Figure 23.1 Structure of β‐farnesene (left side) and α‐farnesene (right side...

Figure 23.2 MVA pathway for the conversion of acetyl‐CoA into IPP and DMAPP ...

Figure 23.3 MVA pathway for the synthesis of β‐farnesene with pyruvate dehyd...

Figure 23.4 MVA pathway for the synthesis of β‐farnesene with acetyl‐CoA sup...

Figure 23.5 Simplified DXP pathway for the conversion of pyruvate and GAP in...

Figure 23.6 β‐farnesene based on triglycerides/fatty acid with partial perox...

Figure 23.7 Pathway toward farnesene in

E. coli

with enzyme fusion.

Figure 23.8 Process flow diagram for the fermentative production of β‐farnes...

Figure 23.9 Innovative synthesis route toward α‐tocopherol.

Guide

Cover

Table of Contents

Title Page

Copyright

Preface

Introduction

Begin Reading

Index

End User License Agreement

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Pathway Design for Industrial Fermentation

Walter Koch

Author

Dr. Walter KochBASF SECarl‐Bosch‐Str. 38Ludwigshafen67056Germany

Cover Image: © Parilov/Shutterstock

All books published by WILEY‐VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

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Print ISBN: 978‐3‐527‐35275‐3ePDF ISBN: 978‐3‐527‐84393‐0ePub ISBN: 978‐3‐527‐84394‐7oBook ISBN: 978‐3‐527‐84395‐4

Preface

Fermentation technology has often outcompeted organic chemistry in the case of advanced molecular functionality. This refers especially to enzymes and antibodies, though also includes selected organic acids (e.g. lactic acid, citric acid, and itaconic acid), amino acids (e.g. lysine, glutamic acid, and alanine), and various fine chemicals. It offers a great advantage and cost reduction since one fermentation process bundles several steps, which would alternatively be needed with a cascade of single synthesis steps in organic chemistry. The process shortcut is enabled by the multifunctional abilities of the biocatalyst, which handles a complete pathway in one cell. On the other hand, the catalysts applied in organic chemistry, be it a homogeneously or heterogeneously catalyzed process, are usually restricted to single synthesis steps.

The political and societal attempts to replace petrochemistry by solutions with an improved sustainability profile and lower or zero product carbon footprint have drawn management attention toward fermentation products with lower molecular functionality and fuels. These target molecules comprise 3‐hydroxypropionic acid or acrylic acid, butanol, isobutanol, isobutene, 1,4‐butanediol, and building blocks for the PA6 and PA66 value chains such as adipic acid, hexamethylenediamine, caprolactam, or 6‐aminocaproic acid, respectively, and the MDI value chain like anthranilic acid.

The success of fermentative fuels relies on a high energy efficiency in the conversion of feedstock into hydrocarbons with high energy density. Ethanol is the fermentation fuel used as a fuel additive in combustion engines. The rise to a production volume of 100 Mio. t in the last 30 years has been enabled by the national fuel mandates and quota systems and has profited technologically from the existing and highly efficient yeast technology. R&D focus switched to second‐generation approaches using lignocellulosic biomass as the feedstock. Significant R&D resources were also allocated to establish butanol as a fermentative fuel.

This book tries to capture more than 20 chapters, respectively, in the technology competition of organic chemistry versus fermentation for molecules with lower molecular functionality and fuels beyond ethanol. Mature organic chemistry is facing increasing competition since fermentation technology is further enhanced by advancements in molecular biology, enzyme screening and optimization, metabolic engineering, and downstream processing. Furthermore, chemolithoautotrophic metabolism with carbon monoxide, carbon dioxide, and hydrogen as feedstocks has successfully been introduced with an acetogenic host to overcome the limitations of the potential volume of feedstocks of the traditional heterotrophic metabolism.

