Handbook on Small & Medium Scale Industries (Biotechnology Products)


Handbook on Small & Medium Scale Industries (Biotechnology Products)

Author: Dr. H. Panda
Format: Paperback
ISBN: 9788178331713
Code: NI301
Pages: 480
Price: Rs. 1,695.00   US$ 150.00

Published: 2017
Publisher: Asia Pacific Business Press Inc.
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The Indian biotechnology industry is one of the fastest growing knowledge-based sectors in India and is expected to play an important role in small & medium enterprises industries. Biotechnology is not just one technology, but many. There are a wide variety of products that the biotechnology field has produced. Biotechnology as well all know, is the field of combination of various fields such as genetics, environmental biology, biochemistry, environmental, general, agriculture, fermentation, etc.

Biotechnology has a long history of use in food production and processing. It has helped to increase crop productivity by introducing such qualities as disease resistance and increased drought tolerance to the crops. Biotechnology used in processing of wines, beers, Coffee, Tea, Cabbage and Cucumber, etc. Fermentation is biotechnology in which desirable microorganisms are used in the production of value-added products of commercial importance. The products of fermentation are many: alcohol and carbon dioxide are obtained from yeast fermentation of various sugars. Lactic acid, acetic acid and Organic acid are products of bacteria action; citric acid, D-Gluconic acid, Coffee, Tea, Cabbage & Cucumber and Yeasts are some of the products obtained from fermentation.

The worldwide demand for biotech products is the only indication; the speed of its advance is the only set to accelerate. Indian Biotechnology industry is considered as one of the sunrise sectors in India. The industry is divided into five major segments: Bio-Pharma, Bio-Services, Bio-Agri, Bio-Industrial and Bio-Informatics. Biotechnology industry’s growth in India is primarily driven by vaccines and recombinant therapeutics.

The biotechnology sector of India is highly innovative and is on a strong growth trajectory. The sector, with its immense growth potential, will continue to play a significant role as an innovative manufacturing hub. The high demand for different biotech products has also opened up scope for the foreign companies to set up base in India. Today in India there are more than 350 Biotechnology companies in India providing employment for over 20,000 scientists.

The authors cover different aspects of biotechnology such as production of fermented foods, functional foods, enzymes in food processing. The Book contains production of Wines and Beers, Production of Amino Acids, Lactic Acid, Acetic Acid and Organic Acid, Processing of Coffee, Tea, Cabbage, Cucumber, Yeasts and Photographs of Plant & Machinery with Supplier’s Contact Details.

The book provides a better understanding about biotechnology production of value-added products, improve productivity, and enhance product quality in the agro food processing sector. The book is highly recommended to new entrepreneurs, professionals, existing units who wants to start manufacturing business of biotechnology products.

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A. Yeasts
1. Taxonomy, ecology
2. Industrially important yeasts
3. Killer (K) Yeasts
4. Effect of yeasts on the organoleptic character of wines.
1.Composition of grape musts
Nutritional requirements of yeast and their provision in musts
1. Growth cycle of yeasts and kinetics of the fermentation
2. Fermentation problems and their causes
3. Stimulation of the fermentation
4. Concept of the survival factor
1. Primary and secondary products
2. Volatile substances contributing to the aroma of wine
A. Lactic Acid Bacteria of Wines
1. Taxonomy
2. Ecology
3. The role of lactic acid bacteria in vinification
1. Development of lactic acid bacteria during vinification;
    kinetics and biochemistry of the malolactic fermentation
2. Parameters affecting the development of lactic acid bacteria
  in wines
3. Stimulation of bacterial growth and of the malo-lactic
A. Spoilage by Lactic Acid Bacteria
1. Taxonomy, ecology
3. Effect of the metabolism of acetic acid bacteria on the
  quality of musts and wine
A. Grapes and Corrective Measures for the Vintage
1. White wine production
2. Red wine production
A. Sparkling Wines
C. Brandy
Man’s First Alcoholic Drink
Man’s Earliest Brewing
1. Mesopotamia and Egypt
2. Greece and Rome
3. European tribes
4. Africa
5. China
6. India
7. South America
C. Ancient Brewing and Nutrition
D. Sanitary Considerations
E. Large-Scale Brewing
A. Classical Beer Types
B. Beer-Like Beverages
C. Beer Production in the world
A. Water
B. Alcohol
C. Carbohydrates
D. Nitrogen Compounds
E. Inorganic Constituents
F. Organic Acids
G. Carbon Dioxide
H. Other Compounds
1. Water sources
2. Water purity
3. Water minerals
4. Heavy metals
1. Barley
(a) Harvest and Storage
(b) Weathering
2. Malt
(a) Earliest Malt
(b) The malting process today
C. Brewing Adjuncts
D. Hops
1. Earliest use of hops
2. The Hop Family
3. Hop Utilization
4. Hop Chemistry
E. Brewer’s Yeast
2. Cell morphology and Physiology
3. Yeast Metabolism
(a) Carbohydrate Metabolism
The Pasteur and Crabtree Effect
(b) Metabolism of Nitrogenous Compounds
(c) Lipid synthesis
(d) Sterol Synthesis
(e) Sulfur Compounds
(f) Miscellaneous Metabolic Reactions
A. Brew house Operations
1. Milling
(a) Malt Milling
(b) Wet Milling
(c) Adjunct Milling
2. Mashing
(a) Infusion Mashing
(b) Decoction Mashing
(c) Malt Conversion
(d) Adjunct Conversion
(e) Enzyme Activity During Mashing
3. Lautering
(a) The Lauter Tub
(b) Run Off
(c) Sparging
(d) Wort Filtration
(e) Spent Grain Removal
4. Wort Boiling
(a) Heating
(b) Function of Wort Boiling
(c) Hop Extraction and Conversion
5. Wort Cooling/Trub Removal
(a) Hot Trub
(b) Wort Cooling
(c) Cold Trub Removal
(a) General Practices
(b) Microbiological Precautions
1. Carbohydrates
2. Nitrogenous Compounds
3. Inorganic Constituents
4. Vitamins
5. Polyphenols
6. Hop Compounds
7. Melanoidins and Phenolic Pigments
8. Lipids
1. Cold Wort Aeration
2. Yeast Pitching
(a) Yeast Examination
(b) Yeast Population Count
(a) Lager Fermentation
(b) High Gravity Brewing
(c) Ale Fermentation
(b) Reuse of Yeast
(c) Yeast Disposal
(d) Continuous Fermentations
(e) CO2 Recovery
(a) Washing and Preparation
(a) Culture Propagation
(b) Laboratory Checks
(a) Flavor Maturation
(b) Carbonation
(c) Standarization
(d) Chillproofing and Stabilizing
(e) Clarification
A. Bacterial Contaminants
1. Gram Positive Bacteria
(a) Lactobacillus
(b) Pediococcus
(c) Miscellaneous Cocci
2. Gram Negative Bacteria
(a) Acetic Acid Bacteria
(b) Zymomonas
(c) Enterobacteriaceae
(d) Miscellaneous Wort Organisms
1. Beer Spoiling Yeasts
2. Yeast Spoilage Flavors
3. Killer Yeasts
4. Wild Yeast Control Measures
A. Bottling Operations
1. Filling
2. Pasteurization
3. Light Struck Beer
1. Can Filling
2. Pasteurization
3. Shelf Life of Packaged Beer
(a) Oxygen
(b) Temperature
1. Cooperage
2. Racking
A. Physical and Chemical Measurements
1. Cleaning and Sanitation
2. Raw Materials Acceptability
3. Biological Survey of Beer “in process”
4. Analysis of the Finished Beer
B. Flavor Measurements
1. Tasting beer
C. Tastable Beer Defects
1. Diacetyl
2. Metallic Tastes
3. High Air Beer
4. Light Struck Beer
5. Old, Oxidized Beer
6. Medicinal Odors
7. Grainy, Harsh, Astringent, Bitter
8. Flavor Depression
A. Production of Amino Acids by Wild Strains
B. Production of Amino Acids by Auxotrophic Mutants
C. Production of Amino Acids by Regulatory Mutants
D. Production of Amino Acids from Biosynthetic Precursors
A. Hydrolytic Enzymes
1. L- a -Amino-e-caprolactam hydrolase
2. 2-Amino-D2-thiazoline-4-carboxylate hydrolase
3. Hydantoinase
B. Ammonia Lyases
1. Aspartase
2. Phenylalanine Ammonia Lyase
C. Arginine Deiminase
D. Pyridoxal 5'-Phosphate Enzymes
1. Asparate b-decarboxylase
2. b-Tyrosinase
3. Tryptophanase
4. Cysteine Desulfhydrase
5. Tryptophan Synthase
6. b-Chloro-D-alanine hydrogenchloride lyase
7. L-Methionine g-lyase
8. Serine Hydroxymethyltransferase
9. L-Threonine Aldolase
E. Other Enzymes
1. Amino Acid Dehydrogenases
2. Glutamine Synthetase
IV. Enzymatic Resolution of Racemic Amino Acids
A. Introduction
B. Enzymatic Methods
1. Resolution by Enzymatic Asymmertic Derivatization
2. Resolution by Asymmetric Hydrolysis
(a) Esterase Method
(b) Amidase Method
(c) Aminoacylase Method
A. Use for Food
B. Isolation and Characterization of Microorganisms from Zaire Coffee
A. General
B. Are Plant Enzymes Involved in Coffee Fermentation?
C. Are Microbial Enzymes Involved in Coffee Fermentation?
A. Origins of Tea
B. Types of Tea
C. Physical and Chemical Characteristics of Tea Leaves
A. Harvest of Tea Shoot Tips
B. Withering
C. Tissue Maceration (Rolling)
D. Fermentation
E. Firing
F. Grading and Storage
A. Introduction
B. Cabbage Varietals
1. Crop Distribution
2. New Hydbrids
C. Mechanical Operations
1. Mechanical Harvester
2. Grading
3. Core Removal
4. Trim
5. Shredding
6. Salting
7. Conveyance
8. Fermentation Tanks
  Tank Closure
D. Fermentation
E. Product Defects
1. “Off” Flavor
2. Color Defects
3. Processing Defects
1. Bulk Sauerkraut
2. “Hot Fill” Method
3. Chemical Preservatives
A. Production and Consumption
B. Varietals and Harvesting
C. Grading
D. Fermentation
1. Salt Stock
(a) Development of Flora
(b) Defects
(c) Controlled Fermentation
2. Dill Pickles
3. Spoilage
4. Preservation
A. Fermentation
B. Isolation
C. Economic Aspects
A. General
B. Bases of Acetic Acid Fermentation
C. Raw Materials
D. Water for Processing
E. Nutrients
A. Summary and Basic Problems of Classification
B. Industrially Used Strains
A. Ethanol
B. Sugar
C. Acetate
D. Carbon Dioxide
E. Nitrogen
F. Growth Factors
A. Oxygen Demand and Total Concentration
B. Lack of Ethanol
C. Specific Growth Rate
D. Specific Product Formation
E. Changes in Concentration
F. Overoxidation
A. Submerged Vinegar Fermentation
1. The Frings Acetator
2. Other Processes
3. Abandoned processes
B. Surface and Trickling Processes
1. History and Surface Process
2. Older Trickling Processes
3. The Frings Generator
C. Production of Concentrated Acetic Acid
Itaconic Acid
Malic Acid
A.2-Oxogluconic Acid
B.5-Oxogluconic Acid
C.2,5-Dioxogluconic Acid
D.2-Oxogulonic Acid
A. Succinic Acid
B. Pyruvic Acid
C. 2-Oxogalactonic Acid
D. Kojic Acid
Calcium Gluconate Fermentation
B. Sodium Gluconate Fermentation
Biological Fundamentals
A. Strains
B. Fermentation Medium
C. Other Factors
D. Biochemistry and Enzyme Regulation
A. Production Strains
B. Spore Propagation for Inoculation
C. Raw Materials
D. The Koji Process
E. Surface Process
F. Submerged Process
A. Introduction
B. Reactions from Glucose to Cell Material
Some Early Observations
2. Evaluation of the Reactions from Glucose to Cell Material
Yeast Saccharides
Yeast Protein
Nucleic Acids
Neutral Fat
    Equation for Yeast Growth on Glucose
C. Flux of the Substrate During Yeast Growth
A. Composition and Properties Molasses
B. Compressed Yeast from Molasses
1. The Evolution of Baker’s Yeast Production
2. Aspects of the Biochemistry of Baker’s Yeast
3. Requirements for Baker’s Yeast Production
4. Outline of the Manufacturing Process
5. Analysis and Quality Control of Baker’s Yeast
C. Active Dry Yeast
D. Wine Yeast Cultured on Molasses
E. Feed Yeast from Molasses
A. Spent Sulfite Liquor
B. Biomass from Spent Sulfite Liquor
1. Candida Yeasts
2. Baker’s Yeast from Spent Sulfite Liquor
3. Pekilo Process

