The isolation of aerobic cellulose-decomposing organisms and their action on cellulose and associated plant constituents

104 Pages·2016·7.25 MB·English

by  Fuller

THE ISOLATION OF AEROBIC CELLULOSE-DECOMPOSING
ORGANISMS AND THEIR ACTION CM CELLULOSE
AID ASSOCIATED PLANT CONSTITUENTS ^ ?
by
Wallace H, Puller
A Thesis Submitted to the Graduate Faculty
for the Degree of
DOCTOR OF PHILOSOPHY
Major Subject* Soil Bacteriology
Approved*
f ..
Stni'Sarge Sajor 'Work
laaa of Graduate College
Iowa State College
i942
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UMI Number: DP12706
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■'"TS.
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TABLE OF G0R8KXS
Page
iifiow efioi . . ........................ ............................. ............... 4
IlSfOEICAL ..................... 6
A. Aerobic Mesophilio Cellulose-Decomposing Organisms . ...... 6
1. Methods of isolation and purification . . . . . . . . . . . . . . 6
2. Bacteria ............. 8
B. Decomposition of Pure Cellulose ............. 12
1. Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2. Fungi 15
3. Aettaoayeetes ......................................................... 16
C« Decomposition of Cellulose Ihen in Association Wi-tii Other
Plant Constitusnts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1. Decomposition of cellulose in natural plant
material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2. Decomposition of lignin and its influence on
cellulose decomposition ............ 17
3. Decomposition of the hemieelluloses .............. 20
SXF1RIMESXAL ............................................... 22
A, Plan of Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
B. Methods ©f Procedure ................. 22
1. Isolation of cellulose decomposing organisms
from the soil 22
2. Cellulose dextrine and cornstalk oellulesaa . ....... 24
a. Preparation 24
b. Determination of mean chain length of
cellulose dextrine ............. 26
e. Dee of dextrine and eellulosaas as
biological substrates . . . . . . . . . . . . . . . . . . . . . . . . 27
3. Characterisation of the pure cultures . . . . . . . . . . . . . 27
4. Cellulose decomposition . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
a. Preparation of samples . . . . . . . . . . . . . . . . . . . . . . 29
(1) Cornstalk cellulose . . . . . . . . . . . . . . . . . . . 29
(2) Extracted ecrastalk cellulose law
in oellulosan . . . . . . . . . . . . . . . . . . . . . . . . . 30
(3) Extracted cornstalk cellulose inter­
mediate in oellulosan . . . . . . . . . . . . . . . . . 30
(4} Jute samples . . . . . . . . . . . . . . . . . . . . . . . . . . 31
b. technique of fermentation . . . . . . . . . . . . . . . . . . . 31
e. Analytic procedure . . . . . . . . . . . . . . . . . . . . . . . . . . 35
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Page
mmhts .........................................
56
A. The Use of Cellulose Dextrins . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
1. Determination of mean chain length . . . . . . . . . . . . . . . . 36
3. Suitability of dextrine for culturing some
aerobic mesophilic bacteria ................. 38
3. Suitability of dextrins for Isolating and count­
ing aerobic cellulose organisms from the so il ..... 43
B. the Description and Classification of the Organisms ..... 48
1. Characteristics of the organisms ............... 50
2. Systematic position of the organisms . . . . . . . . . . . . . . 63
C. the Decomposition of Cellulose ........ 66
1. Decomposition of filte r paper . . . . . . . . . . . . . . . . . . . . . 67
2. Decomposition, of cornstalk cellulose . . . . . . . . . . . . . . 69
3. Decomposition of Jute preparations . . . . . . . . . . . . . . . . 74
4. Decomposition of filte r paper cellulose by
various actinomycetes and fungi .......... 83
DISC0SSIOB AMD C08CLUSIG8S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
StJIitABY..................... 96
1ITBRAY8S8 CITBB.......................... 98
Acm m m m m t ............................................... 103
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4
1ITR0BUCTIGH
Cellulose is the major plant constituent entering the soil sad for
this reason the decomposition of cellulose is important to soil fe rtility .
