the production and characterization of alginate produced
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Louisiana State UniversityLSU Digital Commons
LSU Historical Dissertations and Theses Graduate School
1994
The Production and Characterization of AlginateProduced by Pseudomonas Syringae.Richard David AshbyLouisiana State University and Agricultural & Mechanical College
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The production and characterization o f alginate produced byPseudomonas syringae
Ashby, Richard David, Ph.D.
The Louisiana State University and Agricultural ana Mechanical Col., 1994
U M I300 N. ZeebRd.Ann Arbor, MI 48106
THE PRODUCTION AND CHARACTERIZATION OF ALGINATE PRODUCED
BY PSEUDOMONAS SYRINGAE
A Dissertation
Subm itted to the Graduate Faculty of the Louisiana State University and
Agricultural and Mechanical College in partia l fulfillment of the
requirem ents for the degree of Doctor of Philosophy
in
The D epartm ent of Microbiology
byRichard David Ashby
B.S., Brigham Young University, 1987 August 1994
ACKNOWLEDGMENTS
I w ould like to express m y deep est ap p rec ia tio n to m y
advisor, Dr. Donal. F. Day, for his advise, encouragem ent, an d
valuable discussions throughout this work, and also for his help in
the preparation of this dissertation.
S incere ap p rec ia tio n is also ex ten d ed to m y com m ittee
m em bers, Drs. E. A chberger, A. Biel, D. Church, R. Laine, and J.
Larkin for valuable discussions, and suggestions on each aspect of
th is work, an d th e ir helpful insights and constructive criticism
pertain ing to the p repara tion of this d issertation . I would like to
thank Ms. C. Henk for h e r helpful assistance in photo developm ent,
Dr. B. G unn (C hem istry , LSU) fo r h e r he lp in com pu terized
m acrom olecular m odeling, and Ms. D. Sarkar (A udubon Sugar
Institu te, LSU) fo r h e r various help an d suggestions during th is
study. I would also like to thank m y lab collegues, D. Kim, J. Lee,
an d M. Ott for the ir valuable discussion, an d especially thank the
A udubon Sugar Institute for providing the facilities and funds to
complete this research.
Finally, I dedicate this work to my wife, Shelley, m y daughter,
Kailee, and to both sides of my extended family. Their love, patience
and understanding m ade this work possible.
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS.......................................................................... ii
LIST OF TABLES...................................................................................... vi
LIST OF FIGURES..................................................................................... vii
ABSTRACT............................................................................................... xi
INTRODUCTION........................................................... 1
REVIEW OF LITERATURE....................................................................... 3
I. Bacterial Exopolysaccharides: General C haracteristics.. 3
II. Alginate: O verview ................................................................ 51. Compositional Differences,
Seaweed vs Bacterial A lginate................................ 52. Properties...................................................................... 103. Applications.................... 144. Commercial M anufacture.......................................... 15
III. Alginate Biosynthesis........................................................... 211. Pathw ay. .................................................................. 212. Enzymology................................................................... 253. Regulation...................................................... 30
IV. Pseudomonas syringae Alginates.................................... 32
V. Polysaccharide Analysis....................................................... 321. Depolymerization........................................................ 322. Acid Sensitivity............................................................ 36
VI. Goals of this Study................................................................ 38
MATERIALS AND METHODS............................................................... 39
I. Organisms, Growth Conditions, and M aintenance 39
II. Analytical M ethods................................................................ 401. Cell Mass D eterm ination ........................................... 402. Total C arbohydrate Q uantitation............................. 403. Alginate Q uantitation....................... 414. Acetyl Q uantitation..................................................... 43
iii
III. Optimization of Bacterial Alginate Production............. 451. Media Composition...................................................... 45
A. Carbon Source.................................................... 45B. Nitrogen Source................................................ 45
2. pH and Tem perature ...................................... 463. Agar vs Broth Culture................................................. 46
IV. Batch Ferm entations............................................................ 48
V. Purification of Bacterial Alginate......................................... 48
VI. Deacetylation of Bacterial Alginate.................................. 49
VII. Alginate Size and Quantity D eterm inations................. 50
VIII. Sugar Sensitivity to Acid................................................... 501. Thin Layer Chrom atography.................................... 502. Ion Chrom atography.................................................. 51
