Biotechnology Monographs Volume 1 Editors S. Aiba . L. T. Fan . A. Fiechter . K. Schtigerl Israel Goldberg Single Cell Protein With 32 Figures Springer-Verlag Berlin Heidelberg New York Tokyo Israel Goldberg Department of Applied Microbiology Institute of Microbiology The Hebrew University - Hadassah Medical School P.O. Box 1172, Jerusalem 91010 Israel ISBN-13: 978-3-642-46542-0 e-ISBN-13: 978-3-642-46540-6 DOl: 10.1007/978-3-642-46540-6 Library of Congress Cataloging in Publication Data. Goldberg, Israel, 1943-. Single cell protein. (Biotechnology monographs; vol. 1) Bibliography: p. Includes index. 1. Single cell proteins. I. Title. II. Series. [DNLM: 1. Dietary Proteins. 2. Food Supply. QU 55 G618sJ. TP248.S54G65 1985 664'.64 85-2813. ISBN-13: 978-3-642-46542-0 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. Under § 54 of the German Copyright Law, where copies are made for other than private use, a fee is payable to "Verwertungsgesellschaft Wort", Munich. © by Springer-Verlag Berlin Heidelberg 1985 Softcover reprint of the hardcover 1st edition 1985 The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. 2152/3140-543210 Preface In early 1973, I returned to Israel from a post-doctoral fellowship at Harvard University, and was accepted as a lecturer in the Department of Applied Microbiology at the Hebrew University of Jerusalem. Shortly after my return, Professor Richard Mateles, who at that time was head of the Department, suggested that I purchase a good and comprehensive book on single cell protein (SCP) in order to expand my general knowledge in the subject I had started then to work on; that was microbial utilization of one-carbon (C compounds. l) Naturally, I took his advice (after all, he was the Boss) and bought the book, which was the only general book published on this subject at that time, and was based on papers presented at the First International Conference on Single Cell Protein, held at the Massachussetts Institute of Technology (M.I.T.), on October 1967 (Mateles and Tannenbaum, editors) [1]. Through this book I became acquainted with the world's hunger problem that existed in the past, and ways in which it was to be solved by SCP products prepared from CO fossil-based raw 2, materials, and from wastes. Eighteen years have passed since the M.I.T. meeting and, unfortunately, SCP did not provide the world's growing population with an abundant supply of high quality protein. Obviously, the food hunger problem, especially in the developing countries, has not yet emerged as an issue sufficiently pressing to stimulate the world as a whole into action. Thus today, large-scale processes for SCP production are not widespread, mainly because of economic, and not because of technological reasons. Nevertheless, the commercial development of SCP during the past two decades, together with the accompanied basic and applied research, tremendously influenced modern fermentation technology, and more general biotechnology. The object of this monograph is, therefore, to describe the achievements in SCP production and the valuable contributions of SCP research and development. The monograph is intended for all who feel the need for some first orientation in this vast and fascinating field. It aims to give the newcomer an overview of the field without confusing him with too many specifics and thus, to equip him for consulting more specialized scientific literature. Also, the monograph provides details of current basic and applied work which, hopefully will intensify the imagination and innovative skills of all those involved in development of SCP production processes. This monograph would never have been written without the initiative of Springer-Verlag and the editorial committee of "Biotechnology Monographs". I have been fortunate in having colleagues willing and able to help and advise me V during the writing of the book. It is a pleasure to acknowledge the contribution of R.1. Mateles from Stauffer Chemical Company, G. L. Solomons from Ranks, Hovis McDougall Research Limited, L. T. Fan from Kansas State University, B. A. Prior from the University of the Orange Free State, and J. S. Rokem and S. Vecht from the Hebrew University, who read and criticized in detail the bulk, or parts, of the manuscript, and who made many suggestions which have been incorporated into the monograph. I want to express my sincerest thanks to my family, who patiently endured the many evenings and weekends that I devoted to writing and who gave encour agement when it was most needed. Jerusalem, May 1985 Israel Goldberg VI Table of Contents Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Organisms and Substrates 11 1.1 Introduction . . . . . . 