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Sample Chapters

(Following is an extract of the content from the book)
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The use of wine goes back to times immemorial; the bible, Homer’s epics, Egyptian and Assyrian documents mention it. However, one must wait for the time of the middle Ages before the alchemists discover the active principle, ethanol. At the end of the 17th century A. Van Leeuwenhoek describes years in grape musts and beer worts, but he establishes no relationship between yeasts and fermentation. A century later Lavoisier publishes the first scientific work on fermentation which he considers a purely chemical phenomenon. Only in 1836 does Cagnard-Latour prove the role of yeasts, living organisms which cause biochemical transformations.

        In 1866 Pasteur publishes his celebrated work, “Etudes sur le vin,” in which he analyzes the spoilage of wines and prescribes appropriate treatment. This is truly the origin of scientific oeanology, which has continued to progress from that point on.

        Taxonomic and systematic studies on the specific microorganisms of musts and wines have multiplied since the beginning of this century. They have led to a good knowledge of the microflora. Since 1950 the development of chemical, chromatographic, and enzymatic techniques has led to a more sophisticated analysis, to the determination of several hundred components of wine, and, consequently, to a better approach to the biochemical phenomena and to a better understanding of the theory of fermentations. The last decades have also seen a significant mechanization of the production of wines. In contrast the transfer of the knowledge of genetics to the field of enology proceeds more slowly.

        Traditionally the transformation of must into wine remains a “spontaneous event”. That means, it is induced by indigenous microorganisms which are found on grapes and equipment used in wine making. It is sufficient to crush the grapes in order to start the fermentation. This implies that it is difficult to control the agents of fermentation. Nevertheless, the acid pH of grape must constitute a very selective medium in which only a limited number of microbial species can multiply. These belong to three types of microbes: yeasts (preferentially), lactic acid bacteria and acetic acid bacteria.

        The acetic acid bacteria as agents of biochemical change must be inhibited. The yeasts and the lactic acid bacteria, each, participate successively in the two principal stages of the fermentation. In the first stage yeasts transform sugars into alcohol; this is the alcoholic fermentation. The second stage, which occurs sometimes, favors the conversion of malo-lactic fermentation acid to lactic acid by the lactic acid bacteria; this is the malolactic fermentation. The production of certain special wines requires a secondary alcoholic fermentation under anaerobic conditions (sparkling wines) or a secondary growth of yeasts under aerobic conditions on alcohol (sherry).

        The composition of the medium, notably the concentration of must sugars, affects the growth of yeasts; the higher concentration of alcohol and the acid pH of the wines affect the growth of lactic acid bacteria. This and the technological operations impose upon the microorganisms a growth cycle and a particular metabolism which constitute the particular characteristic of this biotechnology.

                Brandy is obtained by distillation of wines. This procedure was already known in ancient times. Avicienna, an arab physician and philosopher, gave the first description and philosopher, gave the first description of a still in the 10th century. Arnaud De Villeneuve is the first to speak of “alcohol”, and one attributes the discovery of the “eau de vie” to one of his students. Throughout the Middle ages the use of brandy remains strictly medicinal, and its production remains quasi secret. It changes from the art of the alchimist to an industrial art thanks to the work of the physician Boerhave during the 18th century. The “brandwine” (burnt wine) diluted with water became during the 17th century the normal Beverage of Dutch sailors. The tonic can be stored in concentrated form without change, and it is indispensible in the fight against the epidemics of tropical countries. A lack of sales caused by wars leads to the discovery that brandy ameliorates during aging. Today there are several kinds of brandy distinguished by their origin, the quality of the base wine, and the nature of the distillation. The best known are undoubtedly cognac and armagnac.

        All prestigious products have been created empirically. They are “the fruit of patient care... . the reputation of the great wine regions having advanced, and greatly thanks to chemical and biological studies.” Therefore one easily attributes the merits of wines to the quality of nature, and sometimes one questions the role of enology.

        Ribereau-Gayon and Peynaud supply a precise answer to this question in their “Traite d. “A good wine or a great wine may be obtained without the aid of modern enology. However, the aid of modern enology is in general required to obtain a better quality regularly and with certainty and with the most efficient means.” And, finally, “enology is more than a specialty. It is central interest around which one  can create and coordinate a comprehensive program of fundamental research which is authentic and of general interest.”



