Immobilized Microbial Cells 9780841205086, 9780841206601, 0-8412-0508-6

Table of contents :
Title Page......Page 1
Half Title Page......Page 3
Copyright......Page 4
ACS Symposium Series......Page 5
FOREWORD......Page 6
PdftkEmptyString......Page 0
PREFACE......Page 7
The Approach......Page 9
Process Variables......Page 11
Conclusion......Page 17
Literature Cited......Page 19
2 Pore Dimensions for Accumulating Biomass......Page 20
Determination of Microbial Loadings(Biomass).......Page 21
The Immobilization of Bacillus subtilis by Adsorption.......Page 23
Bioaccumulation of Yeast.......Page 24
Bioaccumulation of Streptomyces olivochromogenes.......Page 27
Results and Discussions......Page 29
Literature Cited.......Page 35
I. The Surface Physics of Adhesion......Page 36
II. The Biophysics of Cell Adhesion......Page 47
III. Conclusions......Page 60
Literature Cited......Page 61
Penicillin G Production by Immobilized Whole Cells......Page 65
Bacitracin Production by Immobilized Whole Cells......Page 68
α-Amylase Production by Immobilized Whole Cells......Page 72
Literature Cited......Page 77
Biological denitrification......Page 79
Biological removal of heavy metals......Page 80
Results......Page 81
Conclusions......Page 90
Literature Cited......Page 91
6 Synthesis of Coenzyme A by Immobilized Bacterial Cells......Page 93
Synthesis of CoA by Free, Dried Cells (Cell Method, 4, 5, 7,12)......Page 94
Synthesis of CoA by the Immobilized Cell Method (2)......Page 96
Litrature Cited......Page 105
7 Phenol Degradation by Candida tropicalis Whole Cells Entrapped in Polymeric Ionic Networks......Page 107
Experimental Procedures......Page 108
The Phenol Degradation Reaction......Page 111
Catalytical Activity and Transport Limitation......Page 114
Catalytic Stability and Cell Viability......Page 118
Acknowledgement......Page 121
Abstract......Page 122
Literature Cited......Page 123
Hydrous Metal Oxides as Supports......Page 125
Applications of Living Immobilised Cells to Fermentations......Page 129
Abstract......Page 136
Literature Cited......Page 137
Pantothenate Production by Cells of E. coli ATCC 9637......Page 138
Properties of Frozen-thawed Cells......Page 139
Discussion......Page 140
Literature Cited......Page 142
Production Process......Page 143
Diffusion-Controlled Rate Model......Page 145
Reactor Column Hydraulics......Page 148
Literature Cited......Page 150
Mathematical model......Page 151
Experimental......Page 158
Evaluation of the model......Page 160
Abstract......Page 174
List of symbols......Page 175
Literature cited......Page 176
Support Characteristics......Page 177
Enzyme Bonding Procedure......Page 178
Analysis of Diffusional Limitations......Page 179
Kinetic Evaluation......Page 180
Half-Life Study......Page 182
Discussion......Page 186
ABSTRACT......Page 189
Literature Cited......Page 190
1. Production of L-aspartic acid using immobilized Escherichia coli [3, 4, 5]......Page 191
2. Production of L-malic acid using immobilized Brevibacterium ammoniagenes [6, 7]......Page 193
1. New matrix,κ-carrageenan, for immobilization of microbial cells......Page 194
2. Production of L-aspartic acid using immobilized Esherichia coli......Page 196
3. Production of L-malic acid using immobilized Brevibacterium flavum......Page 198
4. Summary of к-carrageenan method......Page 199
1. Production of ethanol using immobilized yeast cells......Page 201
2. Production of L-isoleucine using immobilized Serratia marcescens......Page 203
Literature Cited......Page 205
14 Application of Immobilized Whole Cells in Analysis......Page 207
BOD-sensors......Page 208
Poison guards......Page 209
Analysis of antibiotics......Page 211
Bioassay of specific substances with the use of immobilized mutants.......Page 213
ASSAY OF SINGLE SUBSTRATE SPECIES.......Page 214
IMMOBILIZED BACTERIA USED AS SORBENTS IN ANALYSIS......Page 217
CONCLUDING REMARKS......Page 219
LITERATURE CITED......Page 222
Microbial Electrode Sensor for Cephalosporins......Page 225
Microbial Electrode Sensor for Glucose......Page 231
Other Microbial Electrode Sensors......Page 238
Literature Cited......Page 240
16 Hollow Fiber Entrapped Microsomes as a Liver Assist Device in Drug Overdose Treatment......Page 241
Results and Discussion......Page 243
ACKNOWLEDGEMENTS......Page 250
LITERATURE CITED......Page 251
B......Page 252
C......Page 253
F......Page 254
I......Page 255
P......Page 256
S......Page 257
Z......Page 258

Citation preview

Immobilized Microbial Cells

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

Immobilized Microbial Cells K. Venkatsubramanian,

EDITOR

H. J. Heinz Company and Rutgers University

Based on a symposium jointl the ACS Divisions of Microbial and Biochemical Technology, Agricultural and Food Chemistry, and Carbohydrate Chemistry at the 176th Meeting of the American Chemical Society, Miami Beach, Florida, September 14,

ACS

1978.

SYMPOSIUM

SERIES

106

AMERICAN CHEMICAL SOCIETY WASHINGTON, D. C.

1979

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

Library of CongressCIPDat Immobilized microbial cells. (ACS symposium series; 106 ISSN 0097-6156) Includes bibliographies and index. 1. Industrial microbiology—Congresses. 2. Micro-organisms, Immobilized—Congresses. 3. Micro-organisms, Immobilized—Industrial applications—Congresses. I. Venkatsubramanian, K., 1948. II. American Chemical Society. Division of Microbial and Biochemical Technology. III. American Chemical Society. Division of Agricultural and Food Chemistry. IV. American Chemical Society. Division of Carbohydrate Chemistry. V. American Chemical Society. VI. Series: American Chemical Society. ACS symposium series; 106. QR53.I45 ISBN 0-8412-0508-6

660'.62 ACSMC8

79-15794 106 1-258 1979

Copyright © 1979 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each article in this volume indicates the copyright owner's consent that reprographic copies of the article may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating new collective works, for resale, or for information storage and retrieval systems. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. PRINTED IN THE UNITED STATE OF AMERICA

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

ACS Symposium Series M. Joan Comstock,

Series Editor

Advisory Board Kenneth B. Bischoff

James P. Lodge

Donald G. Crosby

John L. Margrave

Robert E. Feeney

Leon Petrakis

Jeremiah P. Freeman

F. Sherwood Rowland

E. Desmond Goddard

Alan C. Sartorelli

Jack Halpern

Raymond B. Seymour

Robert A. Hofstader

Aaron Wold

James D. Idol, Jr.