May 2023

Walter Koch

Introduction

Fermentation as Production Technology

Fermentation is to be conceived as a production technology competing with natural extraction, organic chemistry, and biocatalysis. All three technologies to access chemical products have their own advantages and disadvantages.

Raw material extraction, which may be regarded as the first production technology, uses natural crops as reactors with controlled cultivation to make chemical products. The production technology is facing limited arable land that could alternatively be used for food production, and an expansion is critical. The cultivation period might be long and define a critical hurdle in case a period of several years is required. Raw material extraction often bears a high extraction cost since the product is mixed with various other crop ingredients. The low content of the target molecule in the biomass might be increased to improve production economics. Subject of raw material extraction might be a natural variety (wild type), a non‐natural variety derived by breeding, or a genetically modified variety obtained by plant biotechnology (Chawla 2003; Kempken and Kempken 2004). Most of the agronomic varieties applied for industrial food production were obtained by breeding and possess a higher product content and thereby provide a higher yield per ha than the original natural varieties. Given the inherent disadvantages, natural extraction has in many instances been supplemented or been replaced by organic chemistry, like for natural dyes (Wittke 1984), flavors, and fragrances.

Organic chemistry offers a broad range of potential reaction conditions with respect to temperature, pressure, pH, and organic solvents. It often facilitates high yield (turnover × selectivity), high productivity (space–time‐yield), and reasonable investment efforts. Accordingly, the technology has seen a tremendous development and is broadly applied in the petrochemical and chemical industry (Arpe 2007). Organic chemistry has allowed a significant reduction of production costs and enabled the synthesis of many molecules and polymers or plastics, respectively, not found in nature. It has often replaced, supplemented, or at least partially pushed back natural extraction into niche markets, like e.g. in the case of terpenes (Breitmaier 2005) and vanillin (Banerjee and Chattopadhhyay 2018; Kaur and Chakraborty 2013).

Biocatalysis uses enzymes as catalysts and is favored in case an enantioselective synthesis is needed to derive, e.g. chiral alcohols or amines (Hilterhaus et al. 2016). The technology is in some instances applied in high‐volume applications, such as the isomerization of glucose into fructose for the soft drink industry (Schmid 2016), but it is often a niche technology for complicated molecular functionality not accessible via organic chemistry within a reasonable number of steps (Faber 2011). Biocatalysis is favored in case the needed raw material conversion is merely an isomerization or a hydrolysis since no cofactors are required. Thus, most of the industrial enzymes applied in the food, feed, or detergent industry are hydrolases, which exclusively rely on water as a cofactor (Sahm et al. 2013). On the other hand, if the needed raw material conversion is a redox reaction in which electrons are transferred, the biocatalyst depends on cofactors like NADH or FAD, which are very costly. For the synthesis of low‐volume fine chemicals with sufficient value perspective, suitable cofactor regeneration systems are available (Wu et al. 2021; Grunwald 2018), but synthesis routes for low‐value commodity products are often not commercially feasible.

Fermentation has historically covered the microbial conversion of carbohydrates in the absence of oxygen. The term was originally introduced by Louis Pasteur: “La fermentation c'est la vie sans l'air” (Schegel 1992). Fermentation initially happened accidentally if non‐sterilized carbohydrate containing food or biomass was stored in the absence of oxygen and was then culturally developed as an on purpose conservation technology. The cultural history of e.g. alcoholic beverages mirrors the long‐term development of fermentation technology. The conceptual restriction of fermentation as an exclusively anaerobic process was later given up, and the meaning of the term now comprises the anaerobic and the aerobic conversion of chemical feedstocks by prokaryotic or eukaryotic hosts (Schegel 1992). The application field of fermentation may be differentiated into “White Biotech,” the production of fuels and chemical intermediates, “Red Biotech,” the production of recombinant proteins and human antibodies for pharma application (Lee and Kildegaard 2020); and “Industrial Enzymes,” the production of enzymes for application in industrial processes. The focus of this book in on “White Biotech.”