A. Yeasts

1. Taxonomy, ecology

        Numerous studies of wine yeasts have been undertaken in the vine growing regions of the entire world. Their identification is based on Lodder’s “The yeasts” (1970) supplemented by the work of Barnett. It rests basically on a consideration of morphological and physiological characteristics, the metabolism of certain sugars, and the assimilation of diverse nitrogenous compounds.

        Wine yeasts belong to the class of Ascomycetes (sporogenous) and Deuteromycetes (asporogenous). Ample documentation shows that the number of species which occur with a frequency surpassing 1% is no greater than about 14. The presence of about 40 other species is fortuitous. In their ecological niches yeasts are often associated with molds, lactic acid bacteria, and acetic acid bacteria. Their isolation and specific counting is done at an acid pH and in the presence of mold inhibitors Identification techniques have been simplified by the use of the “Api system”.

                Soil yeasts are spread by wind and insects. Fruit flies and bees play a predominant role in their dissemination. Yeasts are present on vines from the start of ripening. The populations reach a maximum at the time of maturity. Yeasts are not numerous on stems and leaves where they form a pseudomycelium. They colonize all exudates of the openings of stomata and injuries on the surface of the grapes. Growth of cells stops at the point of contact with the cuticular wax. On scanning electron photomicrographs yeast cells can be recognized by their shape, the absence of ornamentations of their surface, and by their bud scars.

condition of the vintage, and climatic conditions, mainly the temperature. The effect of the grape variety is minor certain chemicals applied to fight various parasites of the vines can also effect their distribution.

Each medium is solidified by the addition of an equal volume of 2% agar solution.

        On grapes one finds essentially molds, the yeast Aureobasidium pullulans, and yeast species with oxydative metabolism (Rhodotorula) or with little fermentation activity. Among the latter, Hanseniaspora apiculata  and its imperfect form, Kloeckera apiculata predominate (99% of the isolated yeasts). Metschnikowia pulcherrima, Pichia membranaefaciens, and Hansenula anomala are less frequent; and other species may occur fortuitously.

        All studies confirm the extreme rarity of the occurence of Saccharomyces cerevisiae on grapes. The essential yeast microflora on other supports (beams,vaults,cellar walls) consists of Hanseniaspora uvarum, Hansenula anomala, Metschnikowia pulcherrima, Pichia fermentans, and P.membranaefaciens.

        During subsequent manipulations of grapes the yeasts with a strong fermentative capacity develop preferentially and invade the raw material.

        In white wine production a selective effect in favour of S.cerevisiae is due to the elimination of the pomace, the clarification of the must (which decreases the total population substantially), as well as to sulfiting of the crushed grapes or the must.

During the first hours of the fermentation one encounters large populations of Hanseniaspora uvarum and Kloeckera apiculata which are rapidly superseded by Saccharomyces yeasts. The species Torulopsis stellata seems to be characteristic of certain regions with”noble rot”(Sauternais). All authors agree on the preponderance of S.cerevisiae several days after the onset of the spontaneous fermentation. S.bayanus often considered the “finishing yeast”exists–often from the start-with S.cerevisiae. It is favoured by the presence of residues from some chemical treatment of the vine.

        Under particular circumstances one can induce the growth of Schizosaccharomyces or of Saccharomyces rosei.

        In wines, populations of Zygosaccharomyces, saccharomycodes ludwigii, and (as surface growth) species belonging to the genera Pichia and Candida can predominate accidentally. The growth of certain strains of Saccharomyces bayanus as film forming yeasts has been considered for the production of special wines.

2.  Industrially important yeasts

        The principal yeasts fermenting must and responsible for biochemical changes in wines can be identified on the basis of a limited number of criteria (morphological and physiological). The effect of their occurrence on the essentials of wine quality is known. For the sake of simplicity LODDER has combined in one species some yeasts closely related on the basis of some physiological characteristics, however, distinct on the basis of their interest for enology.


        Saccharomyces cerevisiae (syn.: S. cerevisiae var. ellipsoideus, S. ellipsoideus, S.vini). This is the wine yeast par excellence. 80% of the yeasts isolated from fermenting musts in the Gironde belong to these species, as well as 98% of the spices identified at the end of the fermentation. Its shape is globular. The yeast starts fermentations rapidly and has a good resistance to ethanol (8–15 vol%). It disappears rapidly from wines during storage. Certain so called aromatic strains have been selected for their ability to form higher concentrations of esters and higher alcohols. According to Maugenet such yeast can modify the organoleptic properties of wines made from less aromatic vintages.

                Saccharomyces bayanus (syn.: S.oviformis, S.beticus, S.cheriensis, S.rouxii). This is the finishing yeast thanks to its capacity to form high concentrations of alcohol, up to 19 vol%. It is found mainly towards the end of the fermentation. The use of S. bayanus has been prescribed for the refermentation of residual sugars in stuck fermentations and for the inoculation of musts from over-ripe grapes. However, its resistance to  SO2 (up to 100mg/L) is the reason for almost all of the re-fermentations of sweet, white wines by this yeast. In addition it has the disadvantage of forming relatively important concentrations of acetic acid in the course of the alcoholic fermentation. Recent taxonomic studies by Belin include S.bayanus in the species S.cervisiae. It seems to us that the quite distinct physiological properties of the two species, notably the ability of S. bayanus to grow in films and is inability to degrade galactose, justify the maintenance of two separate species.

        Cultures of S. beticus and S. cheriensis are used in the production of sherry. They develop on the surface of the base wine (fortiffed with alcohol) because of their respiratory metabolism of ethanol. They liberate large concentrations of acetaldehyde and esters and degrade acetic acid.


        Saccharomyces rosei (syn.: Torulasporarosei). This is a sphercial yeasts of slow growth. It is found on grapes and in musts undergoing spontaneous fermentation particularly in hot climates. Its  alcohol producing capacity varies from strain to strain, but is generally quite feeble (6-10 vol%) which explains its absence at the end of the fermentation. The fermentation of acetic acid by S. cerevisiae increases notably at low pH values and at elevated sugar concentrations. In contrast S. rosei produces  only  small concentrations of acetic acid (0.04g/L) regardless of the composition of the medium. This property can be utilized in the technique of successive fermentations to lower the volatile acidity of wines from grape musts which are too acid. At the end of the growth phase of S. rosei (4th day of the fermentation) one inoculates with a strain of S. cerevisiae which ferments all of the remaining sugar. More recently, use of this yeast has been suggested for the vinification of over-ripe grapes which have been invaded by Botrytis cinerea. The lowering of the concentration of acetic acid in such successive fermentation is the result of two phenomena: (a) S.rosei produces little acetic acid; (b) its growth produces conditions which decrease the rate of the glycero-pyruvic fermentation during the succeeding fermentation by S.cerevisiae or S.bayanus in this medium. Saccharomyces capensis is closely related to S.rosei in its physiological properties.

        Saccharomyces uvarum (syn. S.carlsbergensis). Frequently found in beer worts this yeast is also found in musts. Its alcohol producing ability is intermediate. Its growth is readily inhibited by actidione. A certain number of strains of S. uvarum can be found among yeasts capable of liberating small quantities of SO2 during the fermentation.

                Saccharomyces chevalieri (syn. S.fruturum, S.lindneri). this yeast approaches S.cerevisiae in its physiological properties, and it  is frequently found in fermenting musts. It is distinguished by its inability to ferment maltose.


                Saccharomycodes ludwigii. The large cells of this apiculate yeast can attain a length of 25 mm and show bi-polar budding. A single species is known. Rarely seen on grapes this yeast occurs sometimes in sulfited musts. It produces up to 16.8% vol% ethnol and is highly resistant to SO2 (600 mg/L). It is strongly acetogenic and can produce up to 200 mg/L of acetaldehyde during anaerobic fermentations. 70% of the cultures of T. stellata isolated by Domercq in the Gironde come from areas with “noble rot”, and it also occurs frequently in Italian vineyards. It occurs side by side with Hanseniaspora uvarum during the first days of the spontaneous fermentation. It relatively feeble alcohol producing capacity (on the average 10 vol% EtOH) accounts for its disappearance towards the end of the fermentation. This osmophilic yeast also tolerates relatively high temperatures (30-35°C). It ferments fructose faster than glucose.

        Hanseniaspora uvarum and its imperfect form Kloeckera apiculata. The small cells are lemon shaped, and may also be recognized by their bippolar budding. They constitute about 95% of the microflora of grapes. Although these yeasts are responsible for the spontaneous fermentation of fresh musts, they are quickly supplanted by Saccharomyces yeasts because of their low alcohol tolerance (3.7-6.4 vol%). They form important concentrations of acetic acid, 1 g/L on the average, and ethyl acetate, 125-374 mg/ L as well as amyl acetate and glycerol. The desirability of these species is controversial. Some authors think that they produce in wine a particular fruity aroma. Others consider them undesirable because of the formation of excessive concentrations of ethyl acetate. A technique of vinification called “la fermentation superquatre” has been suggested for their elimination. Their sensitivity to SO2 explains their small number in sulfited musts, that is, in the majority of cases.