IX. Identification of the Acid Hydrolysis Product of L-Gulose................................................................................... 52
1. Thin Layer Chrom atography ........................... 522. Stability of 1,6 Anhydro p-L-Gulopyranose 53
X. Alginate Reductions.................. 53
XI. Alginate Compositions......................................................... 54
XII. Properties of Bacterial Alginates and Effects of Acetylation............................................................................. 55
1. Viscosity.......................................................................... 552. W ater Holding Capacity.............................................. 563. Surface Tension.................................................. 564. Precipitation by Metal Ions....................................... 57
RESULTS................................................................................................... 58
I. Production of Bacterial Alginate...................................... 581. Media Compositions and Conditions ........ 582. Agar vs Broth Culture................................................. 62
II. Characterization of Bacterial Alginate.............................. 691. Recovery................................................................. 692. Molecular W eight......................................................... 693. Composition.......................... 71
iv
III. Functional Properties......................................................... 761. Viscosity.......................................................................... 762. Physical Effects.............................................................. 853. Cation Precipitation by Alginates.............................. 94
DISCUSSION............................................................................................ I l l
REFERENCES............................................................................................ 125
VITA........................................................................................................... 141
v
LIST OF TABLES
Page
Table 1. The structures of some bacterial exopolysaccharides. 6
Table 2. Some food applications of alginates................................ 16
Table 3. Some industrial applications of alginates...................... 17
Table 4. Estimates of U. S. consum ption and price ofpolysaccharides........................................... 20
Table 5. Effects of carbon source on alginate yield fromP. syringae ATCC 19304..................................................... 60
Table 6 . Alginate yield by P. syringae ATCC 19304 ondifferent m edia.................................................................... 67
Table 7. Molecular weights of alginates......................................... 70
Table 8 . Thin layer chrom atography (Rf values)........................ 74
Table 9. The energetics and stability of 1,6 anhydrop-L-gulopyranose................................................................ 79
Table 10. Effects of calcium concentration on the waterholding capacities of alginate gels.................................. 86
Table 11. Effects of acetylation on bead surface tension 91
Table 12. The precipitation of Macrocystis alginate andacetylated and deacetylated P. syringae alginate by m etal ions........................................................................ 1 1 0
vi
LIST OF FIGURES
Page
Figure 1. Structures of the uronic acids of alginates................... 8
Figure 2. The block structures of alginate..................................... 11
Figure 3. The "egg box" model for Ca4f induced gelation ofpoly a-L-guluronate..................... 13
Figure 4. Flow diagram for the extraction of sodium alginatefrom seaweed.................................................................... 19
Figure 5. The proposed biosynthetic pathway of bacterialalginate in Azotobacter vinlandii and Pseudomonas aeruginosa............................................... 22
Figure 6 . A com parison of the overall routes ofincorporation of fructose and glucose in alginate produced by Pseudomonas aeruginosa....................... 24
Figure 7. The relative location of the alginate (alg) geneson the chrom osom e linkage m ap of Pseudomonas aeruginosa................................................. 26
Figure 8 . The m echanism of acid hydrolysis of glycosides 34
Figure 9. The relationship between cell mass andalginate accum ulation by P. syringae ATCC19304 with time in shake flask culture...................... 59
Figure 10. The effect of the initial am m onium concentrationon cell mass, and total alginate accum ulation by P. syringae ATCC 19304 at 48 hours...................... 61
Figure 11. The effect of the initial am m onium concentrationon specific yields of alginate by P. syringae ATCC 19304 a t 48 hours............................................................ 63
Figure 12. The effect of initial pH on cell mass, andalginate accum ulation by P. syringae ATCC 19304 a t 48 hours........................................................................ 64
vii
Figure 13. The effect of tem perature on cell mass, andalginate accum ulation by P. syringae ATCC 19304 at 48 hours....................................................... 65
Figure 14. The effect of initial ferric ion (Fe^+) concentration on the specific yields of alginate by P. syringae ATCC 19304 a t 48 hours............................... 68
Figure 15. Thin layer chrom atograph of HC1 hydrolyzedD-mannose....................................................... 72