Gunter Zweig

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

FOREWORD The ACS SYMPOSIUM SERIES was founded in 1974 to provide a medium for publishin format of the Series parallels that of the continuing ADVANCES IN CHEMISTOY SERIES except that in order to save time the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable since symposia may embrace both types of presentation.

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

PREFACE "D iochemical processing with immobilized microbial cells represents a novel approach to biocatalysis. Such a system offers a number of unique advantages over traditional fermentation processes as well as the more recent immobilized enzyme processes. Although this concept is still relatively new, a few immobilized cell systems have already been commercialized. This, in turn, has triggered a surge of research activity in this exciting and rapidly growing field. Numerous conferences and symposia have been held on the subject of enzyme engineering in recent years. Although they contai microbial cells, no single conference was devoted to covering this subject matter exclusively. Therefore, we organized a symposium on immobilized microbial cells as part of the 176th Annual Meeting of the American Chemical Society held at Miami Beach in September 1978. This volume contains most of the papers presented at the symposium. In addition, several chapters written by leading experts in the field have also been included. Several important aspects of immobilized microbial cell technology are discussed here: carriers for immobilization, methods of cell attachment, biophysical and biochemical properties, reactor design, and process engineering of bound cell systems. A number of applications in the food, pharmaceutical, and medical areas—including those commercialized already—have been described. In essence, this is a comprehensive single volume state-of-the-art presentation of immobilized microbial cell systems. Thefirstchapter by Vieth and Venkatsubramanian provides a broad overview of the subject matter including the rationale for immobilizing microbial cells, the advantages and disadvantages of such an approach, and the overall prospects and problems of a technological development based on bound cell systems. The chapter by Messing and associates discusses the critical pore dimensions needed for fixing microorganisms inside various inorganic matrices. This is followed by an interesting discussion on the adhesive forces that come into play in fixed microbial systems. A series of biochemical processes mediated by fixed cells are described next. They vary in complexity in terms of the number of individual enzymatic reactions, and coenzymes involved. Included in this section are descriptions of immobilized cell systems for producing coenzyme A, pantothenic acid, antibiotics, and extracellular enzymes. In ix

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

addition, waste treatment applications such as phenol degradation and denitrification are outlined. Several important industrial applications are discussed next, starting with two commercial processes for the conversion of dextrose to fructose. The chapters by Bungard and co-workers and Roels and his associates describe two different approaches to this interesting commercial problem. Because of the commercial importance of this process, we have also in­ cluded a paper by Goldberg on the use of glucose isomerase enzyme (as opposed to the whole organism containing the enzyme immobilized on a porous polymeric matrix). Chibata discusses several industrial applica­ tions of immobilized microbial cells as practiced in Japan. The chapters by Mattiasson and Suzuki and his associates discuss many interesting analytical applications of immobilized cell systems. Thefinalchapter by Kastl describes a process for immobilizing isolated organelles and use of such a system in detoxifying drugs. I am indebted to al meet a tight publication schedule, and to the reviewers for their prompt responses. Many thanks are due to Charles Cooney and George Charalambous of the Microbial and Biochemicl Technology Division and the Agricultural and Food Chemistry Division, respectively, for encouraging me to organize this symposium, and to John Whittaker for serving as cochairman of the symposium. I am thankful to the ACS Books Depart­ ment for its assistance. Finally, the impeccable secretarial help of Diane Otto is gratefully acknowledged. H. J. Heinz Company Pittsburgh, Pennsylvania April 10, 1979

K.

VENKATSUBRAMANIAN

χ

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1 Immobilized Microbial Cells in Complex Biocatalysis WOLF R. VIETH and K. VENKATSUBRAMANIAN

1

Department of Chemical and Biochemical Engineering, Rutgers—The State University, New Brunswick, NJ 08903

Continuous heterogeneous catalysis by fixed microbial cells represents a new approac Immobilization of isolate cells mediating simple, monoenzyme reactions has already been reduced to industrial practice. However, the development of immobilized cell systems to carry out complex fermentation processes--characterized by multiple reactions and complete reaction pathways involving coenzymes--is still in its infancy. Drawing upon our rather concerted effort in this area over the past several years, we are appraising the prospects and problems of such a technological advancement in this brief communication. The Approach In earlier papers from this laboratory, we have proposed the terms "Controlled Catalytic Biomass" and "Structured Bed Fermentation" to describe immobilized cell systems effecting complex biocatalysis (1,2). The meaning of these terms is obvious when one considers the biocatalyst in relation to its microstructure, predesigned catalytic reactor design, and controlled catalytic activity vis-a-vis cellular reproduction. Some of the potential advantages of such a catalytic system are summarized in Table I. Examining the character of microbial cells in classical fermentation, i t is clear that they possess the desired catalytic machinery in a highly structured form. The controlled conditions of fermentation permit retention of this meticulous structural 1

Also with: H.J. Heinz Company, World Headquarters, P.O. Box 57, Pittsburgh, Pennsylvania 15230.

Presented at the Symposium on "Immobilized Cells and Organelles," ACS National Meeting, Miami Beach, September, 1978.

0-8412-0508-6/79/47-106-001$05.00/0 © 1979 American Chemical Society In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2

IMMOBILIZED MICROBIAL CELLS

TABLE 1 POTENTIAL ADVANTAGE OVER

1.

Placement o f Fermentation on Heterogeneous Basis

C a t a l y s i s Design

2.

Higher Product Y i e l d s

3.

A b i l i t y t o Conduct Continuous Operations As Opposed t o T r a d i t i o n a l Batch Fermentation

4.

Operation a t High D i l u t i o n Rates Without Washout

5.

A b i l i t y to Recharge System by Inducing Growth and Reproduct i o n o f Resting C e l l s

6.

Decrease o r E l i m i n a t i o n o f Lag and Growth Phases f o r Product Accumulation A s s o c i a t e d With the Non-Growth Phase of the Fermentation

7.