                Hansenula. This genus include 25 species. Hansemula anomala is often found on grapes and it has been isolated from musts in many wine growing areas. On a liquid medium this species develops a wrinkled film. The ascospores are hat shaped. Its fermentation capacity is small, and it liberates contaminates poorly maintained cellers and is one of the most feared spoilage yeasts. Its development in wine bottles is characteristic.

Amino Acids


        An amino acid is generally defined as a compound that possesses amino and carboxyl groups. Some amino acids, however, are iminocarboxylic acids, such as proline, and others are aminophosphonic acids. In nature, there are about 20 amino acids which form a huge variety of complex copolymers, the proteins; 8 of them, the essential amino acids, are required for human growth. All the amino acids are L-a-amino (or imino) carboxylic acids except glycine, which is achiral. In addition to these proteinaceous amino acids, various non-protein amino acids containing D-amino acids and w-amino acids occur naturally in free and bound forms, and play important roles in metabolism. Amino acids are of importance not only as nutrients, but also as seasonings, flavorings, and starting materials for pharmaceuticals, cosmetics and other chemicals.

                Amino acids may be prepared by isolation from natural materials, by microbial or enzymatic procedures, or by chemical synthesis. The first two procedures give optically active (usually L-) amino acids, whereas the chemical methods in general produce the racemates, and an additional optical resolution step is necessary to obtain optically active amino acids. Originally amino acids were exclusively obtained by the hydrolysis of plant proteins. L-Glutamate was identified as a taste component of Konbu (kelplike seaweed), used for seasoning in Japan and had been produced up to the 1950s from wheat gluten, soybean and corn proteins, and from sweet turnip molasses by separation of the acid hydrolysate. Other amino acids were also obtained from proteins in a similar way. With the increasing demand for various amino acids, chemical procedures were used for their syntheses. For example, in the Dupont process DL-glutamate is synthesized from acetylene with the intermediate formation of acrylic acid ester and the methyl ester of b-formyl propionic acid:

HC = CH   CO2 ROH        CH2 = CH—COOR     CO2 H2


                                    Strecker Synthesis          DL-glutamic acid

        In the Ajinomoto process acrylonitrile is converted to b-cyanopropionaldehyde and then to DL-glutamate in a high yield:

CH2 = CH – C = N       CO2H2           OHC—CH2—CH2—C=N

                                Strecker Eynthesis                 DL-glutamic acid

        The enzymatic and chemical methods for the optical resolution of DL-amino acids have been developed simultaneously with the progress made in their chemical synthesis.

        The investigation into the accumulation of L-glutamate in bacterial cultures by Kinoshita opened the door to the microbial production of amino acids.  The amino acid producing microorganisms were improved genetically to yield amino acids more efficiently. The production of amino acids, e.g., L-lysine, using auxotroph and regulatory mutants has been developed to industrial scales. Numerous studies on the microbial production of amino acids have been carried out, and an increasingly large number of optically active amino acids are now produced using bacteria, with the exception of a few, such as methionine.

        On the other hand, the enzymatic procedures for amino acid production have been extensively studied, since Kitahara reported the efficient conversion of fumarate and ammonia into L-aspartate with bacterial aspartase. Several amino acids have been produced industrially with microbial enzymes. The microbial and enzymatic methods have their merits and demerits. In the microbial production (amino acid fermentation), amino acids can be synthesized from simple and cheap raw materials such as glucose, acetate or molasses, and ammonium sulfate or urea, but the production is  time consuming and the required amino acids have to be separated from the other amino acids formed and from various impurities including microbial cells. The waste may cause water pollution especially when molasses is used as a carbon source. A few amino acids can be synthesized bacterially from biosynthetic precursors produced chemically; L-tryptophan and L-serine are produced effectively from anthranilate and glycine, respectively.

        The enzymatic procedure surpasses the microbial one for the following reasons: it is less time consuming and usually more efficient, and does not produce complex impurities and wastes. However, the enzymatic method is not suitable for the production of amino acids from simple materials. The substrates are either precursors, or other chemicals related to amino acids and are generally more expensive in comparison with the starting materials required in the microbial methods. The enzymes used are neither cheap nor stable. Immobilization of enzymes has been developed to diminish these demerits of the enzymatic methods. The microbial, enzymatic and chemical methods have rapidly advanced in competition with each other to supply us with various amino acids.


        A variety of microorganisms accumulate amino acids in the culture fluid. However, only bacteria have sufficient productivity to warrant the commercial production of amino acids. Since amino acids are essential components of microbial cells and their biosyntheses are teleologically regulated to maintain an optimal level, they are normally synthesized in limited quantities and subject to negative feedback regulation. The importance of “preferential regulation” in amino acid biosynthesis has also been recognized. Therefore, it is necessary to overcome the regulation to achieve overproduction of amino acids. Microbial amino acid over-production can be achieved using the following procedures: 1. stimulation of the cellular uptake of the starting materials, 2. hindrance of the side reactions, 3. stimulation of formation and activity of the enzymes for the biosynthesis, 4. inhibition or reduction of the enzymes concerned with the degradation of the amino acids produced, and 5 stimulation of the excretion of the product into the extracellular space. The above requirements have been attained by introducing mutation techniques.

A. Production of Amino Acids by Wild Strains

        Since the isolation of L-glutamate producing strains by Kinoshita a number of bacterial strains producing more than 30 g/ L of L-glutamate from carbohydrates have been reported. These bacteria have been assigned to various genera and species: Corynebacterium glutamicum, Brevibacterium flavum, Brevibacterium thiogenitalis and Micro bacterium ammoniaphilum. In their microbiological characteristics all these bacteria resemble the strain of Corynebacterium glutamicum that Kinoshita isolated first: Gram positive, non-spore forming non-motile, coccal or rod like, and all requiring biotin for growth. Most of them can utilize acetate or ethanol as a carbon source for L-glutamate production, but neither methanol nor n-paraffins.

                The nutritional requirement for biotin, and the lack or very low activity of a-ketoglutarate dehydrogenase complex are important factors in the accumulation of L-glutamate. SHIIO et al. observed an overproduction of L-glutamate (2.3 g/L) in an a-ketoglutarate dehydrogenase defective mutant of Escherichia coli. having no requirement for biotin. The parent strain could not accumulate L-glutamate, and therefore, a lack of the enzyme was regarded as the prerequisite for L-glutamate production. The presence of suboptimal amounts of biotin (2.5-5.0 µg/L) causing maximum growth led to efficient L-glutamate production, but in the presence of an excess of biotin (25-30 µg/L), L-glutamate was produced poorly, and other compounds such as lactate and succinate were formed. Oleate was found to be a substitute for biotin, not only for bacterial growth, but also for the accumulation of L-glutamate. An oleate requiring mutant of Brevibacterium thiogenitalis accumulated L-glutamate in a biotin-rich medium with a limited supply of oleate. The utilization of b-lactam antibiotics such as penicilin and cephalosporin C and of surfactants or of C16 - C18 saturated fatty acids also allowed the production of L-glutamate even in a biotin-rich medium, e.g., in media containing cane or beet molasses. Biotin, oleate or C16 - C18 saturated fatty acids cause changes in the fatty acid composition of the cell membrane, particularly in the content of oleate and palmitate, resulting in an alteration of the permeability barrier of the cell membrane to L-glutamate. This phenomenon can be fully explained by the action of acetyl-CoA carboxylase, which contains biotin as its prosthetic group. The enzyme participates in the biosynthesis of oleate and of other fatty acids, and is inhibited by C16 - C18 saturated fatty acids. In contrast, the effect of penicillins on the cell permeability to L-glutamate cannot be ascribed to a decrease in the oleate content. After the addition of penicillin many cells swell or elongate. Therefore, the action of penicillin is thought to cause an incomplete biosynthesis of the cell wall, which results in glutamate excretion. On the other hand, the biotin auxotrophs and the oleate auxotrophs cannot be employed for L-glutamate production from n-paraffins. The degradation pathways of glucose and of n-paraffins are different as shown in Fig. 1. The excretion of L-glutamate in the presence of penicillin by n-paraffins assimilating strains is accompanied by the excretion of phospholipids and of UDP-N-acetyl-glucosamine derivative. Nakao isolated a glycerol auxotroph having a defective L-glycerol-3-phosphate; NADP oxidoreductase, from n-paraffins assimilating Corynebacterium alkanolyticum, in which about 40 g/L of L-glutamate is produced from n-paraffins by addition of 0.01% of glycerol in the absence of penicillin. The extracellular accumulation of L-glutamate by the mutant was found to be dependent on the presence of glycerol, and the maximum production was obtained at low levels of cellular phospholipids. These results suggest that the permeability of L-glutamate through the bacterial cell membrane is controlled not only by the cellular content of unsaturated fatty acids, especially of oleate, but also by the cellular content of phospholipids.