Rising Interest in Fermentation as Production Technology

Production costs and price, respectively, have been up until now, for chemical high‐volume products the most important and, in some cases, the single decisive purchase criterion. The products are regarded as “commodities,” opposed to “specialties”, with low‐volume demand, higher molecular functionality, and potentially customization to meet the defined requirements of single customers. The marketing of high‐volume chemicals appeals to molecular functionality and specification. The low‐cost level of the still dominant petrochemistry is mainly enabled by the circumstance that oil refining to make gasoline for automotive combustion engines generates byproducts like naphtha, which is handed over to chemical companies to feed a cracker (Baerns et al. 2013; Jess and Wasserscheid 2013). The cracker reassembles the C—C bonds of naphtha to derive the main four petrochemical aliphatic intermediates, i.e. ethylene, propylene, isobutene, butadiene, and the three aromatic intermediates, i.e. benzene, toluene, and xylene.

Fermentation is the technology of choice for producing pharma proteins, antibodies (“Red Biotech”) and enzymes (“Industrial Enzymes”). Though there is an increasing interest in fermentation technology for high‐volume chemicals with much less advanced molecular functionality (“White Biotech”), which receives promotion in national industry development plans. The US bioeconomy strategy pursues the direction of “moving beyond fuels toward biobased and bio‐enabled production of chemicals and other products” (A Bioeconomy Strategy 2022). The growing importance of fermentation as production technology and the increasing share of “white biotechnology” to replace petrochemistry are due to several reasons (Rosales‐Calderon and Arantes 2019; Tsuge et al. 2016; Jang et al. 2012).

Biobased raw materials and product carbon footprint considerations are gaining importance, although production costs are still the most important sourcing criterion for chemical products. Chemicals might additionally be marketed with reference to their biobased origin if the raw material is derived from natural sources and the integrated carbon has originally been derived from carbon dioxide captured out of the atmosphere via photosynthesis. The interest in biobased products represents a rising trend for about 20–30 years. Biobased origin was traditionally an advantage for chemicals applied in the food or cosmetic industry with high proximity to the final consumer, which achieved a price premium in the market (Ravenscroft 2019; Ravenscroft 2013). The price premium is required since the production starting with natural extraction and some chemical conversion steps is often more costly than a petrochemical synthesis. It can be observed that in parallel to food ingredients and cosmetics also markets for biobased chemical intermediates are developing, and petrochemistry is facing competition from alternative feedstocks. The reason of this trend reflects the growing customer demand for natural products and the concern that the high carbon dioxide emissions associated with petrochemistry need to be overcome for climate protection. The trend is enforced by national and international regulation and shifting customer attention as well.

The claim of a biobased origin might be achieved via the direct use of a natural feedstock or mediated via a mass balance approach in which the biobased carbon origin of natural feedstocks is allocated to a petrochemically generated product. Fermentation technology profits from the trend toward biobased feedstocks since glucose and sucrose are biobased and derived from, e.g. corn, wheat, sugar beet, sugarcane, or cassava. Fermentation might be the technology providing the lowest production cost, such as in the case of ethanol, lactic acid, citric acid, glutamic acid, and lysine, and has become the leading technology irrespective of its conversion of biobased feedstocks. On the other hand, in case fermentation incurs higher cost than petrochemistry, a price upside in the market is required. Biotech companies and startups were often inclined to overestimate the biobased price premium. Fermentation technology was often able to provide a sound production process, though the marketing success was limited given a cost level above petrochemistry. It turned out that the market success of fermentative products often requires cost equivalence or functional advantages:

“Customers in the electronic and automotive industries will not pay a premium for biobased products unless there is a technical advantage,” M. Scheibitz, specialist at BASF automotive (Ravenscroft 2013).

“A cost‐advantaged and more sustainable route to adipic acid is expected to offer significant value for the company‘s performance materials business,” J. Iademarco, DSM's Vice President Biobased Chemicals (Coons 2011).