11 1.1.1 Microorganisms Used in SCP Production. 11 1.1.2 Carbon Sources for SCP Production. . . 14 1.2 Carbon Sources and Microorganisms Used in SCP Production. 20 1.2.1 Autotrophic Growth on CO2 . • . . . • • 20 1.2.1.1 SCP from Bacterial Photosynthetic Reactions 22 1.2.1.2 Photosynthetic Algal SCP . 23 1.2.1.3 Chemolithotrophic Bacteria. . . . 26 1.2.2 Fossil Mass . . . . . . . . . . 26 1.2.2.1 Crude Oil, Gas-Oil, Hydrocarbons. 26 1.2.2.2 One Carbon (Ct)-compounds. . . 33 1.2.2.3 Ethanol . . . . . . . . . . . . 45 1.2.3 Renewable Resources as Carbon Substrates for SCP Production . 48 1.2.3.1 Saccharide Substrates . . . . . . . . . . . . . . . 49 1.2.3.2 Polysaccharide . . . . . . . . . . . . . . . . . . 54 1.3 Other Nutritional Requirements for Biomass Production 64 1.3.1 Energy Sources . . . . . . . 64 1.3.2 Sources of Nitrogen . . . . . 65 1.3.3 Sources of the Minor Elements 65 1.3.4 Vitamins ......... . 66 2 Fermentation Processes for Microbial SCP Production 67 2.1 Introduction . . . . . . . . . . . . . . . . . 67 2.2 Physiological and Engineering Process Aspects . . 68 2.2.1 Batch and Continuous Cultures in SCP Production 68 2.2.1.1 Kinetics of Microbial Growth in a Batch Culture . 68 2.2.1.2 Aspects in Continuous Culture Operation of Relevance to SCP Production. . . . . . . . . . . . . . . . . . . 69 2.2.1.3 Advantages of Continuous Culture over Batch Fermentation 71 VII 2.2.1.4 The Applications of Continuous Culture in Research Relevant to SCP Production. . . . . . . . . . . . . . 72 2.2.2 Improvement of Media for Biomass Production. . . . .. 74 2.2.3 Growth Yields of Microorganisms. . . . . . . . . . .. 79 2.2.3.1 Theoretical Biomass Yields for Different Carbon Substrates. 80 2.2.3.2 Experimental Cellular Yields . . . . . . . . . . . . .. 82 2.2.3.3 Maintenance Energy Requirements and "True Growth Yield" Values. . . . . . . . . . . . . . . . . . . . 83 2.2.3.4 Productivity . . . . . . . . . . . . . . . . . 84 2.2.3.5 Relationship Among Cell Yield, Oxygen Demand, and Heat Load . . . . . . . . . . . . . . . 85 2.2.4 Physiological Problems Associated with Scale-up 87 2.2.4.1 The Effect of Oxygen . . . . . . . . . . . . 87 2.2.4.2 The Effect of Carbon Dioxide . . . . . . . . 88 2.2.4.3 Variations in the Concentration of Limiting Nutrients 89 2.2.5 Sterilization . . . . . . . . . . . . 90 2.2.6 "Structured" Mixed Cultures . . . . . 91 2.2.6.1 Complex and Multiple Carbon Sources 91 2.2.6.2 Defined and Simple Carbon Sources . 95 2.2.7 Recycling . . . . . . . . . . . . . 10 1 2.2.8 Recovery of the SCP Product. . . . . 102 2.2.9 Description of Selected Pilot Plants and Industrial Processes for SCP Manufacture. . . . . . . . . . . . 103 2.2.9.1 ICI's "Pruteen" Process (Methanol-Bacteria) 103 2.2.9.2 The "Pekilo" Process (SSL-mold) 109 2.2.9.3 High-Rate Algal Ponds (HRAP) 111 2.2.9.4 "Mycoprotein" for Human Food 112 2.2.9.5 Yeast SCP from n-Paraffins 112 2.2.9.6 The "Waterloo" Process-Production of SCP from Waste Biomass. . . . . . . . . . . 114 2.2.10 Improvement of the Process for SCP Production 116 2.2.10.1 Type of Fermentor . . . . . . . . . . . . . 116 2.2.10.2 Computer-Controlled SCP Fermentation Processes. 117 2.2.10.3 Genetic Manipulation Techniques . . . . . 119 2.2.10.4 Thermophilic Microorganisms . . . . . . 123 2.3 Economic Considerations in SCP Production 125 3 The SCP Product 129 3.1 Introduction . . 129 3.2 Composition of the SCP Product 129 3.2.1 Protein . . . . 131 3.2.2 RNA and DNA . . . . . . . 133 3.2.3 Lipids. . . . . . . . . . . . 133 3.2.4 Other Constituents of the SCP Product. 135 3.3 Changes in the Composition of the SCP Product 136 VIII 3.3.1 Changes in Macromolecular Composition 136 3.3.1.1 Protein and Nucleic Acids 136 3.3.1.2 Lipids . . . . . . . . . . . . . . . . 142 3.3.1.3 Carbohydrates . . . . . . . . . . . . 144 3.3.2 Environmental and Genetic Control of Protein Composition 144 3.3.2.1 Environmental Conditions . . . . . . . . . . . . . .. 145 3.3.2.2 Genetic Control of Protein Composition . . . . . . . .. 146 3.4 Toxicological Status and Nutritional Value of SCP Products. 148 4 Concluding Remarks and Epilogue . . . . . . . . . . . . . 153 5 References . . . . . . . . . . . . . . . . . . . . . . . 161 6 Subject Index. . . . . . . . . . . . . . . . . . . . . . 