Cabbage & Cucumber Processing

General Introduction

        From a historical point of view the fermentations of cabbage (Brassica oleracea) and cucumber (Cucumis sativus) had their inceptions in the Far East, notably China and India. As civilization began to develop and expand into new hemispheres, the art of using lactic acid fermentations became firmly entrenched as an ideal method for preserving fruit and vegetable products.

        The use of acidic fermentations by both the home and commercial food producer has been perpetuated because properly fermented products possess distinct and unique flavor characteristics. They are incapable of supporting the growth of microorganisms of public health significance and furthermore, the method permits commodities to be stored for prolonged periods of time without seriously impairing the physical and nutritional qualities of the product.

        The spectrum of horticultural commodities that undergoes acidic fermentations is quite extensive (green beans, beets, brussel sprouts, cabbage, carrots, cauliflower, celery, cucumber, olives, onions, peppers, green tomatoes, turnips etc.); however, only a few of these commodities are consumed in quantities of sufficient magnitude, to warrant their production on an extensive industrial scale. For example, The United States only three commodities (cabbage, cucumbers and olives) provide significant contributions to the overall production volume of the fermented food industry.


A. Introduction

        The transformation of shredded cabbage to sauerkraut is, from a mechanical point of view, a very simple operation; however, from a biochemical and microbiological viewpoint the fermentation is enveloped in an array of complexities.

        A schematic diagram depicting the steps involved in producing commercial sauerkraut is shown in Fig. 1, and the functions of the respective operations will be discussed in the ensuing paragraphs.

B. Cabbage Varietals

1. Crop Distribution

        Cabbage used for sauerkraut production is generally considered to be a “cold crop”, i.e., its hardiness, proper development and maturity occur under those climatic conditions found within the geographical latitudes of, or equivalent to, those associated with the Northern United States. This area is comprised chiefly of the states of New York, Ohio, Michigan, Wisconsin, Colorado, and Oregon. More than 200000 tons (metric) of fresh cabbage for sauerkraut are harvested annually, and nearly 60% of this yield is produced in New York and Wisconsin.

2. New Hydbrids

        Although the per capita consumption of sauerkraut, 0.64-0.72 kg. has remained quite constant throughout the past decade, the varietals of cabbage used to produce the final product have undergone significant changes. Many of those cultivars (Glory, Old Glory etc.) used for fermentations 10 to 15 years ago have been replaced by more vigorous species that area more amenable to current harvesting and ecological practices. For example, the need to tolerate the physical abuse incurred during mechanical harvesting and the environmental demands to reduce the generation of excessive volumes of spent fermentation brines has been resolved in part, by developing new hybrids that are compatible with the requisites of conservation. This is especially evident with the development of the “hi solids” species, i.e., cultivars genetically derived to contain lesser amounts of native water. These varietals contain at least 20% more dry weight than their predecessors and provide similar increments in the yield of marketable product. Decreasing the moisture content of those varietals currently used in commerce has no adverse effects upon product quality; however, it markedly reduces the discharge volumes of waste effluent.

        It should be noted, however, that some experimental hybrids have been developed that will not support an adequate lactic acid fermentation. Apparently these derived hybrids contain growth inhibitors or are deficient in nutrients essential for the growth of lactic acid bacteria.

C. Mechanical Operations

1. Mechanical Harvester

        The modern mechanical harvester, capable of harvesting 7 to 14 tons of cabbage per hour, has essentially replaced the laborious process of cutting cabbage by hand. In New York more than 97% of the cabbage used for sauerkraut product is harvested by machine. Mechanical harvesting provides the processor with a continuous and ample supply of fresh cabbage and has thereby eliminated the need to stockpile cabbage for anticipated usage.

2. Grading

        Once on the processor’s premises the cabbage is subjected to rather intensive inspections. The criteria used for grading purposes include: type and extent of pest and insect include: type and extent of pest and insect damage; head size (minimum diameter: 14 cm); shape and firmness of head; length of internal core; presence of internal “spotting” and rot; and the amount of unusable leaf material (waste material).

3. Core Removal

        Following the grading critique the bulky outer leaves (wrapper leaves”) are removed in part by the mechanical delaying machine consisting of a series of horizontal counter-rotatory rollers.

        Following partial defoliation the heads are conveyed to the coring station The coring machine is equipped with automatic vertical augers “drills” and removes the dense core matter. The removal of the tough core material is essential for producing a uniformly shredded cut.

4. Trim

        Upon removal of the core, the heads proceed to the final trim line. At this point the skilled operators cut and remove extraneous green leaves, a step required for producing a sauerkraut that a devoid of dark brown spots arising from chlorophyll blemish. In order to achieve this latter goal, 26 to 42% of the gross weight of fresh cabbage delivered to the processing plant is ultimately discarded as waste material.

5. Shredding

        The shredded cabbage is prepared by passing the trimmed head through a mechanical slicer equipped with rotating knife edges affixed in a horozintal plane. A single shredding machine fitted with a circular blade 80 to 90 cm in diameter, is capable of producing 8 to 10 tons of sliced cabbage per hour. The length and thickness of the shreds are determined by the distal setting of the blades and can be adjusted to those specifications established by the individual processor, usually about 0.10 cm in thickness.

6. Salting

        The next step, the application of crystaling salt (food grade) to the shredded cabbage, is an extremely important factor because it directs the course of the fermentation and influences the quality of the final product. Salt serves as an important determinant because it:

        •    rapidly extracts from the plant cells those nutrients required to support microbial growth,

        •    inhibits the growth of some undesirable microorganisms,

        •    contributes to maintaining optimum textural properties,

        •    serves as a flavor ingredient in the final product.

        The significance of salt as a vital adjunct for the commercial production of sauerkraut in the United states was recognized at the turn of this century and the establishment of standards of quality by the United States Department of Agriculture in 1925 clearly states that sauerkraut must be produced in the presence of not less than 2% nor more than 3% salt. This declaratory definition remains in effect today.

        The methods used for salting cabbage have, however, been more modernized. The fluming of cabbage in a brine solution has been totally abandoned and the “salting by hand” technique has been replaced for the most part, by automatic salting devices. These automatic salters are designed to dispense granular salt a top a thin layer of shredded cabbage. In principle the changing mass of cabbage passing a fixed point in the conveyor line in continuously measured and monitored by electronic sensors that subsequently proportion the salt on a differential weight basis. Under commercial processing conditions, salt is applied at the rate of 0.9 to 1.1 kg per 45.5 kg of shredded cabbage.

Organic Acid of Minor Importance


        In this section the production of certain organic acids by microorganisms will be described. The demand for these acids is however, low because of their very specialized fields of application or because the acid can be produced more economically synthetically than by fermentation. It is possible that this trend will change in the future due to rising costs of petrochemicals and the discovery of new applications.


Itaconic Acid

        CH2-COOH                     MW         130.10


        C-COOH                         mp           161-162°C


        CH2                                 D             1.6

        Itaconic acid is at present the most important of the acids considered in this section.

        This metabolic product was discovered by Kinoshita in 1929 in a culture of Aspergillus, thereafter named Aspergillus itaconicus. In the following period several other species of Aspergillus were found, which were also able to produce itaconic acid.

        As far as is known the biosynthesis follows the Embden-Meyerhof pathway; with the subsequent decarboxylation of the formed cis-aconitate. As in other fermentations for organic acids the pH of the medium is very important for the formation of the desired product. At pH 2.1 all the glucose is metabolized to itaconic acid, at pH 6.0 other acids are formed instead. Results of a two-stage fermentation showed that if the preculture was grown at pH 2.1 the main culture resulted in a good production of itaconic acid, independent of the pH (2.1 or 6.0). On the other hand, no taconic acid was produced in the main culture at either pH if it was inoculated with a preculture grown at pH 6.0. The Northern Regional Research Laboratories, Peoria, Illincis, USA described a production process in the mid-forties. At first a surface culture in aluminum pans was the method chosed (Lockwood and Ward, 1945). The yield of itaconic acid was 30-50% of the added sugar. This was a rather good yield if one considers that theoretically 100 g glucose are transformed to 72 g itaconic acid. A subsequently developed submerged process, however, is of greater interest. The stock cultures of Aspergillus terreus NRRL 1960 were sustained on malt agar: 2.5% malt extract 0.1% peptone, 2% dextrose and 2% agar. For the preculture and the main culture a medium of the same composition was used: 6% glucose monohydrate, 0.27% ammonium sulfate, 0.08% MgSO4 7H2O and 0.18% corn steep liquor. In this case the best yields were reached at pH 5.0. The inoculation volume was kept at 11%, the temperature held at 37 °C, the pressure in the fermenters was 1 bar, the aeration rate 0.25 v/v/min and the agitation speed 115 rpm (propeller type). After 3 days the maximum yield was reached. On an average, 100 g glucose gave about 60 g itaconic acid, which corresponded to 80% of the theoretical yield. After separation of the mycelium, the filtrate was vacuum concentrated. The crystals were isolated by centrifugation and washed. Treatment with activated carbon and recrystallization led to a product with a purity of 99%.