P o s s i b i l i t y o f A c c e l e r a t e d Reaction Rates Due t o Increased C e l l Density

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1.

viETH

AND VENKATSUBRAMANIAN

Complex Biocatalysis

3

i n t e g r i t y but ,the r e s u l t i n g c e l l u l a r suspensions are u s u a l l y at low c o n c e n t r a t i o n . Considering f r e e enzymes d e r i v e d from these c e l l s , i t i s p o s s i b l e t o concentrate them by e x t r a c t i o n processes, but l a c k i n g the a n c i l l a r y s t r u c t u r e which s t a b i l i z e s them i n the c e l l , they are r e l a t i v e l y unstable. Some s t r u c t u r a l r e c o n s t i t u t i o n i s p o s s i b l e by immobilization, l e a d i n g to higher concentrat i o n and b e t t e r s t a b i l i t y but one i s then r e s t r a i n e d t o c o n s i d e r a t i o n of s i n g l e step or two-step r e a c t i o n s . With immobilized c e l l s , one has the concentrated form, there i s s t r u c t u r a l p r e s e r v a t i o n and s t a b i l i t y together with the p o s s i b i l i t y of improved r e a c t o r design, based upon the c h a r a c t e r i s t i c s of the c a r r i e r . Thus, immobilized c e l l systems c o n s t i t u t e an important o p t i o n w i t h i n the framework of biochemical technologies (Table 2). The o v e r a l l r a t i o n a l e f o r whole c e l l immobilization i s o u t l i n e d i n Table 3. In a l l our work, w c o l l a g e n as the c a r r i e , c o l l a g e n , o f f e r s a number of unique advantages as a support f o r m i c r o b i a l c e l l immobilization. Other p u b l i c a t i o n s from our l a b o r a t o r y describe these advantages as w e l l as the procedures to prepare f i x e d c e l l s i n d e t a i l (3, 4). We have attached many d i f f e r e n t microorganisms i n t h i s manner; some of the complex r e a c t i o n s mediated by such f i x e d c e l l p r e p a r a t i o n s are shown i n Table 4. Process V a r i a b l e s Several important c o n s i d e r a t i o n s i n the p r e p a r a t i o n and use of collagen-bound c e l l systems are adumbrated here with c i t r i c a c i d production by immobilized A s p e r g i l l u s n i g e r as an example. The c o l l a g e n membrane must be c r o s s l i n k e d t o make i t s t r u c t u r a l l y strong enough to withstand the shear f o r c e s i n r e a c t o r o p e r a t i o n . I t was found that post-tanning the c o l l a g e n - c e l l membrane by exposing i t t o a 5% glutaraldehyde s o l u t i o n f o r one minute r e s u l t e d i n an optimal r e t e n t i o n of c a t a l y t i c a c t i v i t y which was a l i n e a r f u n c t i o n o f the c e l l l o a d i n g . We can l o a d the s t r u c t u r e up t o 70% c e l l s (by dry weight) and the amount of expressed a c t i v i t y i n batch assay i n c r e a s e s p r o p o r t i o n a t e l y . However, the mechanical strength drops o f f too d r a s t i c a l l y , and a good compromise i s 50% c e l l s on a dry weight b a s i s . In the course of these s t u d i e s , we came t o r e a l i z e t h a t the dehydration of c e l l s i s d e l e t e r i o u s ; even under r e f r i g e r a t e d c o n d i t i o n s c e l l a c t i v i t y could reduce s i g n i f i c a n t l y . T h i s has l e d us to new d i s p e r s i o n techniques and/or d r y i n g or s o l i d i f i c a t i o n techniques to preserve these f r a g i l e s t r u c t u r e s which can so e a s i l y denature (6). Maximal c a t a l y t i c a c t i v i t y of the c e l l s i s r e t a i n e d upon immobilization when the c e l l s are i n the proper p h y s i o l o g i c a l s t a t e . T h i s corresponds to an optimal i n d u c t i o n o f enzyme a c t i v i t i e s p a r t i c i p a t i n g i n the d e s i r e d r e a c t i o n sequence;

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

BIOCONVERSION NETWORK

TABLE

2

4χ

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

VIETH AND VENKATSUBRAMANIAN

Complex

BiOCdtalysis

TABLE 3

RATIONALE FOR WHOLE CELL IMMOBILIZATION

1.

Obviates Enzyme E x t r a c t i o n / P u r i f i c a t i o n

2.

G e n e r a l l y Higher O p e r a t i o n a l S t a b i l i t y

3.

Lower E f f e c t i v e Enzyme Cost

4.

High Y i e l d o f Enzyme A c t i v i t y on Immobilization

5.

C o f a c t o r Regeneration

6.

Retention of S t r u c t u r a l and Conformational

7.

Greater P o t e n t i a l f o r Multi-Step Processes

8.

Greater Resistance t o E n v i r o n t a l P e r t u r b a t i o n s

Integrity

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

Klebsciella pneumoniae

Mammalian erythrocyte

11.

Anacystis nidulans Anacystis nidulans Streptomyces griseus Pseudomonas aeruginosa

Serratia marcescens Acetobacter sp. Corynebacterium lilium Aspergillus niger Chloroplast

10,

9.

6. 7. 8.

4. 5.

2. 3·

1.

Microorganism

Nitrogen

Water Nitrate Glucose

Sucrose Water

Ethanol Glucose

Glucose

Substrate

Ammonia

Oxygen Ammonia Candieidin

C i t r i c acid Oxygen

2-Keto g l u c o n i c acid Acetic acid Glutamic a c i d

Product Comments

Model s t u d i e s of in_ v i v o enzyme a c t i o n

M i c r o b i a l f i x a t i o n of atmospheric n i t r o g e n

Primary metabolite Immobilized o r g a n e l l e ; f i r s t step i n b i o p h o t o l y s i s of water Immobilized a l g a l c e l l s B i o l o g i c a l nitrogen f i x a t i o n A n t i b i o t i c synthesis; secondary metabolite Concentration of plutonium from waste waters (bioadsorption)

Multi-enzyme; c o f a c t o r Pathway (primary metabolite)

Multi-enzyme

COLLAGEN-IMMOBILIZED CELL SYSTEMS

TABLE 4

1.

