KINETICS OF AEROBIC UTILIZATION OF MIXED SUGARS BY HETEROGENEOUS MICROBIAL ...

489 Pages·2008·44.16 MB·English

KINETICS OF AEROBIC UTILIZATION OF MIXED SUGARS
BY HETEROGENEOUS MICROBIAL POPULATIONS
A THESIS
Presented to
The Faculty of the Graduate Division
by
Sambhunaih Ghosh
Ir. Partial Ful.filimeut
of the Requirements for the Degree
Doctor of Philosophy
In the School of Civil PUgineering
Georgia Ins+itute of Technology
November, 1 9 69
KINETICS OF AEROBIC UTILIZATION OF MIXED SUGARS
BY HETEROGENEOUS MICROBIAL POFUIATIONS
Approved:
Chairman ^ *' _
Date approved by ©teerirmari: Dec. 5j 19'^(
ii
ACKNOWLEDGMENTS
The author gratefully acknowledges Dr. W. E. Gates for his
continued interest in the research project, his encouragement, and his
advice. The author is indebted to Dr. Gates for stimulating his inter­
est in the area of biokinetics. Special thanks are due to Dr. F. G.
Pohland for his advice and untiring help in thoroughly reviewing and
constructively criticizing the entire manuscript. Thanks are also due
to Dr. P. B. Sherry for kindly serving as a member of the Reading Com­
mittee and for making constructive criticisms regarding presentation
of the research findings.
This work was partly supported by the Federal Water Pollution
Control Training Grant No. 5T1-WP-62-03 to 05 • Thanks are due to Pro­
fessor C. E. Kindsvater for making this traineeship available to the
author. Sincere appreciation is extended to Dr. W. M. Sangster for all
of his help and encouragement. Thanks are accorded to Dr. A. W. Hoadley,
Mr. E. E. Ozburn, Dr. J. D. Westfield, Mr. B. G. Milton, and other mem­
bers of the faculty, staff, and fellow graduate students for various
forms of help and assistance. The author would like to express his
sincere appreciation to Miss N. G. McKinney for her valuable help in
locating reference materials not easily available. Her help contributed
significantly toward a comprehensive review of the literature.
The author expresses his deep appreciation to his parents for their
patience and encouragement, and to his wife, Anima, for her cooperation
and understanding during the last four years in which much of his efforts
Ill
were directed toward completion of this work; she is also especially
thanked iV r her material help iii typing the rough draft of the volumi-
n< lis mariusc ?ip4'..
Permissi n has been granted by tie Graduate Division for special
pagination and margins in order to enable this dissertation to be pub-
.ished as a report of the Water Resources Center, Georgia Institute of
technology. The titles of" full-page figures are given in the standard
'typography for the field«
iv
PREFACE
The influents to a system employed for the biological stabiliza­
tion of organic wastewaters frequently contain multiple sources of
carbon and energy necessary for microbial metabolism. Each of these
sources of carbon and energy (hereafter called substrate) has the po­
tential of controlling the microbial growth rate at one stage or another.
It is generally assumed that a microbial culture assimilates all of the
substrates simultaneously. However, the pioneering work of Monod with
pure cultures revealed that the presence of a specific substrate con­
stituent (such as glucose) caused competition between the substrates
for controlling the rate of growth. Under these circumstances, the
microorganisms preferentially assimilated one substrate at a time while
temporarily preventing metabolism of the others. Therefore, the corres­
ponding batch growth curves were characterized by sequential growth
cycles with each cycle corresponding to the exclusive utilization of a
specific substrate of the mixture in separate phases.
A few studies have been reported in the past ten years showing
that phasic uptake of competing substrates can also be brought about
by heterogeneous cultures employed in biological waste treatment pro­
cesses. However, the conclusions of the investigators have been contra­
dictory. Some have shown that glucose and galactose are sequentially
removed by a heterogeneous batch culture whereas others have found these
same substrates to be simultaneously assimilated in a batch process
employing organisms having their origin in waste treatment plants.