        A survey of the literature shows that this process has been modified. Other carbon sources have been used, for example, high-test molasses, molasses and sucrose, the pH has been varied, and modified strains have been developed by mutagenic treatment. Other naturally occurring microorganisms have also tested, e.g., a species of Rhodotorula, however, with little success.

        Itaconic acid is solely used in the plastic and paint industry. Apart from few manufacturers in Western Europe, Russia and Japan, Pfizer is the main producer of this acid.

Epoxysuccinic Acid

                                                      MW      132.08



                                                     mp         180-185°C (decomp.)

        During the search for few raw materials epoxysuccinic acid was found as a metabolic product in cultures of Paecilomyces sp. and Aspergillus fumigatus. Application of isotopic tracers showed that it was formed by direct oxidation of fumaric acid. With mutated strains and sucrose as substrate a 39% yield was obtained.

        Further metabolism by means of other microorganisms did not lead to the desired D(+)-tartaric acid, but to mesa tartaric acid, which is of no economic interest. Presumably the trans-configuration of epoxysuccinic acid was used, because according of Kamatani cis-epoxysuccinate should give the naturally occurrign D(+)-tartaric acid.

Malic Acid

   COOH                         MW         134.09


H—C—OH                      mp           100°C


H—C—H                         [a]D18 =    – 2.3°C


    COOH                         D             1.595

        Malic acid, used as an acidifying agent in the food industry, is usually chemically synthesized as a racemate. In nature only L(–) malic acid is found, which can also be produced biotechnologically. However, this method has to complete with the cheaper enzymatic conversion, called transcrystallization. Starting with fumaric acid and with whole cells of microorganisms or enzymes isolated from them the transformation proceeds with good yields.

        Degen used microorganisms of the genus Paracolobactrum for a fermentative process. The medium contained 2% fumaric acid, 2% glucose, 0.1% yeast extract, 0.5% NaNO3, 0.2% K2HPO4, 0.05% MgSO4 - 7H2O, and 0.1%, Tween 80. At pH 7 and 30 °C with agitation and aeration 75% of the added fumaric acid was converted into L(–)-malic acid within 88 hours.

        In a similar medium, but without glucose and Tween 80, it was possible to grow cells at 30°C with agitation and aeration (0.1 v/v/min) for 23 hours to yield 10 g wet biomass per liter. These cells could be used directly or after treatment with acetone or ultrasonic vibration. The yield of malate amounted to over 70% of added fumarate, within 6-12 hours.

        Over 90% of malate was obtained from calcium fumarate using fumarase from cultures of Lactobacillus brevis.

        Immobilized fumarase prepared from pig heart or from cultures of Paracolobactrum aerogenoides was used by Morisi and Chibata. The enzyme was immobilized with cellulose or with polymers of acrylate. In this procedure the enzyme could be reused several times if it was separated from the reaction mixture (90% yield) or by the application of column reactors. The turnover of fumarate to malate was 60-85%. In the column method a turnover rate of 80% was obtained over a period of 35 days.

        The reaction mixture consisting of sodium fumarate and malate was acidified with HCI, the precipitated fumaric acid filtered off and, by addition of CaCO3 to the filtrate, Ca-malate was recovered in crystalline form, Pure malic acid could be obtained by treatment with sulfuric acid and purification by ion exchange.

        Recently, procedures have been described using carbon sources other than sugar or fumarate for he production of malate. It was possible to obtain yield of 72% of L9-) malic acid from n-paraffins in a two-stage procedure, the first using Candida hydrocarbofumarica and the second using Candida utilis. Similar high yields of L(–)-malate could be achieved with ethanol as substrate using Schizophyllum coommune.

Oxogluconic Acids

A. 2-Oxogluconic Acid

       COOH                    MW         194.14











        Several bacteria are able to produce 2-OXO-D-gluconic acid from glucose or gluconate. Several species of Acetobacter and Pseudomonas belong to this group. In the first step glucose is oxidized to gluconate. The oxidative enzyme system seems to be bound to the plasma membrane. A separate enzyme system further oxidizes the gluconic acid to oxogluconate.

        Misenheimer described a method by which nearly 100% yield was obtained with aerated cultures of Serratia marcescens NRRL B-486. Yields depended on the amount of glucose used as substrate. The best yields were achieved in a 20 liter batch under the following conditions: 12% glucose, 0.19% ammonium sulfate, 0.05% sodium sulfate, 0.04% MgSO4 7 H2O, 0.5%, KH2PO4, 0.004% NaCI, 0.004% MnSO4.4H2O, 0.005% ferroammonium sulfate and 3% CaCO3. The medium was inoculated with 5% v/v of bacterial suspension, cultivated at 30 °C under aeration (0.75 v/v/min) and agitated at 400 rpm. Within 16 hours the yield reached 95-100%. Larger amounts of glucose could be used if they were added either stepwise of continuously. In this way it was possible to obtain yields of 95-100% within 24 hours with 180 g glucose per liter, or 85-90% within 32-40 hours with 240 g glucose per liter.

        The 2-oxogluconic acid is recovered by filtration, concentration and crystallization and serves as an intermediate in the production of erythorbic acid (D-araboascorbic acid).


B. 5-Oxogluconic Acid

       COOH                   MW         194.14


  H— C—OH




  H— C—OH





        Similarly to the formation of 2-oxogluconic acid, this oxidation proceeds in two steps: first the glucose is oxidized to gluconate, then further oxidized at the 5-position. The composition of the medium and the selection of the bacterial strain determine whether the oxidation occurs at the 2- or 5-position. Strains of Acetobacter suboxydans are well suited for the synthesis of 5-oxogluconate.        

        The procedure was described  in the  forties and has hardly been altered since then In this process Acetobacter suboxydans was cultivated at 25 C in several two day steps .                                        

        The yield of the calcium salt with 2½ mol water of crystallization was up to 90% of the theoretical value. The upper limit of the starting concentration of sugar was 12% higher concentrations caused the crystallization of Ca-gluconate, which is more soluble than Ca-5-oxogluconate.

        This procedure has never been used commercially, but it could be useful for the production of D(+)-tartaric acid, where 5-oxogluconate could be used as the starting material.

C. 2,5-Dioxogluconic Acid







  H— C—OH





D-Gluconic Acid


        D-Gluconic acid and its d-lactone are simple dehydrogenation products of D-glucose. Production by microorganisms was discovered in 1880 by Boutroux who observed the formation of a ‘sugar acid’ by acetic acid bacteria. Molliard in 1922 first detected gluconic acid in cultures of the filamentous fungus, Sterigmatocystis nigra, now known as Aspergillus niger. Subsequently the formation of gluconic acid was demonstrated with various members of the Pseudomonadaceae (esp. Gluconobacter, Pseudomonas, Acetobacter) as well as a number of fungi - particularly various species of Penicillium and Aspergillus, and numerous others.

        Systematic studies revealed that Aspergillus niger can convert glucose to gluconic acid in high yields when the acid produced is neutralized, preferably by the addition to calcium carbonate. It was found, however, that this pH-dependence is much less pronounced in Penicillia, indicating some correlation between the amounts and time-dependent appearance of acids (gluconic, citric, and oxalic acids) formed under different conditions. A similar pH dependence was also observed in the above mentioned bacteria.

        Although Bernhauer’s findings were patented, large scale utilization of fungal gluconic acid production was first performed in the USA following the pioneering studies of Currie (1917) on citric acid fermentation which encouraged the Chas. Pfizer Co. to undertake industrial production of this acid in 1923. Technological research on gluconic acid production started in the US Department of Agriculture in 1926.

        May et al. in 1927 first reported on a shallow pan process utilizing organisms of the Penicillium luteumpurpurogenum group which was optimized to yields of about 60% of the theoretical.