vrjETH AND VENKATSUBRAMANIAN

Complex Biocatalysis

7

i t i s manifested i n peak product s y n t h e s i s r a t e i n the fermentat i o n . For c i t r i c a c i d p r o d u c t i o n with A. n i g e r , i t turns out to be 72 t o 96 hours i n batch fermentations. Of course, i n a t y p i c a l fermentation process one has to repeat t h i s p a t t e r n each time. A b e t t e r a l t e r n a t i v e , i t would seem, would be t o harvest the c e l l s at t h e i r peak a c t i v i t y , followed by t h e i r immobilization so as t o r e t a i n them i n a v i a b l e s t a t e f o r reuse u n t i l t h e i r s t a b i l i t y has decreased t o an uneconomical p o i n t . Once immobilized, the c e l l s must be kept i n a v i a b l e s t a t e i n the membrane without f u r t h e r excessive r e p r o d u c t i o n . T h i s i s necessary t o channel the s u b s t r a t e i n t o the d e s i r e d product r a t h e r than t o a d d i t i o n a l c e l l mass. Besides, i t would minimize c e l l e l u t i o n from the c a r r i e r matrix as w e l l as preserve the mechanical i n t e g r i t y of the c a r r i e r . We have found t h a t one way to accomplish t h i s i s the e s s e n t i a l n u t r i e n t c o n c e n t r a t i o n . An i n d i r e c t b e n e f i t o f t h i s approach i s lowering the growth o f contaminating organisms. Ease o f r e a c t o r scale-up i s an important process engineering c o n s i d e r a t i o n ; maximizing the e f f i c i e n c y o f c o n t a c t between the c a t a l y s t and i t s s u b s t r a t e i s an e q u a l l y c r i t i c a l i s s u e . We have determined t h a t where the bound-cell membrane can be r o l l e d i n t o a s p i r a l wound r e a c t o r c o n f i g u r a t i o n (Jo) , i t provides e x c e l l e n t contact e f f i c i e n c y . The c o l l a g e n membrane i s wound together with a polyolefin Vexar spacer m a t e r i a l . The r e s u l t i n g open m u l t i channel system promotes p l u g flow contact with very low pressure drop even when o p e r a t i n g with p a r t i c u l a t e s u b s t r a t e matter which would cause p l u g g i n g problems i n the conventional type o f f i x e d bed o p e r a t i o n . Fermentation s u b s t r a t e s are o f t e n c h a r a c t e r i z e d by p r e c i s e l y t h i s type o f substrates; so t h i s i s a l a r g e p l u s f a c t o r i n f a v o r o f t h i s type o f design. Furthermore, i t i s p o s s i b l e t o d e s i g n - i n high a c t i v i t y p e r u n i t volume, as a r e s u l t of the c o i l i n g o f a l a r g e amount o f membrane i n t o a confined volume. The b a s i s f o r scale-up becomes then simply the membrane surface area. Presented i n F i g s . 1 and 2 are data r e l a t i n g t o e x t e r n a l and i n t e r n a l mass t r a n s f e r f o r the case o f c i t r i c a c i d s y n t h e s i s . The e f f e c t o f l i n e a r v e l o c i t y on the observed r e a c t i o n r a t e (Fig. 1) shows, f o r t h i s case, the presence o f a s i g n i f i c a n t boundary l a y e r r e s i s t a n c e below a flow r a t e o f 235 ml/min. The existence o f n o n - n e g l i g i b l e pore d i f f u s i o n a l r e s i s t a n c e i s ded u c i b l e from F i g . 2, i n which the dependence o f observed r e a c t i o n r a t e on f i l m t h i c k n e s s i s d e p i c t e d . O v e r a l l the immobilized c e l l s e x h i b i t e d about 50% o f the s p e c i f i c a c t i v i t y o f the f r e e c e l l s (in fermentation) toward the p r o d u c t i o n of c i t r i c a c i d . With regard t o other s i g n i f i c a n t f a c t o r s , oxygen t r a n s f e r can be s i n g l e d out as o f paramount importance. To enhance t h i s t r a n s p o r t step, we operated the s p i r a l wound r e a c t o r counterc u r r e n t l y . In other words, a s p e c i a l p r o v i s i o n was incorporated i n t o the r e a c t o r design t o allow flow o f pure oxygen countercurrent

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

8

IMMOBILIZED MICROBIAL CELLS

X

50

10

SUBSTRATE

Figure 1.

RATE

( ML / MIN

)

Dependence of reaction rate on linear velocity

.

I WET

Figure 2.

FLOW

5 MEMBRANE

.

a__

10 15 THICKNESS (MILS)

1

20

Effect of membrane thickness on citric acid production rate. (O) Shake flask, (Q) reactor.

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1.

viETH

AND VENKATSUBRAMANIAN

Complex Biocatalysis

9

to the flow of s u b s t r a t e ; i n t h i s sense, the o v e r a l l system operated as a combined absorber-reactor. D i s s o l v e d oxygen conc e n t r a t i o n s of 80 to 90% of s a t u r a t i o n value were maintained throughout the course of the r e a c t o r runs. R e f e r r i n g back t o F i g . 2, the i n c r e a s e d s p e c i f i c a c t i v i t y of the c a t a l y s t observed i n the r e a c t o r compared to t h a t i n a shake f l a s k i s a t t r i b u t a b l e , at l e a s t i n p a r t , to improved oxygen t r a n s f e r i n the r e a c t o r . Thus, the simple, e f f e c t i v e , f l e x i b l e use of the membrane form i n t h i s type of reactor.has demonstrated s e v e r a l a d d i t i o n a l p o s i t i v e features. From a p r a c t i c a l standpoint, the two most important chara c t e r i s t i c s of an immobilized c e l l c a t a l y s t are i t s a c t i v i t y and i t s operational s t a b i l i t y . The l a t t e r parameter i s u s u a l l y expressed i n c a t a l y s t h a l f - l i f e . The amount of a c t i v i t y , say i n I n t e r n a t i o n a l U n i t s (I.U.) would be a f u n c t i o n of c e l l - t o c a r r i e r r a t i o . As mentione found to be optimal. A s p e r g i l l u s niger c e l l s attached t o c o l l a g e n e x h i b i t good a c t i v i t y r e t e n t i o n , as shown i n Table S · Please note t h a t r a t e comparisons have been made on the b a s i s of maximal'rate. I f one uses an i n t e g r a t e d average r a t e obtained over the e n t i r e p e r i o d o f the batch fermentation c y c l e , the comparison becomes even more favorable f o r the immobilized c e l l system, s i n c e i t experiences a very small l a g p e r i o d preceding c i t r i c a c i d s y n t h e s i s . In a d d i t i o n t o s p e c i f i c p r o d u c t i v i t y r a t e s , i t i s a l s o necessary to examine the r e l a t i v e concentrations of the product i n both cases, as the t i t e r value i s very c r u c i a l with regard t o product i s o l a t i o n and p u r i f i c a t i o n . Data obtained thus f a r i n d i c a t e t h a t bound c e l l s y i e l d 8 t o 40% of the f i n a l c o n c e n t r a t i o n obtainable i n fermentation. H a l f - l i f e of the c a t a l y s t was 138 hours. Chromatographic a n a l y s i s of r e a c t i o n products of c i t r i c a c i d synthesized by f i x e d c e l l s r e v e a l s the presence of products generated from s i d e r e a c t i o n s . They i n c l u d e i s o c i t r i c a c i d , o x a l i c a c i d and t r a c e q u a n t i t i e s o f g l u c o n i c a c i d . I s o c i t r a t e i s perhaps the major one, amounting to as much as 15 t o 20% of citrate. O x a l i c a c i d formation i n c i t r i c a c i d fermentations i s reported t o be dependent both on pH and on the extent of a e r a t i o n . By proper c o n t r o l of pH and d i s s o l v e d oxygen l e v e l s , i t might be p o s s i b l e t o reduce the formation of oxalate. Conclusion Immobilized c e l l and o r g a n e l l e systems o f f e r a great d e a l of promise i n mediating many r e a c t i o n schemes to produce commercially important products. U n l i k e bound mono-enzyme systems, c a t a l y s i s by f i x e d c e l l s i s q u i t e complex and many b a s i c aspects are yet to be understood. However, t e c h n i c a l f e a s i b i l i t y of r a t h e r elaborate immobilized c e l l processes, as exemplified by c i t r i c a c i d production through