Moreover, glucose and sorbitol have been shown, to be assimilated simul­
taneously or in separat: phases in batch cultures depending on the "age"
of the culture, Some researchers have also investigated the fate of
competing substrates in continuous biological processes. Continuous
culture studies have shown that competing substates can be simultan­
eously assimilated by heterogeneous microbial populations although these
same substrates were utilized sequentially (i.e., in separate phases)
daring batch growth. Clearly, the results of continuous culture studies
we re both in agreement with and contradicted by the results of the batch
culture studies. Uiifcrtuoately, few efforts have been directed toward
resolution of these contradictions. The conditions under which compet­
ing substrates will be sequentially or concurrently assimilated have
on teen established. Furthermore, information regarding kinetics of
o/owhh and. removal of Jnn competing substrates is not available.
'The causes and. kinetics of sequential and/or concurrent utiliza-
to n of competing substrates ate of great importance for prediction of
n.e performas ;es as well as f r the design and control of biological
treatment processes*. Since such information has not become clearly
establisheo., the objectives of this research, were:
1. to i lives M ga( e t he Trie of the environmental, biochemical, or
^+ter fa tors responsible for the occurrence of phasic or concurrent
as similar.:- u . x two competing substrates;
2. to obtain basic information necessary for formulation of
mathematical m lets describing the kinetics of assimilation of the
..- mpetiug sucstiates; ana
vi
3« to identify the probable cellular mechanisms that regulate
the pattern (sequential or concurrent) of substrate assimilation.
vii
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS . . , . . „ . . . „ . . II
PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . .. iv
LIST OF TABLES. xi
LIST OF ILLUSTRATION'S . . . . . . . . . . . . . . . . . . . . . .. xili
SUMMARY . . . . . . . . . . . . . . . . . . .. . . .. xix
Chapter
I. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . .. 1
Biological Cultures 1
Biological Cultures in Sanitary Engineering. . . . . . .. 7
Sanitary Engineering Processes 9
Process Kinetics „<> . . . . . . . . . . . . . . . . .. . 11
Substrate Interaction and Diauxie. . . . . . . . . . . .. 15
Cellular Mechanisms for Regulated Uptake
of Competing Substrates. . . . . . . . . . . . . . . .. 18
Substrate Interactions in Sanitary
E .gineering Pr< cesses. . . . . . . . .. . 21.
Significance ir. Sanitary Engineering 22
S< pe i f Furthes Studies 23
REVIEW OF
II. LITERATURE . . . . . .. . . . . . . . . . .. . 26
General. . ... . . . . . . . . . . . . . . . . . . .. . 26
Biologi :;al Processes . . . . . . . . . . . . . . . . . .. 26
Cellular Transport of Nutrients. . . . . . . 28
Enzymes a; .a. Metabolism . . . . . . . . . . . . . . . . .. 30
Grovth . . . . . . . . . . . . . . . . . . . . . . . . .. 33
Evolutionary Development of Microbial
Response Mechanisms to Environment . . . . . . . . . .. 55
I ,'. c.. al Opera''• 10 c of Cellular Processes
and diauxie Phenomena. 61.