Cellulose may he found in peat and the F-layer of forest soil in small quanti­
ties, hut when, incorporated in most soils, i t rapidly decomposes and dis­
appears. Exactly how th is sudden loss occurs and the contribution cellulose
makes to the soil and to the soil mioroflora are not clearly known.
Most of the attention given to the decomposition of cellulose has been
focused on investigations using filte r paper or using whole plant m aterials.
In the latter ease little attention has hem given to the Influence of the
major associated plant constituents on the cellulose decomposition, this
disregard may he largely attributed to a lack of knowledge of the chemical
composition and structure of these associated constituents and the absence
of means of Isolating them without accompanying undesirable changes.
All cellulose cannot justifiably be represented by filte r paper and
Indeed, cellulose undoubtedly varies with the plant and, quite likely, with
the stage of maturity of the plant. The relative proportion of the other
plant constituents to cellulose is known to vary in different plants and a
relative change of one plant constituent may profoundly alter the rate of
decomposition of another. Coir fiber, for instance, has about the same
cellulose content as cornstalks, but is proportionately higher in lignin
«id is far less easily decomposed. Past methods of attack have not made
clear what exact effect a certain associated plant constituent has on the
decomposition of cellulose.
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the fact that the bacteriology of the cellulose-decomposing organisms,
and particularly of the aerobic mesophilic bacteria, 1* incomplete and some­
what obscure has also contributed to the lack of a better understanding as
to the fate of cellulose in the soil. Both the fibrous nature of cellulose
and its insolubility in water* which make the isolation end separation of
the cellulose attacking bacteria difficult* are* in part, tee cause of the
unsatisfactory nature of the information in this field.
In tee following work certain objectives were set in order better to
understand the fate of cellulose in the so il. First* cellulose dextrins
were prepared and used for the isolation of seme mesophilic organisms from
tee soil. Second* tee characteristics of tee cellulose decomposing organisms
were examined, third* pure-culture* pure-substrate studies were made to de­
termine the specific nature of the decomposition of cellulose derived from
different soirees. Fourth, pure-culture* aaixed-substrate experiments were
conducted to determine the effeot of tee various plant constituents on the
decomposition of cellulose.
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6
HISTORICAL
A, Aerobic Mesophilic Cellulose Decomposing Organisms
1* Methods of isolation md purification
Iren thought i t has long been known that cellulose disappears rapidly
when in contact with the so il, the specific agents causing the aerobie de­
composition are not well understood, except for some of the fungi and some
specialised bacteria, notably the cytephagae* The cellulose-deeomposing
fungi* and to a lesser extent the actinomycetes, offer little difficulty in
isolation and purification since they produce colonies on cellulose that
are easily distinguishable and grow well on a wide variety of media* This
is not true of the bacteria. The physiologically variable character of the
baoteria and the inapplicability of cellulose to the ordinary methods of
plate technique make their isolation and purification a problem.
A survey of methods used in the isolation and purification of cellulose-
decomposing organisms has recently been made (M)* The methods have been
divided into two major categories! those that use some form of purified
cellulose and those that use regenerated cellulose* The latter is cellulose
that has been dissolved and reprecipitated, a procedure that causes the
cellulose to lose appreciably the orientation of the micelles* Only the
methods that are suitable for the isolation of aerobic mesophilic baoteria
w ill be briefly reviewed here*
The first methods used to determine the kinds of organisms responsible
for cellulose decomposition consisted of the examination of decayed areas
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of plant materials or bits of linen or paper tbnt had been inserted into
the soil (§)* Gmeliansky (58) preferred inoculating, with grains of soil,
a mineral nutrient solution to which had been added cellulose in the fern
of filte r paper* Most of the growth is observed on the cellulose at the
vicinity of the w ater-air interface in such cultures. Relatively pure cul­
tures may be obtained by repeatedly transferring bits of the decayed portion
to other mineral nutrient solutions containing filte r paper, either in strips
or in the shape of inverted cones in flasks, the former is essentially the
method used by Dubes (7).