“Products won't sell themselves just because they are based on renewables resources […] Companies need to understand how to bring new biopolymers to market,” J. Ravenstijn, bioplastic consultant (Ravenscroft 2013).

“The success of biobased chemicals will rest on competitive economics or premium and uses,” M. Hackett, senior manager at IHS Chemicals (Ravenscroft 2013).

“We are not in a situation where […] any company manufacturing diapers at this moment is ready to pay a 15% premium because it is a biobased raw material […] This is because no consumer is ready to pay a premium for the diaper” (Tullo 2013).

The second marketing advantage for fermentation technology refers to product carbon footprint considerations (Carruthers and Lee 2022). The original proposal for enhanced sustainability is transforming more and more into the request of politics that the petrochemical and the chemical industry, and the corresponding big consumer brands, need to implement a carbon‐neutral business perspective until 2050 (Boulamanti and Moya 2017). Triggered by climate change and the enhanced regulatory burden of carbon dioxide emissions, the conviction was born that no »fresh« carbon in the form of oil and gas must be allowed as feedstock for the petrochemical and chemical industry, and the concept of biobased feedstocks and chemical recycling is gaining momentum (vom Berg et al. 2022). The “endgame” of petrochemistry might have been kicked off. This would imply that the carbon footprint of chemicals needs to be reduced more and more until finally a carbon neutral industry – with net zero carbon dioxide emissions – is established. Ultimately, the carbon content of chemicals, which usually cannot be substituted, needs to be gained via recycling or will be derived from biobased raw materials. Fermentation does often, though not always, allow a lower product carbon footprint than petrochemistry in case the biogenic uptake is considered. It will be interesting to see if fermentation succeeds in providing an alternative supply for the main petrochemical intermediates given the limited availability of carbohydrate as a carbon and energy source (SYSTEMIQ 2022)..

Technical Comparison of Fermentation Versus Organic Chemistry

The fermentation process has several restrictions and disadvantages, respectively, though also decisive advantages compared to organic chemistry. The benchmark of both production technologies is discussed with respect to defined criteria:

Coverage of reactions: The outstanding advantage of fermentation technology compared to organic chemistry is the ability of the fermentation biocatalyst to carry out several chemical reactions in one production step without the isolation and purification of intermediates. The substrate is modified by several enzymes within the host cell and channeled through a whole pathway. Fermentation is usually the preferred technology if several steps toward fine chemicals with high molecular functionality can be bundled and the alternative order of steps in organic chemistry is avoided (Vandamme and Revuelta 2016). The coverage of >100 reactions that otherwise would need to be pursued in separate steps of organic chemistry – with yield loss and purification efforts in each step – explains that enzymes or antibodies are more or less exclusively produced via fermentation. The complete field of “Industrial Enzymes,” i.e. the production of enzymes for application in industrial processes, and “Red Biotech,” i.e. the production of recombinant proteins and human antibodies for pharma application, is dominated by fermentation technology (Lee and Kildegaard 2020).

Feedstock: Fermentation processes usually apply carbohydrates, i.e. glucose as a monosaccharide or more complex carbohydrates, as their sole carbon and energy source. There is a limited number of feedstocks available that can be used as carbon and energy sources by the major industrially used hosts such as Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Aspergillus niger. These comprise, e.g. the monosaccharide glucose (C6) and glycerol (C3). Pending on the specific host also disaccharides or complex carbohydrates may be used. Sucrose, a common table sugar, can usually be metabolized in case an efficient glycosidase is integrated. Otherwise, a chemical hydrolysis is needed. The use of cellulose and hemicellulose requires specific treatment procedures to ensure accessibility of the C5 and C6 sugars. The metabolism of the shorter C5 sugars D‐xylose and L‐arabinose derived via the digestion of lignocellulosic biomass usually requires the addition of specific pathway genes. Fermentation technology with acetogenic bacteria as hosts using carbon monoxide and hydrogen (Mock et al. 2015; Molitor et al. 2016; Müller 2019) as energy sources is available and industrially applied. The methanization of carbon dioxide with exclusively hydrogen as an energy source is evaluated at pilot scale (Bernacchi et al. 2014a, b). Furthermore, methanol as a potential feedstock for fermentation processes is under development, though commercial maturity has not yet been derived. On the other hand, organic chemistry is much more flexible with respect to feedstock: nearly all organic compounds may be regarded as educt. A comparison of fermentation and organic chemistry with respect to defined criteria reveals the main differences (Table 1):