In 1933 Currie et al. obtained a patent covering the production of gluconic acid by Penicillium or Aspergillus employing the modern technique of submerged culture of the molds with stirring and aeration of the fermentation medium. Yields as high as 90% in 48 to 60 hours were claimed for both A.niger and P.luteum.

        This important achievement was not sufficiently recognized, as Miall pointed out; the reason may be seen, at least in part, in the fact that at about the same time another method of submerged fermentation was developed at the US Department of Agriculture using a rotating drum. This apparatus was difficult to handle, but it was claimed to be advantageous for fermentations under higher pressures up to 4 bar. Attempts to utilize Penicillium chrysogenum in this process failed due to the inability of the mold to produce sufficient quantities of conidia for inoculation. Finally a strain of A. niger (strain 67) was obtained by selection which could easily be handled. In pilot plant studies yields of up to 95% of theoretical yields in glucose solutions of 150 to 200 g/L within 24 hours fermentation time were obtained. Elevated air pressure (2 to 4 bar) and neutralization with calcium carbonate were applied. Furthermore, it was found that the process can be run semicontinuously by reuse of the mycelium. If the mycelium was recovered by filtration or centrifugation it could be repeatedly employed up to nine times.

        In the 1940s it was discovered that the concentration of glucose can be increased up to 350 g/L by the addition go boron compounds as complexing agents (0.5, 1.0, 1.5, and 2.5 g/L in solutions of 200, 250, 300, and 350 g glucose per L, resp.). Addition of borates prevents the precipitation of calcium gluconate, but at these concentrations it detrimental to the growth of A. niger. It was necessary of overcome this effect by selecting specially resistant strains and by adding the borates only during a later phase of fungal growth. On a technical scale, glucose solutions of 250 g/L were converted with an efficiency of more than 95% with reuse of mycelium in cycles of 24 hours each.

        In 1952 Blom et al. developed a process for the production of sodium gluconate in which the acid produced during fermentation was neutralized by queues NaOH to pH 6.5. This process nowforms the basis of modern plant operations in deep tank fermentation.

Biological Fundamentals

        The conversion of glucose to gluconic acid involves rather simple reactions. In fungi the first step is catalyzed by the enzymeglucose oxidaste (b-D-glcose: oxygen 1-oxidoreductase, E.C. according to the following scheme

        As may be seen, this involves the direct consumption of one mole of glucose resulting in the formation of hydrogen peroxide, which is then cleaved by the fungal catalase. The glucononlactone is formed as the d-lactone, and can be converted to gluconate by spontaneoushydrolysis. The presence of specific lactone-hydrolyzing enzymes has also been demonstrated in Aspergilli. Glucose oxidase (GOD) was first isolated by Muller from a press juice obtained from Aspergillus niger. It is a flavoprotein, containing FAD as a prosthetic group, with rather high specificity for the oxidation of the b-anomer of D-glucose. In the microorganism perhaps the presence of a mutarotase (E.C. is required in order to facilitate the formation of the b-form, whose oxidation is favored about 150 fold over the a-anomer.

        Interestingly enough several fungi are able to excrete glucose oxidase into the culture medium, particularly those organisms which are less sensitive to low pH values of the medium, for example, some of the Penicillia. In the forties several authors reported on the isolation of “powerful antibiotics” from the culture fluids if Penicilla, esp. P. notatum, which were designated as “Penicillin B”, “Penatin” or “Noratin”. Very soon, however, it was recognized, that these preparations were identical with the enzyme glucose oxidase, and that the bactericidal properties were merely due to the disinfective action of the hydrogen peroxide formed in the presence of glucose. In the meantime, glucose oxidase has found various practical applications, especially in the quantitative determination of glucose in body fluids such as blood and urine, and it is also widely used in the food industry. Several procedures, therefore, have been elaborated is isolated the enzyme involving either recovery from the fermentation broth or extraction from the mycelium following cell disruption.

        Relatively little is known with regard to the regulation of the synthesis of glucose oxidase and formation of gluconic acid. It is taken for granted that there is a quantitative relationship between enzyme formation, i.e., the quantity of glucose oxidase, and acid production. The ability to build up glucose oxidase appears to be widespread among molds of the two groups of Penicillium and Aspergillus. Apparently, enzyme synthesis is inducible by glucose in the medium and by elevated oxygen levels. The situation is complicated by the fact that the physiological role of gluconic acid formation by glucose oxidase is still unclear. Van Dijken and Veenhuis

have demonstrated that in Aspergillus niger glucose oxidase is located in the peroxisomes. Thus some function in intracelular hydrogen (electron) transfer in accordance with the peroxisome concept could be considered. Apparently more knowledge of the evolution of oxidative pathways involving flavoproteins capable of directly reducing molecular oxygen would be required.

        For practical purposes, the following conditions have been found to be mandatory for successful fermentations with molds esp. with A.niger:

        1.     High concentrations of glucose-110 to 250 g/L

        2.     Low concentrations of the nitrogen source-in the range of about 20 mM nitrogen

        3.     Equally low concentrations of phosporus

        4.     Sufficient quantities of trace elements, esp. manganese, which are in general supplied e.g. by corn steep liquor. This was well documented by Bernhauer (1928), was found that in mycelial mats that had lost their ability to produce gluconic acid this could be restored by the addition of 0.7 to 13 mM manganease-11-sulfate.

        5.     The pH value of the medium should be in the range of 4.5 to 6.5. Below pH 3 glucose oxidase is inactivated and this results in increased glycolysis triggering the metabolism to citric acid formation.

        6.     High aeration rates-preferably by the application of elevated air pressure (up to 4 bar).

        Considering that gluconate production is directly correlated with the activity of glucose oxidase, the conditions listed above basically resemble the factors to be observed when designing a process for the production of glucose oxidase.

        Similar considerations may be valid when bacteria are used for the oxidation of glucose to gluconic aid. In various bacteria, but also in several molds and yeasts different glucose dehydrogenases have been detected with varying specificities, esp. with respect to coenzyme requirements.

        Gluconic acid formation is found in most of the genera of the family Pseudomonadaceae and in most of the species of Acetobacter. It is most pronounced in the genus Gluconobcter, which owes its designation to this ability, and in the genus Pseudomonas. Gluconate thus formed is frequently oxidized further: formation of 2-oxogluconate has been observed with pseudomonas spp., whereas Gluconobacter and some Acetobacter spp. are able to produce, in addition to 2-oxogluconate (at neutral pH), 5-oxogluconate (at an acid pH), 2.5-dioxogluconate and g-pyrones. As these oxidations may in part proceed simultaneously with glucose oxidation, this must be taken into consideration when industrial utilization of this potential is attempted. Most of these oxidation products can be introduced into the normal pathways of carbohydrate catabolism by phophorylating and reducing steps at several levels as outlined in Fig. 1.

        These oxidative transformations of glucose are frequently designated as gluconate pathways indicating physiological functions in circumventing enzyme and regulation deficiencies.

Fermentation Processes Involving

Calcium Gluconate Fermentation

        Based on the pioneering work at the US Department of Agriculture laboratories during the thirties and forties a standard procedure has evolved for the production of calcium gluconate, which may be out-lined as follows:

        Inoculum for the production stage may be either a conidial suspension obtained from a potent culture grown on a solid or liquid sporulation medium or precultured mycelium from liquid culture in a medium lower in glucose and higher in nitrogen and phosphorus compounds than the production medium.

        The production medium consists of glucose of medium purity (e.g. corn sugar) at a maximum concentration of 150 g/L. This is limited by the fact that the calcium gluconate can form supersaturated solutions up to this concentration with minimum risk of precipitation of the product. The solubility of calcium gluconate is normally about 3 to 4% w/v at temperatures of 20 to 30ºC.

        A typical medium as described in the literature and checked by the present authors is composed as follows (g/L):

        Glucose (e.g. corn sugar)                              110...150

        (NH4)2HPO4                                                            0.388

        KH2PO4                                                                   0.188

        MgSO4.7H2O                                                          0.156

        CaCO3 (separately sterilized)                                    26

        Fermentation is usually carried out in conventional stirred bioreactors with intensive aeration (0.25 to 1 vvm), preferably applying elevated air pressures of up to 3 bar. The temperature is held at 30ºC by an appropriate cooling system. An inoculum is preferably provided as mycelium from good fermentation obtained by fluttering or centrifuging and resuspending immediately in fresh medium; alternatively vegetative growth mycelium is prepared from a conidia culture. Amounts of 5 to 30% of the final volume of the fermentation batch are usually employed. Calcium carbonate is added as a steam sterilized slurry in increments according to the course of acid production keeping the pH of the broth at values above 3.5. In order to avoid precipitation of calcium gluconate the total amount of calcium carbonate should be only about two thirds of the stoichlometric requirement.