Several procedures for the preparation of slllca-ge! plates for isola­
tion purposes have been proposed (4) (56) (€3), Although this method may be
used in Isolating and identifying cellulose-decomposing organisms, it is not
well adapted to their purification.
The use of regenerated cellulose, a product made by dissolving cellulose
in cuprammonium solution and repreoipitating in acid solution, was first
advocated by Xellerasn and MeSeth (19)* The product probably has net under­
gone much reduction in molecular size, but is considerably changed both physi­
cally and structurally. These la tte r changes make it suitable for incorpora­
tion in an agar medium and, therefore, this form is well adapted to the ordin­
ary dilution plate technique. A more strongly hydrolyzed product, prepared
by treating cellulose with zinc chloride (21) or ferrio chloride (36), has
been employed with some measure of success in the separation of cellulose
organisms.
A hydroeellulose or cellulose hydrate was prepared by Scales (43) using
sulfuric acid. F ilter paper was dissolved in 00 per cent sulfuric acid at a
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temperature of 60-66°C, sad Immediately precipitated in water, the product
is easily incorporated in agar medium and has the added advantage of being
relatively easy to prepare, the exact chemical nature of this hi^ily hydrat­
ed material is not known, although it differs tram, natural cellulose in many
respects. Most of the regenerated cellulose products are somewhat laborious
to prepare and non© of them have been described as being capable of support­
ing growth of sob© of the less versatile organisms that ©an use only cellu­
lose as a carbon sourcet namely, the oytophagas.
The use of cellulose dextrins prepared by cold acid hydrolysis has met
with considerable success in the isolation and separation of aerobic meso-
phillc bacteria including the oytophagas (9) (10),
In general the oeilulos© derivatives are not suitable for isolation
work primarily because of their resistance to microbial attack, Xrzemiemiew-
ska (22) showed that cytophaga could grow on cellophane and used this sub­
strate to aid in studying the morphological characteristics of members of
this genus,
2. Bacteria
Considerable attention has been focused oathe specialised group of
baoteria whose principal capability is that of attacking only cellulose. In
addition to the more specialised forms, there are many cellulose-decomposing
bacteria quite versatile and capable of using many differ mat sources of oar-
bon (34),
She fact that cellulose is decomposed aerobically was only slowly
recognized, though anaerobic decomposition had long been known, Tan Iterson
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9
(53) was the first to emphasise and establish this fact by the isolation of
an aerobic culture -which, ho named Bacterium ferruginiua. Even though the
culture was of questionable purity, it did serve to demonstrate that cellu­
lose is decomposed aerobically and his work stimulated research in this direc­
tion. Cultures of aerobic baoteria of unquestionable purity were f ira t
isolated by Kellersan and his associates (19) (20), Twenty species are lis t­
ed in Sergey’s Manual (2) as belonging to the genus Oellulamonas. They are
described as short reds with rounded ends, either motile or nom-motile with
peritrichous flagella. Some produce pigment and all attack cellulose. They
are somewhat variable with respect to their ability to attack various other
carbon and nitrogen sources. A later detailed study by Stapp and Bertels
(51) of some of these organisms showed that they are not specific for cellu­
lose as a carbon source. It was also pointed out that when grown under
laboratory conditions or cultured on medium not containing cellulose the
baoteria soon appeared to lose their ability to attack cellulose.
Since the isolation of Splroohaeta cytophaga by Hutchinson and Clayton
(15) workers on aerobic cellulose decomposition seem to have been preoccupied
with this organism. This is probably due to its wide-spread distribution in
soils, its activity on pure cellulose, and its interesting and characteristic
morphology. It was first described as a long, thin, filamentous rod that is
accompanied by a round "sporoid* form in old cultures. The former cells
vary from 2 to 10/a in length and 0.3 to 0*4/~ in width, whereas the latter
are approximately l*B/< in diameter. They stain weakly by the usual methods.
The m otility is not by means of flagella, but by an undulatory or rotatory
action. A b rillian t yellow pipw at is produced. Only cellulose is utilised
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