Table 1 Comparison of production technologies for chemical molecules.

Fermentation

Organic chemistry

Feedstock

Glucose, xylose, glycerol, CO/CO

2

/H

2

In principle all chemical molecules

Enthalpy

Exothermic overall

Exothermic or endothermic

Temperature

25‐60 °C

Up to 800 °C

Pressure

1 barg up to 10 barg

Vacuum up to 300 bar

Reaction medium

Liquid phase

Gas phase or liquid phase

Yield

max. slightly above 90% (including biomass)

Dependent on reaction (may achieve values 99.0‐99.9%)

Product titer

10‐200 g/l

»200 g/l

Productivity

0.001‐0.01 kg/lxh

1–10 kg/lxh heterogen. catalysis 0.01–1 kg/lxh homogen. catalysis

Catalyst

Biocatalyst/Host required

With or without catalyst

Reactor

CSTR/Airlift reactor

Broad portfolio of reactors

Investment

<1000 €/t anaerobic>1000/5 000 €/t aerobic

<1000 €/t commodity >1000/5 000 €/t fine chemical

Temperature and pressure: The fermentation temperature is usually between 25 and 35 °C for the common biocatalysts since the enzyme activity of the host is very sensitive to temperature. A tight window does also apply with respect to pressure, and a fermentation with E. coli or S. cerevisiae is usually applied at ambient pressure. In exceptional cases, boundary conditions beyond this window are selected. E.g. the fermentative hydrogenation of carbon dioxide with hydrogen is pursued at a temperature of 60–65 °C and a pressure of 4–9 bar (Rusmanis et al. n.d.). Reaction temperatures outside the defined temperature window of the specific host would negatively interfere with the process productivity, which usually cannot be restored. Higher temperatures trigger the partial denaturization of enzymes, which lose their individual three‐dimensional structure important for functionality. Since the overall fermentation process is exothermic, sufficient cooling equipment needs to be installed to align with the exothermic heat release of the main reaction. On the other hand, organic chemistry offers a much higher flexibility in the selection of the most suitable temperature and may be pursued at temperatures up to 800 °C in case catalyst and product stability are ensured. However, it is often preferred to avoid very high temperatures to extend the catalyst lifetime, to reduce the investment, and to limit maintenance efforts. Additionally, the chemical reaction may be pursued under vacuum or high pressure to improve kinetics, yield, or selectivity.

Yield: The yield may be defined as the molar ratio between the obtained product amount and the used educt amount and is indicated as percentage. The realistic yield achieved in industrial processes is in almost all cases <100% since the turnover is often not complete (despite recycling loops of non‐converted educt) and a compromised selectivity allows by‐products whose formation cannot completely be suppressed:

Yield (%) = Turnover × Selectivity/100

With respect to fermentation, the yield is often expressed as “carbon yield”, given by the ratio of the mass of the obtained product and the mass of the applied glucose (Arpe 2007). However, this value is especially misleading in case fermentation products with a higher reduction status and higher energy content are considered. The maximum carbon yield in the fermentative production of ethanol amounts to 0.51 t ethanol per ton of glucose (2 × 46 /180 g/mol), which might seem low. However, the industrial process achieves a carbon yield of about 0.46 t ethanol per ton of glucose, which corresponds to about 90% of the theoretical maximum molar yield and is thereby quite high since the formation of biomass is included. Since one glucose derives maximum two mol of ethanol, the theoretical maximum molar yield is 2.0. Thereof 90% or 1.8 mol ethanol per mol glucose are achieved 90%:

Accordingly, the “yield” referred to in this book is defined as the ratio between the achieved molar yield and the theoretical maximum molar yield. Hereby the formation of biomass is included which, pending on the biomass titer and the potential recycling of biomass as inoculum, also consumes a share of the feedstock.