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

10

IMMOBILIZED MICROBIAL CELLS

TABLE 5

CITRIC ACID SYNTHESIS BY IMMOBILIZED CELLS

Sample

Maxiumum s p e c i f i c productivity (g a c i d / g dry c e l l s - h )

R e l a t i v e maximum specific p r o d u c t i v i t y (%)

Fermentation

0.0043

100

Resting C e l l s

0.0045

104

Immobilized C e l l s

0.0021

48.4

Fermentation data obtained from 5-Z s t i r r e d fermentor; others from shake f l a s k s . Sucrose a t 40—£ was used as the s u b s t r a t e i n a l l cases.

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1.

viETH

AND VENKATSUBRAMANIAN

Complex Biocatalysis

11

i n t a c t f u n c t i o n o f the TCA c y c l e enzymes, has been demonstrated. I n v e s t i g a t i o n o f the b a s i c problems o f structured-bed fermenta­ t i o n systems ( c e l l p h y s i o l o g y , c e l l v i a b i l i t y , t r a n s p o r t r e s i s ­ tances, oxygen t r a n s f e r , m i c r o b i a l contamination) i s now being pursued i n our c u r r e n t work. Perhaps the g r e a t e s t p o t e n t i a l f o r immobilized c e l l systems l i e s i n r e p l a c i n g complex fermentations such as secondary m e t a b o l i t e p r o d u c t i o n . Some o f the f u r t h e r developments i n t h i s f i e l d should c l e a r l y be steered i n t h i s direction.

Acknowledgements The authors are g r a t e f u l t o the c o n t r i b u t i o n s o f Mr. Charles B e r t a l a n ; some o f the r e s u l t s reported here have been drawn from h i s t h e s i s . area was p r o v i d e d i n p a r (AER 7618816), E t h y l Corporation and H.J. Heinz Company.

Literature

Cited

1.

Venkatasubramanian, Κ., and Vieth, W.R., Progress In Industrial Microbio. (in press).

2.

Vieth, W.R., Annals New York Acad. S c i . (in press)

3.

Vieth, W.R., and Venkatasubramanian, Κ., Methods Enzymol. 34, 243 (1976).

4.

Venkatasubramanian, Κ., Vieth, W.R., and Constantinides, A. i n E.K. Pye and H.H. Weetall (Editors), Enzyme Engineering, Vol. 3, Plenum Press, New York (1978) pp. 29-42.

5.

Vieth, W.R., and Venkatasubramanian, Κ., Enzyme Engineering (Vol. 4), G. Broun and G. Manecke (editors), Plenum Press, New York (in press).

6.

Vieth, W.R., Gilbert, S.G., Wang, S.S., and Venkatasubramanian, Κ., U.S. Patent 3,809,613 (1974).

RECEIVED February 15, 1979.

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2 Pore Dimensions for Accumulating Biomass R. A. MESSING, R. A. OPPERMANN, and F. B. KOLOT Corning Glass Works, Sullivan Park, Corning, NY 14830

Such processes as the production of cells (single cell proteins), fermentatio and waste conversio cells. Generally speaking, to produce a large number of cells such as is required f o r single cell proteins, one must pass through the lag phase and operate within the log phase. The greater the number of cells per unit volume, the more progeny will be produced per volume provided that the cells are neither nutrient l i m i t e d nor gas l i m i t e d . The requirement for high concentration of cells or accumulations of biomass i n the production of secondary metabolites i s even more apparent than for cell production. Secondary metabolites are generally produced i n the stationary phase; the greater the concent r a t i o n s of cells, the greater the production of secondary metabolites per unit volume and per unit time. The accumulation and r e t e n t i o n of biomass lends itself r e a d i l y to the employment of continuous single pass reactors such as plug-flow or fluidized-bed reactors. When high q u a n t i t i e s of biomass are retained, greater t o t a l q u a n t i t i e s of nutrients may be delivered and greater q u a n t i t i e s of waste products may be removed per unit time. Any mechanism that can be offered to r e t a i n the c e l l s i n high concentrations, d e l i v e r the nutrients r a p i d l y , and remove waste products should o f f e r a h i g h l y e f f i c i e n t reactor. We have found a r e l a t i o n s h i p between the accumul a t i o n of stable and v i a b l e biomass and the pore morphology of a dimensionally stable inorganic c a r r i e r . That r e l a t i o n s h i p i s dependent upon the mode of reproduction of the s p e c i f i c microbe. 0-8412-0508-6/79/47-106-013$05.00/0 © 1979 American Chemical Society In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

14

IMMOBILIZED MICROBIAL CELLS

M a t e r i a l s and Methods. The c o n t r o l l e d - p o r e c e r a m i c s and the f r i t t e d g l a s s e s employed i n t h e s e s t u d i e s were manufactured by C o r n i n g G l a s s Works. The b o r o s i l i c a t e g l a s s i s a p r o d u c t o f C o r n i n g G l a s s Works. A DuPont Biometer, Model 760, was employed t o determine the v i a b l e microbe count f o r a l l organisms e x c e p t Streptomyces olivochromogenes and Pénicillium chrysogenum. We employed a p r o t e i n d e t e r m i n a t i o n u t i l i z i n g the F o l i n r e a g e n t f o r d e t e r m i n i n g the l o a d i n g s o f the l a t t e r two m i c r o b e s . f