181 IX Introduction Since the mid-1950's, there has been much emphasis on world's food shortage (world's hunger crisis) and possible ways to reduce it. A growing awareness for the acute food needs of the world's expanding population, especially in the de veloping countries, has led to the examination of a variety of methods to enhance agricultural and animal food sources and to produce "unconventional" food sources (microbial protein) as potential additions to the world's food supply (see the reviews of Altschul [2], Brown [3], Munro [4], Scrimshaw [5], Borgstrom [6], Litchfield [7], Solomons [8], and Mellor and Adams [9]). The most important factor contributing to world's hunger is the accelerated population growth rate. World's population is growing faster than ever, and to describe this rate, the term "population explosion" was coined (Fig. 1). Although growth takes place everywhere, the population growth rate of the developing countries (Asia, Africa, and Latin America) is double that of the developed coun- Fig. I. Population explosion (an artist's view) tries. According to a recent report issued by the World Bank (Mexico, August 1984) the present world's population of 4 billion people will be doubled in just 45 years, with the highest increase in population in the developing countries. Between 1961 and 1980, food production in the developing world (including 105 countries) increased at an average annual rate of 2.6%. This was slightly greater than the corresponding average annual population growth rate of 2.4%. However, on a per capita basis, food production in the developing countries as a whole increased only 0.2% a year. Moreover, in countries like North Africa, the Middle East, and Sub-Saharan Africa, the annual growth of major food crop production was lower (between 1.7 to 2.4%) than the annual population growth (about 2.8%) [9]. In the developed countries there is ample production of major food commo dities such as corn, wheat, soya, potatoes, milk, fish and eggs. These countries provide about one third of the world population with nutritious food. In develop ing countries, comprising large parts of the world, the opposite is true. In these countries, the inadequacies are not only in the low quantity of food, but also in its quality. The diets of people in these countries are generally low in calories and unbalanced in other nutrients, because the bulk of the diet consists mainly of ce reals (mostly rice) and starchy food. Owing to the lack of variety in the diet, and particularly to the shortage of foods such as milk, eggs, fish and meat, this diet fails to provide adequate quantities of vitamins, minerals and especially high quality protein, such that the food problem is largely a protein deficit [6]. About two-thirds of the world's population live in squalid unhygenic condi tions which enhance sickness. This factor in conjunction with a poor diet, effects gross malnutrition. It is estimated that presently more than 450 million people, most of whom are living in developing countries, are hungry and undernourished [9]. Recently, an increasing demand for high-quality protein has been observed in developed countries, as overall living standards have improved. Concom itantly, in the developing countries, the rising per capita income has further con tributed to the ever-increasing demand for protein. The developed countries have the funds and technical expertise to cope with an increased domestic demand for food, and thus have improved the level of protein production, thereby widening the food production gap, more accurately called "the protein hunger gap", be tween the "have" and "have not" countries [3, 6]. The principal methods of maximizing food availability in the developing countries are by enlarging the area of cropped land, increasing crop yields, and by maximizing imports of surplus commodities from the developed countries [9]. It is highly unrealistic to expect developing countries to raise agricultural produc tion to that of developed countries, as these advanced countries have superior re sources in soils, forests, fertilizers, pesticides, agricultural equipment, water, and above all, capital. Rising food transportation costs suggest that food export ar rangements from developed countries are temporary measures which cannot be relied upon indefinitely to furnish substantial or continuing relief to needy areas [6]. Presently, even with the efforts of most of the developing nations, together with help from international agencies, developed countries and private philan thropic foundations, production (or importation) offood is failing to keep in pace with the population growth rates. It would appear, therefore, that the hunger cri- 2