The turnover of the applied glucose might be close to 100% since the reaction is often stopped after the glucose feed in the medium has completely been consumed. A weak selectivity may be caused by competing pathways that channel intermediates into unwanted by‐products and the formation of biomass. The carbon yield usually includes the formation of biomass, which may be added as inoculum at the beginning of the reaction or may be formed during the fermentation. The most developed industrial fermentation processes for ethanol and lactic acid achieve a yield of slightly >90%, including the formation of biomass (Jansen et al. 2017; van Maris et al. 2004).

Product titer and productivity: A high yield is important for the reduction of raw material costs. Titer and productivity are important to limit the reactor volume and the scope of the downstream process to reduce the investment. Hereby, it is to be considered that the glucose molecule – the conventional feedstock – needs to be absorbed out of the broth, is converted within the cell by passing a metabolic pathway, and the respective product secreted into the broth. Hereby, specific uptake rates for each host do apply. With respect to the likely most important bacterial host, E. coli, the maximum glucose uptake rate for the transfer through the prokaryotic cell wall is about 20 mmol per g DCW (dry cell weight) per h (Yim et al. 2011) and defines a threshold for productivity at a given cell concentration. For in silico simulations, a reasonable average glucose uptake rate of 10 mmol per g DCW per h may be set (Table 2):

Table 2 Maximum glucose uptake (qS) rates for selected hosts.

Host

Glucose uptake rate

E. coli

20 mmol/g DCW × h (maximum threshold, Ahn et al.

2020

; Liebal et al.

2018

; Chae et al.

2017

)

E. coli

10 mmol/g DCW × h (reasonable average value Ahn et al.

2020

; Liebal et al.

2018

; Chae et al.

2017

)

Corynebacterium glutamicum

4.6 mmol/g DCW × h (Becker et al.

2008

)

C. glutamicum

3.6 mmol/g DCW × h (Buschke et al.

2013

)

Vibrio natriegens

21 mmol/g DCW × h (aerobically growing cells; Thoma and Blombach

2021

)

V. natriegens

43 mmol/g DCW × h (anaerobically growing cells; Thoma and Blombach

2021

)

V. natriegens

6 mmol/g DCW × h (anaerobically resting cells; Thoma and Blombach

2021

)

The value for C. glutamicum refers to a lysine producing strain C. glutamicum lysCfbr with feedback resistant aspartate kinase (Becker et al. 2008). For comparison, the xylose uptake rate in C. glutamicum is with about 4.0 mmol/g DCW × h in the same range (Buschke et al. 2013).

High figures have been reported for Vibrio natriegens, which have triggered several product‐specific R&D projects with the target to exceed productivities of the established mainstream hosts. However, the commercial relevance of V. natriegens is still limited.

The resulting productivity for an industrial fermentation process may display a level of up to 10 g/l × h, which may be achieved in efficient periods with respect to ethanol or lactic acid (Jansen et al. 2017; van Maris et al. 2004). For laboratory scale experiments, higher values are reported in exceptional cases (Table 3):

Table 3 Very high productivity values achieved in lab‐scale.