Determination of M i c r o b i a We were not a b l e t o employ the c o n v e n t i o n a l p l a t e c o u n t i n g t e c h n i q u e s t o determine l o a d i n g s due t o the f a c t t h a t m i c r o b i a l l o a d i n g s (biomass a c c u m u l a t i o n s ) i n v o l v e d measuring t h e number o f microbes bonded w i t h i n the p o r e s o f v a r i o u s porous s u p p o r t s . I n s t e a d , t h e microbe counts or the r e l a t i v e q u a n t i t y o f b i o mass were determined by employing the Biometer which d e t e r m i n e s the r e l a t i v e number o f v i a b l e microbes o r v i a b l e biomass based upon the amount o f ATP p r e s e n t i n a g i v e n sample. The a c t u a l p r o c e d u r e employed was as f o l l o w s : t o a p p r o x i m a t e l y 10-20 mg o f i m m o b i l i z e d microbe composite, 0.5 ml o f 90% d i m e t h y l s u l f o x i d e i n water was added and the s u s p e n s i o n was mixed v i g o r o u s l y f o r 10 seconds. The s u s p e n s i o n was a l l o w e d t o e x t r a c t f o r 20 minutes and then 4 ml o f 0.01M morpholinopropane s u l f o n i c a c i d b u f f e r , pH 7.4, was d e l i v e r e d and t h e s u s p e n s i o n was mixed t h o r o u g h l y and s t o r e d i n i c e u n t i l p r e p a r a t i o n s were complete f o r the ATP d e t e r m i n a t i o n s . P r i o r t o t h i s d e t e r m i n a t i o n , t h e l u c i f e r i n - l u c i f e r a s e m i x t u r e was p r e p a r e d a c c o r d i n g t o the Biometer p r o c e d u r e ! . A f t e r 0.1 ml o f enzyme-substrate m i x t u r e was d e l i v e r e d t o t h e r e a c t i o n c u v e t t e , 10 m i c r o l i t e r s o f t h e above DMSO e x t r a c t was added t o t h e c u v e t t e c o n t a i n i n g the l u c i f e r i n - l u c i f e r a s e m i x t u r e . The l i g h t e m i s s i o n measurement was t h e n made and t h e c o r r e l a t e d v a l u e t o v i a b l e c e l l q u a n t i t y was r e c o r d e d . S i n c e i t i s i m p o s s i b l e t o r e l a t e i n d i v i d u a l c e l l s of microbes that generate m y c e l i a t o ATP p r e s e n t , we e l e c t e d t o r e p o r t t h e mass o f ATP i n femtograms ( 1 0 " gms) as r e p r e s e n t a t i v e o f 1 5

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2. MESSING ET AL.

15

Biomass Accumulation

the v i a b l e biomass w i t h r e s p e c t t o l o a d i n g / g r a m f o r each i n d i v i d u a l c a r r i e r . The d e t e r m i n a t i o n o f biomass p r o t e i n f o r both S. ο1ivochromogenes and P. chrysogenum was performed on 0.5 gms o f c a r r i e r which c o n t a i n e d t h e i m m o b i l i z e d biomass. A f t e r t h e l o a d i n g s and/or subsequent growth, the 0.5 gm o f c a r r i e r sample was washed 3 t i m e s w i t h 10 ml o f phosphate b u f f e r , pH 7.0. The c a r r i e r was then e x t r a c t e d w i t h 3 ml o f phosphate b u f f e r by g r i n d i n g t h e r e w i t h . Subsequently, 3 ml o f 1 Ν sodium h y d r o x i d e were added, t h e temperature o f t h e m i x t u r e was r a i s e d t o 60° and m a i n t a i n e d a t t h a t temperature f o r 1 hour i n o r d e r t o h y d r o l y z e t h e m y c e l i a . T h i s procedure r e s u l t s i p r o t e i n from t h e m y c e l i a To f u r t h e r e x t r a c t p r o t e i n , 3 ml o f e t h y l a l c o h o l were added t o t h e m i x t u r e i n t h e case o f S.. o l i v o c h r o mogenes w h i l e 2 ml o f t h e a l c o h o l were added i n t h e case o f P.. chrysogenum and t h i s was a l l o w e d t o r e a c t at room temperature f o r o n e - h a l f hour. After centrif u g a t i o n , t h e s u p e r n a t a n t f l u i d was decanted and t h e p r o t e i n c o n t e n t o f t h e f l u i d was determined a c c o r d i n g t o t h e p r o c e d u r e o f H i l l e t a L ? and H a u s c h k a l . Table I C a r r i e r Parameters Average Carrier Pore Number Diameter (u) 1 2 3 4 5 6 7 8 9 10 11

1.1 3.0 3.1 3.5 4.5 10 13 19 40 195 non-porous

Pore Diameter( μ) Distribution 0.8-1.8 1.5-6 1.5-4 1.5-4.5 3-6 2-19 8-20 17-35 18-100 170-220

Carrier Composition F r i t t e d glass Cordierite Fritted glass Fritted glass F r i t t e d glass Cordierite F r i t t e d glass Z i r c o n i a Ceramic Fritted glass F r i t t e d glass Borosilicate glass

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

16

IMMOBILIZED MICROBIAL CELLS

Bioaccumulation of E s c h e r i c h i a

coli.

Samples o f Ε . c o l i ( h a v i n g major dimensions o f 1-6μ) were i m m o b i l i z e d t o t h e v a r i o u s c a r r i e r s , u s i n g s t e r i l e t e c h n i q u e on b o t h a v i r g i n c a r r i e r s u r f a c e and a s u r f a c e t h a t had been c o n v e r t e d t o a t e r m i n a l amine by s i l a n i z a t i o n . The bonding by a d s o r p t i o n t o t h e v i r g i n s u r f a c e was a c c o m p l i s h e d by r e a c t i n g 3 gm p o r ­ t i o n s o f 18-25 mesh p a r t i c l e s o f t h e i n d i c a t e d c a r r i e r s f o r 3 hours a t 22°C w i t h 20 ml o f s u s p e n s i o n o f Ε . coli cells. The bonding t o t h e t e r m i n a l amine s u r f a c e was i n i t i a t e d f i r s t by r e a c t i n g 2 gms o f 18-25 mesh p a r t i c l e s w i t h 20 ml o f 10% γ-aminopropyltriethoxys i l a n e i n water f o c a r r i e r was s u b s e q u e n t l o f s u s p e n s i o n o f E. c o l i c e l l s o v e r n i g h t a t 22°C. B a c t e r i a l l o a d i n g s (number/gram o f support) were t h e n r e c o r d e d v i a ATP measurement a p p r o x i m a t e l y 18 hours a f t e r the p r e p a r a t i o n o f t h e i m m o b i l i z e d m i c r o b e s . The r e s u l t s a r e summarized i n F i g u r e s l a and l b . B i o a c c u m u l a t i o n o f S e r r a t i a marcescens by P o l y i s o c y a nate

Coupling.