As pointed out in the introduction, higher concentrations of glucose, up to 350 g/L, can be employed with an excess of calcium carbonate


Citric Acid

Biological Fundamentals

        Since early studies it has been obvious that citric acid fermentation is extremely complex. A successful process depends both on an appropriate strain and on optimal fermentation parameters. Many investigations are from academic institutions and are performed under conditions that are of little significance with respect to industrial practice. On the other hand, the industrial experience acquired has more or less been kept secret. Only in few cases has it been possible to reproduce results obtained with a certain strain and/or under certain experimental conditions in other laboratories and it has been even less possible to transfer results to point plant or industrial operation.

A.  Strains

        Citric acid, as product of primary metabolism, is not likely to be excreted under natural conditions in noticeable amounts. Thus any appreciable exertion must be regarded as a result of some severe irregularity of metabolism caused by genetic deficiencies or by drastic metabolic imbalances. It is for these reasons that only few classes or even genera of microorganisms-bacteria and/or fungi-have been reported to excrete substantial amounts of citric acid into the fermentation medium under certain conditions.

        As already mentioned, Thom and Currie established concisely that citric acid production in varying amounts is fairly common among members of the genus Penicillium. The failure of such fermentations at the time was mainly due to contaminations under neutral pH conditions. In 1961 new strains,—Penicillium janthinellum Biourge var, kuensanii and P. restricum Gilman and Abbott var. kucnsani–were described in a patent granted to Knosthita et al. with appreciable yields and productivities of citric acid being claimed. In this context, the work of Sakaguchi ad Beppu, patented by Kyowa Hakko Kogyo K.K., on the fermentative production of Lallo-iso-citric acid should be mentiond. This is a diastereomer of naturally formed isocitric acid which was hiterto believed isocitric acid which was hiterto believed not to occur in nature. The organisms which produce this are Penicillium purpurogenum Still var. rubri-sclerotium Thom and several related strains.

        Apart from Aspergillus niger, which will be treated in Sect. III. A., the following species of Aspergillus have been found to produce citric acid: A. awamori, A. clavatus, A. fenicis, A. fonsecaeus, A. fumaricus, A. luchensis, A. saitoi, A. usumii, and A. wentii. From these only A. wentii has been applied in a well documented process. In all other cases the performance of strains does not justify further treatment.

        Other molds recorded as producing relatively large amounts of citric acid are Botrytis cinerea, Mucor piriformis, and Trichodema viride. Only the latter deserves special mention since it is described in a patent taken out by Kinoshita et al. for Kyowa Hakko Kogyo K.K. Owing to the fact that Trichoderma viride possesses a wide spectrum of enzymes of the utilization of cellulose and other polysaccharides its application has been claimed as being advantageous. Several other fungi and bacteria are described as excreting citric acid, but this may only be the case for scientific reasons or they may have been listed for patent coverage.

        Citric acid is produced in substantial amounts also by some coryneform bacteria (e.g. Arthrobacter), and related organisms, e.g., Actinomycetes, when cultivated on n-alkanes.

        With regard to yeasts the genus Candida has almost exclusively been utilized. This applies mainly to the following species: C. lipolytica, C. tropicalis, C. guiliermondii, C. intermedia, C. parapsilosis, C. zeylanoides, C. fibriae, C. subtropicalis, C. oleophila. Several of these strains are novel isolates obtained in the course of studies on citric acid production on n-alkanes and are described in the current patent literature.

B.  Fermentation Medium

        In their pioneer work with the traditional ATCC strain of Aspergillus niger, Shu and Johnson (1948b) presented the first systematic study on the influence of medium composition on citric acid production. Both the effects of macro components and of trace elements were taken into consideration. It is generally agreed that in order to achieve an abundant excretion of citric acid, growth of the production strain must be restricted. The precondition for sufficient critic acid production is a medium deficient in one or more essential elements which can be realized by limiting the concentration of one of the nutrition elements, phosphorous, manganese, iron onzine, phosphorus serves as a macronutrition satisfying the demand of growing cells for syndrodizing nucleotides and other phosphorylated compounds. The role of the trace metals mentioned has not been sufficiently elucidated, but there is general agreement that in many strains they drastically influence citric acid production. The medium constituents which have been found to have an effect on citric acid fermentation are listed in Table 1, and will be discussed in detail in this section.


Table 1. Conditions Favoring Citric Acid Production

        High sugar concentration

        Low concentrations of phosphate

        Low pH, below 2.0

        High oxygen tension

        Absence of trace metals                                  Mn2+, Fe2+, Zn2+

        Sugars. In general only sugars which are rapidly taken up by the fungus are useful carbon sources for citric acid fermentation. In most cases sucrose or molasses are used but glucose (from hydrolysis of polysaccharides) or fructose have also been found to be applicable. Polysaccharides are not generally applicable since their hydrolysis by the organism seems to be rate limiting for the sugar transport and therefore, the glucose would be taken up too slowly for an overflow metabolism. The concentration of sugar in the medium has been shown to be important, and maximum citric acid production is usually achieved at sugar concentrations as high as 14 to 22% (w/v).

        Several sources of crude carbohydrates have also been used for citric acid production. These are beet and cane molasses, unrefined surcose, cane juice, citrus molasses and various starch hydrolysates In addition to the problem of the metal ion content of these substrates, several authors have also reported on the presence of inhibitory substances, especially in molasses. These have to be reminded prior to fermentation, e.g., acetic acid and certain peptides. On the other hand, stimulators have also been found although their chemical nature has not been elucidated.

        Nitrogen. Conventionally, nitrogen is supplied in the form of ammonium sulfate or nitrate. Physiologically ammonium compounds are generally preferred, since during their consumption the pH decreases, which is a prerequisite of citric acid fermentation. This also excludes the use of sodium or potassium nitrates or of urea as sources of nitrogen and thus NH4+ is at present almost exclusively employed.

        In general the concentration of ammonium ions during citric acid fermentation should not be limiting and thus varies over a wide range (0.3 to 1.5 g NH4+/L); moreover their addition during citric acid fermentation has been reported to be advantageous. In this case, best results were obtained when ammonium was added at that stage in the fermentation when the production rate of citric acid begins to decreases. This effect is probably related to the regulatory role of the ammonium ion on citric acid accumulation since an addition of NH4+ has no influence on growth.

        Phosphate. Perquin made an extensive study on citric acid production by A. niger in shake cultures and thereby developed the concept of “citirc acid overflow” as a result of phosphate deficiency. As mentioned before, an early patent by Szocs (1944) is based on this observation. Later, however, it was established that under conditions of rigorous metal ion limitation, phosphate does not need to be limiting. Thus, if one calculates the phosphate requirement of the organism from the data of Shu it can easily be seen that their nutrient medium was not limited by phosphate. Under these conditions an increase of the phosphate level results in the formation of certain sugar acid and also simulates growth and decreases carbon dioxide fixation (unpublished observations).

        pH. The maintenance of a low pH value is extremely important for the successful progress of the fermentation. In general at pH values less than 3, citric acid is the major fermentation product, whereas at higher pH values substantial amounts of oxalic and gluconic acids may also be produced. The optimum pH is between 1.7 and 2.0.

Trace elements. The principal requirement of a great number of trace metals for the growth of A. niger was initially shown by Steinberg in a series of papers. In the work of Shu and Johnson it was clearly shown that low phosphate concentrations are essential for good yields when iron and zinc were present in optical concentrations; on the other hand when either the concentration of phosphate, or iron or zinc was below that for optimal growth, citric acid accumulation was achieved. However, at more reduced concentrations growth and sugar utilization were too low for citric acid production. This was confirmed by Tomlinson et al. with surface fermentations indicating that iron, zinc, manganese and copper in distinctly low concentrations are all essential for citirc acid accumulation. The effect of manganese has surprisingly been neglected in industrial patents dealing specifically with iron although was shown by Shu and Johnson that manganese had a most pronounced effect. At manganese concentrations of 20 µg/L, the yield of citric acid is already drastically decreased even in a low-phosphate medium. Schweiger reported the possibility of eliminating the negative effect of iron ions with high concentrations of copper (50-500 ppm) (Table 2). Unpublished experiments from our laboratory have shown that copper also effectively balances manganese impurities in the fermentation medium. Thus, the results of Schweiger are not absolutely clear. The critical nature of manganese instead of iron was also demonstrated by Clark et al.: they treated molasses with potassium hexacyanoferrate to remove the trace metals present. Of a number of trace metals then added, (Table 3) only manganese drastically decreased the citric acid yield whereas iron or zinc were far less effective. The biochemical role of manganese with respect to citric acid accumulation has recently been studied by the present authors.


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