Productivity

Product

Source

DCW

14.0 g/l × h

Butanol

Nguyen et al. (

2019

)

29 g DCW/l

21.3 g/l × h

Succinic acid

Ahn et al. (

2020

)

18 g DCW/l (own estimate)

34.0 g/l × h

Alanine

Hoffart et al. (

2017

)

(resting cells)

The maximization of productivity is in competition with the goal to achieve a high yield since productivity is enhanced by a higher concentration of the fermentation host (i.e. biomass titer). It is obvious that a higher biomass titer, which consumes glucose feedstock, raises the productivity in case the supply of glucose, oxygen, or other media components is not limited. Though then also the ratio of biomass to product is increasing, which compromises the yield because the biomass can often not be recycled (e.g. used as inoculum of the next batch). A process analysis referring to the maximum product titers of ethanol (Jansen et al. 2017) and lactic acid (van Maris et al. 2004) at batch end and an assumed batch duration of 24 h reveals that a productivity between 5 and 10 g/l × h combined with an average glucose uptake rate of 10 mmol glucose per g DCW and h still allows a yield of 90% of the theoretical maximum yield, facilitated by a rather low biomass byproduct factor (Table 4):

Table 4 Process analysis for the fermentative production of ethanol and lactic acid. In case the carbon content of biomass is estimated with 50% the glucose gap given as difference between the theoretical maximum carbon yield and the realistic carbon yield is sufficient to account for the generation of the needed dry cell weight

Fermentation

Ethanol

Lactic acid

Titer at batch end

200 g/l

150 g/l

Batch duration

24 h

24 h

Productivity

8.33 g/l × h

6.25 g/l × h

Carbon yield theoretical

0.51 t EtOH/t Glc

1.00 t LA/t Glc

Carbon yield realistic

90%

90%

Carbon yield realistic

0.46 t EtOH/t Glc

0.90 t LA/t Glc

Glucose consumption

18.1 g/l × h

6.94 g/l × h

Glucose consumption

101 mmol/l × h

38.53742755 mmol/l × h

Glucose uptake assumed

10 mmol/gDCW × h

10 mmol/gDCW × h

Dry cell weight needed

10.1 g/l

3.85 g/l

Dry cell weight

0.05 g/g EtOH

0.03 g/g EtOH

Biomass productivity

0.823 g/g DCW × h

1.6 g/g DCW × h

Process conditions

Anaerobic

Anaerobic

Host

Saccharomyces cerevisiae

Lactobacillus

For a bulk chemical to be produced by fermentation, a yield of 80%–90% of the theoretical maximum, a product titer of 100 g/l, and a productivity of 2.5–3.0 g/l × h are regarded as minimum threshold for a commercial feasibility (Thoma and Blombach 2021; Van Dien 2013). A product titer greater than 50 g/l may be defined as the minimum threshold for an industrial process, though a clear‐cut borderline is difficult to define (Sun and Alper 2015). These performance data are significantly below the value of a heterogeneous or a homogeneous catalysis in organic chemistry (Klemm 2011). The exemplary productivity for organic chemistry in the acetoxylation of ethylene amounts to 0.3–0.6 kg/l × h (Arpe 2007). It is to be kept in mind that a comparison is difficult since organic chemistry needs to proceed step‐by‐step, whereas fermentation technology may allow to facilitate a whole pathway, e.g. >10 chemical conversions, in one process steps. However, in case a high‐value product is generated that is not accessible via organic chemistry, a fermentation productivity ≪1 g/l × h may be acceptable.

Feeding Strategy

The feeding strategy defines the addition of feedstock during the fermentation covering the carbon source, the energy source, trace elements, and other nutrients. The addition of carbohydrates may be regarded as the most essential part of the feeding strategy; trace elements and other nutrients define media composition. Carbohydrates usually function as combined carbon and energy source in most industrial processes. The most straightforward and easiest approach is the batch process, which foresees that carbohydrate is once added at the start of the fermentation and is not replenished. The host splits the glycosidic bond in case a disaccharide (e.g. sucrose, lactose) is applied and converts glucose into product. The batch is stopped immediately after the glucose has been spent. Since the broth may be disregarded after the product has been isolated, an exhaustive uptake and conversion of carbohydrate is decisive for a high yield. Temperature and pH are controlled and kept at the target value, and oxygen is permanently added in case an aerobic process is pursued.