Mechanisms of Controlled Enzyme Synthesis. . . . . . . .. 6k
Role of Regulatory Mechanisms in Controlled
Uptake of Competing Substrates . . . . . . . . . . . .. 72
i.iauxAe i . ie e :c eoc u Culture . . . . . . . . . . .. 75
Co:: Lnuous Culture I!echniq[ue . . . . . . . . . . . . . .. 81
Population ;s and Selection in
By;,ami
Continuous Culture . . . . . . . . . . . . . . . . . .. 9k
viii
TABLE OF CONTENTS (Continued)
Chapter Page
III. FORMULATION OF THE PROPOSED RESEARCH
METHODOLOGY. 97
Introduction 97
Design of Experiment 1 01
IV. KINETIC CONSIDERATIONS . 106
Development of Enzyme and Growth Kinetics 106
Differential Equations for Bacterial and
Substrate Concentrations in Completely-
Mixed Continuous Flow Reactor 1 17
Fractional Use of Assimilated Substrate
for Growth and Maintenance 1 32
Population Dynamics in Continuous Flow Reactors 1 33
Vo DEVELOPMENT AND DESIGN OF THE EXPERIMENTAL
APPARATUS 1^0
Completely-mixed Continuous Flow Reactor
System for Heterogeneous Bacterial Cultures ikO
Operation of the Continuous Flow Reactor System 1 63
VI. DEVELOPMENT OF ANALYTICAL TECHNIQUES 1 70
Introduction . 1 70
Development of an Analytical Procedure for
the Measurement of Organism Concentration 1 70
Analytical Techniques for Determination of
Glucose and Galactose Concentrations 218
VII. EXPERIMENTAL PROCEDURE 2 21
Determination of the Hydrodynamic Characteristics
of the Reactor 2 21
Selection and Design of Buffer Solution 230
Selection of Influent Concentrations of
Substrate Solutions 233
Selection of the Concentrations of
Nutrient Solutions 235
Selection of Flow Rates 237
Continuous Runs 237
Bacteriological Examinations 2^4-0
Problems in the Attainment of Steady States 2^-1
ix
TABLE OF CONTENTS (Continued)
Chapter Page
VIII. PRESENTATION ANT DISCUSSION OF TEE
EXPERIMENTAL DATA, 2^5
Presentation of the Experimental Data-, . . . . . . . .. . 2^5
Discussi'-r- of. the Experimental Data. . . . . . . . . . .. 2U8
IX. CONCLUSIONS. . . . . . . . . .. . . .. . 3M+
U
Continuous Process Kinetics for Utilization
of Single Sugar Substrate. . . . . . .. „ . . . . .. . 3^
Kinetics of Utilizatr ,u of Interacting Substrates. . . . „ 3U7
The Role - f Bi. chemical and Environmental
Factors in Determining the Mode of
Uptake of Competing Substrates . . . . . . . . . . . .. 350
Conclusions of Engineering Significance. . 3 51
X. RECOMMENDATIONS. . . . . . .. . . . . . . . . . . . .. . 353
APPENDICES. . . . . . .. . . . . . . . .. . . . . . . . . .. . 357
I. LESION OF THE CULTURE VOLUME FOR IIE CONTINUOUS
FLOW REACTOR. . . . . • * > , . * . , . . . . . . . . . „ . .. 358
II. HYDRAULIC ANALYSIS OF THE CONSTANT HEAD DEVICE
EMPLOYED FOR THE DELIVERI OF FEED SOLUTIONS TO
THE CONTINUOUS FLOW REACTOR. . .. . . . . . . . . . .. . 362
TIL. DETAILED PROCEDURE FOR THE DEHYDROGENASE TEST. . . . . .. 365
IV O M INITIAL MEDIA FOR BATCH CULTURES . . . . . . . . . . . .. 368
V. PROCEDURE EOR DETERMINATION OF CONCENTRATIONS
OF DRY BACTERIAL SOLIDS BY THE CONTROL FILTER
GRLAVIMETRIC IECHNIQUE . . . . . . . . . . . . . . . . . .. 369
VI. SUMMAI { OF BATCH DATA. . . . . . .. . . . . . . . . . .. . 372
VII. CORRELAT'ION BETWEEN' AB80RBANCES OF DEHYDRO­
GENASE SOLUTIONS WITH ONE CENTIMETEJR. AND
TEN CENTIMETER LIGHT PATHS . . . . .. . . . . . .. . . 3 77
a
VIII0 ENZiTIATT.C METHOD OF THE DETERMINATION
OF GLUCOSE USING WORTHING!ON GLUCOSTAT . . . . . . . . .. 379
DC. ENZYMATIC METHOD OF THE DETERMINATION OF
GALACTOSE USING WORTH.; It TON GAnACIOSIAT. . . . . . . . .. 383