S. marcescens, h a v i n g major dimensions o f 0.6-2μ was c o u p l e d t o g l a s s s u r f a c e s w i t h p o l y i s o c y a n a t e (PAPI 901, Upjohn Company, Kalamazoo, MI) . The c a r r i e r d e r i v a t i v e was p r e p a r e d by s h a k i n g a t 100 RPM 0.5 gms o f c a r r i e r i n 10 ml o f 0.5% p o l y i s o c y a n a t e i n acetone f o r 45 minutes a t room t e m p e r a t u r e . The c o u p l i n g s o l u t i o n was decanted and r e p l a c e d w i t h 10 ml o f a c e l l s u s p e n s i o n c o n t a i n i n g 3 χ 10^ c e l l s / m l . The c e l l s were r e a c t e d w i t h t h e d e r i v a t i z e d c a r r i e r f o r 3 h o u r s f o l l o w i n g which t h e excess c e l l s were poured o f f and t h e c a r r i e r was washed 3 t i m e s w i t h 0.1 M phosphate b u f f e r a t pH 7.2. The r e s u l t s o f t h i s s t u d y w i t h v a r i o u s c a r r i e r s a r e p l o t t e d i n F i g u r e 2. The

I m m o b i l i z a t i o n o f B a c i l l u s s u b t i l i s by A d s o r p t i o n ,

The c u l t u r e c e l l s were s i z e d p r i o r t o i m m o b i l i ­ zation. The major d i m e n s i o n o f t h e c e l l s were found t o be between 3 and 4μ w i t h some l o n g double c e l l s o f a p p r o x i m a t e l y 7μ i n l e n g t h .

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2.

MESSING ET AL.

Biomass Accumulation

17

P r i o r t o use, t h e c a r r i e r s were d r y a u t o c l a v e d . B a c i l l u s s u b t i l i s c e l l s were grown i n 1 l i t e r b r a i n h e a r t i n f u s i o n b r o t h f o r 36 hours, c e n t r i f u g e d , a n d t h e c e l l s were washed 2 times w i t h s t e r i l e phosphate b u f f e r Ten ml o f t h e washed c e l l s u s p e n s i o n was added t o each f l a s k which c o n t a i n e d 0.5 gms o f c a r r i e r . A f t e r 3 h o u r s o f c o n t a c t time, t h e c a r r i e r s were washed 3 times and m a i n t a i n e d a t 8°C o v e r n i g h t . The c e l l mass i m m o b i l i z e d on t h e c a r r i e r s was determined by t h e B i o m e t e r . The r e s u l t s o f t h i s study a r e r e p o r t e d i n F i g u r e 3. Bioaccumulation

o f Yeast

The y e a s t c e l l employe y grown i n shake f l a s k s i n n u t r i e n t b r o t h p l u s 1% dext r o s e f o r a p e r i o d o f 36-40 h o u r s a t room t e m p e r a t u r e . The c e l l s u s p e n s i o n was c e n t r i f u g e d and washed 3 times w i t h sodium-potassium phosphate b u f f e r , pH 7.2. The washed s u s p e n s i o n o f c e l l s was added t o t h e c a r r i e r and a g i t a t e d by s h a k i n g f o r 3 h o u r s o f c o n t a c t t i m e . For a d s o r p t i o n , t h e c a r r i e r s were ground t o an 1825 mesh, s t e r i l i z e d d r y , and p l a c e d i n a 37°C i n c u b a t o r o v e r n i g h t t o produce a d r y c a r r i e r . A q u a n t i t y o f 0.5 gms o f each c a r r i e r was added t o 50 ml f l a s k s . Ten ml of c o n c e n t r a t e d y e a s t c e l l s u s p e n s i o n was added t o t h e c a r r i e r i n the f l a s k . A t t h e end o f 3 h o u r s o f c o n t a c t time, t h e e x c e s s c e l l s were poured o f f , t h e c a r r i e r was washed 3 times with phosphate b u f f e r and s t o r e d i n t h e r e f r i g e r a t o r p r i o r to the loading determinations. The c o u p l i n g p r o c e d u r e i n v o l v e d t h e a d d i t i o n o f 0.5 gms o f each c a r r i e r t o s e p a r a t e 50 ml f l a s k s . The f l a s k s , w i t h c o n t e n t s , were d r y a u t o c l a v e d and p l a c e d i n a 37°C i n c u b a t o r o v e r n i g h t t o m a i n t a i n t h e c a r r i e r dry. Ten ml o f a 0.5% p o l y i s o c y a n a t e i n acetone s o l u t i o n was added t o each c a r r i e r . The f l a s k s w i t h c o n t e n t s were shaken f o r 45 minutes a t room temperature a f t e r which t h e c o u p l i n g s o l u t i o n was d e c a n t e d . Ten ml o f c o n c e n t r a t e d y e a s t c e l l s u s p e n s i o n was then added t o each f l a s k and t h e f l a s k s were shaken f o r 3 h o u r s a f t e r which t h e e x c e s s c e l l s were d e c a n t e d and t h e immobil i z e d p r e p a r a t i o n was washed 3 times w i t h phosphate buffer. The ATP d e t e r m i n a t i o n s o f c e l l l o a d i n g s were p e r formed w i t h t h e Biometer as p r e v i o u s l y d e s c r i b e d .

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

IMMOBILIZED MICROBIAL CELLS

0%

10

0 50 100 150 200 AVG PORE DIAMETER (μ)

b

AVG. PORE DIAMETER {μ)

Figure 1.

(a and b) Bioaccumulation of E. coli

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

Biomass Accumulation

MESSING ET AL.

0%'

J

I

I L

8 16 24 32 190 AVG. PORE DIAMETER (μ)

Figure 2.