The batch process does not allow to operate the reactor at a high productivity since a carbohydrate limitation might apply already at the midpoint of the batch. Accordingly, most industrial fermentation processes pursue the fed‐batch approach (Sahm et al. 2013), which foresees that the feedstock is permanently be replenished after the initial carbohydrate supply at batch initiation. In case the fed‐batch process can be run as a continuous process, the production cost of the product would profit from a high reactor productivity and a high use of capacity of the downstream equipment because the periods with interrupted production for cleaning and batch preparation are minimized. However, most hosts are not suited for a continuous process since a permanent in situ‐extraction of product is usually not possible and the avoidance of contamination is challenging. In case a gas fermentation is pursued the carbon source and the energy source are disconnected. Carbon dioxide or carbon monoxide may be applied as carbon source and hydrogen or carbon monoxide as energy source. Industrial experience in the supply of gaseous feedstock is available (Heijstra et al. 2017), though still limited compared to feeding glucose. It may be assumed that a gas mixture is permanently fed into the reactor and that the non‐converted components are captured in the head space of the reactor and are recycled..

Metabolic Engineering Strategy

A main part of fermentation R&D refers to the engineering of the metabolic pathway. Until 1980, the strain development programs started with natural overproducers for specific products, like S. cerevisiae for ethanol, Lactobacillus for lactic acid, C. glutamicum for glutamate and lysine, and A. niger for citric acid, and improved performance via media optimization, random mutagenesis, and selection. Accordingly, the industrial fermentative production of ethanol, lactic acid, glutamate, lysine, and citric acid follows natural pathways. Since 1980, the toolbox of molecular biology has allowed more and more rational optimization of the existing pathways or the design and the implementation of artificial pathways.

Advanced technological possibilities allowed for innovative recombinant pathways and the development of new industrial processes. The R&D activities of Dupont/Genencor for 1,3‐propanediol, the working group of J. C. Liao for butanol and isobutanol, Genomatica for 1,4‐butanediol, and Amyris with the working group of J. Keasling for terpenes like artemisinic acid and farnesene define landmark projects in industrial fermentation technology.

The metabolic engineering strategy identifies the most suitable pathway toward the defined target product. Different computer models and tools are available. In this context, the “SymPheny” tool of Genomatica is described as example that significantly contributed to the development of a commercial fermentation technology toward 1,4‐butanediol. It consists of five steps: “Reactor operator management” summarizes all potential chemical steps based on known organic chemistry and enzyme classes. The suitable secondary metabolites or cofactors are defined. The toolbox comprises all potential enzymatic steps, even if a suitable enzyme might not yet have been identified. “The Network calculation” starts with the target molecule, figures out potential reaction cascades, and calculates the presumable network reactions. The “Automated integration” adds secondary metabolites, describes charged species at neutral pH, derives balance reactions, and considers thermodynamic feasibility. Unknown biochemical reactions are flagged. In the following module, “Pathway tracing”, the starting molecules and the maximum pathway length are set. Additionally, the overall pathway thermodynamics are calculated. Finally, the module “Pathway ranking” pursues a calculation of the maximum theoretical yield and prioritizes pathways according to defined selection criteria such as thermodynamic feasibility, length or number of steps, coverage by known enzymes, or gaps requiring enzyme evolution (Yim et al. 2011). After the in silico‐based decision on the recombinant pathway, the real experimental work can be initiated. Native genes from other species are screened, optimized, and integrated into a recombinant host to either transfer a natural pathway into a new organism or to create an artificial pathway not found in nature. The new pathway might be obtained by an orchestrated combination of existing, natural genes, or a new reaction might be implemented based on natural promiscuity or the artificial modeling of enzymes.