Bioaccumulation of S. marcescens

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

20

IMMOBILIZED MICROBIAL CELLS

The y e a s t , Saccharomyces c e r e v i s i a e , was s i z e d p r i o r t o i m m o b i l i z a t i o n . The average c e l l dimensions were 4 χ 5.5μ (2.5-4 χ 4-7) and about 20% o f t h e c e l l s were 4.5 χ 7μ. The r e s u l t s o f t h e b i o a c c u m u l a t i o n o f t h i s microbe by a d s o r p t i o n a r e p l o t t e d i n F i g u r e 4. The y e a s t , Saccharomyces amurcae, p r i o r t o immo­ b i l i z a t i o n were found t o have dimensions o f 5.5 χ 7μ (3-7 χ 6-9) i n terms o f s m a l l , s i n g l e c e l l s w h i l e an a d d i t i o n a l p o p u l a t i o n o f l a r g e r , double c e l l s which c o n s t i t u t e d a p p r o x i m a t e l y 75% o f t h e p o p u l a t i o n were found t o be 6-8 χ 13-18μ. These c e l l s were i m m o b i l i z e d by c o u p l i n g w i t h p o l y i s o c y a n a t e and t h e r e s u l t s o f t h i s s t u d y a r e p l o t t e d i n F i g u r e 5. Bioaccumulation of The s p o r e s o f t h i s organism were observed t o range from 3-5μ. These s p o r e s were e l u t e d from a mature growth i n a B l a k e b o t t l e w i t h a s t e r i l e phosphate buffer. The s u s p e n s i o n was made t o 100 ml w i t h b u f f e r . Ten ml o f t h e spore s u s p e n s i o n was added t o each 50 ml m i c r o f e r n b a c k f l a s k which had been d r y a u t o c l a v e d w i t h 1 gram o f c a r r i e r and d r i e d o v e r n i g h t p r i o r t o i t s u s e . A f t e r 3 hours o f s h a k i n g a t room temperature, t h e excess s p o r e s were poured o f f , t h e c a r r i e r was washed 3 t i m e s w i t h phosphate b u f f e r and s t o r e d o v e r n i g h t a t 8°C. The q u a n t i t y o f s p o r e s t h a t was adsorbed by t h e v a r i o u s c a r r i e r s was determined by ATP measurement and the r e s u l t s a r e p l o t t e d i n F i g u r e 6. In o r d e r t o determine t h e optimum pore diameter range f o r m y c e l i a l growth, 0.5 grams o f each c a r r i e r w i t h t h e i m m o b i l i z e d spores was p l a c e d i n 75 ml o f Sabouraud d e x t r o s e b r o t h and t h e composite was a l l o w e d t o shake on a shaker a t room temperature. A t t h e end o f 27 hours, a sample o f each c a r r i e r was taken and t h e amount o f ATP was determined as a measure o f m y c e l i a l growth. The r e s u l t s a r e r e c o r d e d i n F i g u r e 6. B i o a c c u m u l a t i o n o f Streptomyces

olivochromogenes.

E q u a l amounts o f s p o r e s i n phosphate b u f f e r were added t o 0.5 grams o f each o f t h e c a r r i e r s . The s p o r e s and c a r r i e r s were a l l o w e d t o r e a c t t o g e t h e r f o r 48 hours. The n o n - r e a c t e d s p o r e s were decanted and t h e c a r r i e r was washed 3 t i m e s w i t h 2 ml a l i q u o t s o f

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2.

MESSING ET AL.

I0 5 7

^5

Biomass Accumulation

21

^ ^5 (so Î8Ô 210 Figure 6. Bioaccumulation of A. niger AVERAGE PORE DIA. (μ) in controlled pore inorganics

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

22

IMMOBILIZED MICROBIAL CELLS

s t e r i l e phosphate b u f f e r . These n o n - r e a c t e d s p o r e s p l u s the washings were c o l l e c t e d , the volume determined and t h e n a n a l y z e d f o r p r o t e i n c o n t e n t . The q u a n t i t y of p r o t e i n adsorbed i n the c a r r i e r was c a l c u l a t e d as t h e d i f f e r e n c e between t h e i n i t i a l c o n t e n t o f p r o t e i n i n t h e r e a c t i n g volume c o n t a i n i n g the s p o r e s and t h a t c o n t a i n e d i n the n o n - r e a c t e d s p o r e s p l u s t h e washings. The washed c a r r i e r c o n t a i n i n g the s p o r e s was s e p a r a t e l y t r a n s f e r r e d t o a f l a s k c o n t a i n i n g 50 ml o f Emerson b r o t h medium. Mycelium f o r m a t i o n w i t h i n t h e c a r r i e r p o r e s was e v a l u a t e d a f t e r i n c u b a t i o n w i t h s h a k i n g f o r 24 and 48 hours a t room temperature. At the end o f t h e s t a t e d p e r i o d s o f i n c u b a t i o n , t h e c a r r i e r was s e p a r a t e f u g a t i o n and washed b u f f e r , pH 7.0. The p r o t e i n c o n t e n t w i t h i n t h e c a r r i e r was determined by the p r o c e d u r e d e s c r i b e d p r e v i o u s l y . The r e s u l t s o f both the spore a c c u m u l a t i o n and the m y c e l i a l growth w i t h i n t h e c a r r i e r a r e r e p o r t e d i n F i g u r e 7. B i o a c c u m u l a t i o n o f Pénicillium chrysogenum. The t e c h n i q u e s f o r t h e i m m o b i l i z a t i o n o f the s p o r e s and t h e h a n d l i n g o f t h e c a r r i e r m a t e r i a l s was e s s e n t i a l l y the same as t h o s e d e s c r i b e d f o r t h e olivochromogenes e x p e r i m e n t s . The washing o f t h e c a r r i e r s and t h e d e t e r m i n a t i o n o f p r o t e i n c o n t e n t were conducted i n the same manner as p r e v i o u s l y d e s c r i b e d . Other than t h e m o d i f i c a t i o n o f a l c o h o l q u a n t i t y f o r e x t r a c t i n g the p r o t e i n , t h e o n l y o t h e r change was the medium f o r m y c e l i a l growth. In p l a c e o f the Emerson b r o t h , 50 ml o f an aqueous medium h a v i n g a pH o f 6 . 3 w i t h the f o l l o w i n g c o m p o s i t i o n was u t i l i z e d f o r the growth o f P. chrysogenum; 2% l a c t o s e , 1% g l u c o s e , 0.2%KH2PO4, 0.125% N H 4 N O 3 , 0.05% N a S 0 , 0.025%MgSO , 0.002% MnS04, 0.00025% CuSO4, and 0.002% ZnS04. A s i n g l e i n c u b a t i o n of 48 hours was employed. The r e s u l t s o b t a i n e d w i t h P^ chrysogenum a r e r e c o r d e d i n F i g u r e 8. 2

4

4

R e s u l t s and D i s c u s s i o n s Upon p e r u s a l o f t h e f i g u r e s , i t becomes r a t h e r c l e a r t h a t a t l e a s t one optimum i s noted f o r the bioaccumu-

In Immobilized Microbial Cells; Venkatsubramanian, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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