Figure 16. Thin layer chrom atograph of HC1 hydrolyzedL-gulose............................................................. 73
Figure 17. Stability of m onom eric D-mannose and L-gulose in HC1 and H2SO4 under hydrolysis conditions at 100°C, as determ ined by ion chrom atography .. . 75
Figure 18. Thin layer chrom atograph of the acid hydrolyzedproduct of L-gulose com pared to 1,6 an h y d rid e s .. . 77
Figure 19. Com puter derived image of 1,6 anhydrop-L-gulopyranose............................................ 78
Figure 20. Gulose recovered, expressed as a percent of the total sugar present in the HC1 hydrolyzed reduced alginates, from Macrocystis pyrifera, and P. syringae ATCC 19304, as determ ined by ion chrom atography.................................................. 80
Figure 21. Comparative viscosity, (N/No), of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate as a function of alginate concentration................................................ 82
Figure 22. The effects of acetylation on the com parative viscosity, (N/No), of Macrocystis alginate, and P. syringae alginate........................................ 83
Figure 23. The effects of tem perature on the com parative viscosity, (N/N0), of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate................................ 84
viii
Figure 24. Percent difference in w ater holding capacity of Macrocystis alginate gels m ade with ferric iron, and lead, as com pared to calcium alginate g e ls .. . . 87
Figure 25. Percent difference in w ater holding capacity of acetylated P. syringae alginate gels m ade with ferric iron, and lead, as com pared to calcium alginate gels....................... 89
Figure 26. Percent difference in w ater holding capacity of deacetylated P. syringae alginate gels m ade with ferric iron, and lead, as com pared to calcium alginate gels...................................................................... 90
Figure 27. Rate of w ater loss of calcium alginate gels m ade from Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate as a function of incubation time a t 50°C................... 93
Figure 28. Com parison of the w ater loss of ferric iron alginate gels of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate com pared to calcium alginate gels as a function of incubation time a t 50°C.......... 95
Figure 29. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by uranium ( U ^ ) ions.................................. 97
Figure 30. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by copper (Cu2+) ions.................................... 98
Figure 31. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by lead (Pb2+) ions........................................ 99
Figure 32. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by ferric (Fe^+) ions...................................... 100
ix
Figure 33. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by calcium (Ca2+) ions............................. 101
Figure 34. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by strontium (Sr^+) ions.............................. 103
Figure 35. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by zinc (Zn^+) ions........................................ 104
Figure 36. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by m anganese (M n^+) ions......................... 105
Figure 37. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by cobalt (Co2+) ions...................................... 106
Figure 38. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by cesium (Csl+) ions.................................... 107
Figure 39. Precipitation of Macrocystis alginate, acetylated P. syringae alginate, and deacetylated P. syringae alginate by m agnesium (M g2+) ions......................... 108
x
ABSTRACT
A lginate has m any in d u stria l uses because of its u n ique
colloidal behavior, an d its ab ility to thicken, stabilize, emulsify,
suspend, form films, and produce gels. Currently, all com m ercial
alg inate com es from brow n algae, w here it exists n a tu ra lly as a
structural m aterial. Many d ifferen t types of brow n algae produce
alg inate b u t it can only be o b ta ined in sufficient q u an tity an d
quality from a lim ited num ber of species. On the basis of location,
a t least half of the w orld's resources of this polym er are potentially
a t risk due e ith e r to political in stab ility o r industria l pollution.
B acteria p ro v id e a p o ten tia lly u n lim ited a lte rn a te source fo r
alginate. Pseudomonas syringae pv phaseolicola ATCC 19304,
p roduced an acety lated alginate-like polysaccharide with a weight
average m olecu lar w eight (Mw ) o f 1.2 x 1 0 $. This b ac te ria l
polym er was com posed of 82% m annuronic acid and 18% guluronic
acid. Com positional analysis of the red u ced alg inate po lym er
showed th a t L-gulose was m ore sensitive to acid degradation than
D -m annose. The p ercen tag e of gulose reco v e red a t various
hyd ro lysis tim es was ex trap o la ted to zero hyd ro lysis tim e to
account for the loss of gulose. Acetylation affected the solution and
gelling p roperties of the polym er. A cetylated bacteria l alg inate
showed increased viscosity, and w ater holding capacity, and altered
cation precip itability over unacety lated alginates. By controlling
the degree of acetylation on the bacterial alginate, the solution and
gelling p ro p erties of the po lym er can be m an ip u la ted and the
polym er targeted to specific applications.
xi
INTRODUCTION
Alginate is a water-soluble gum used in the food and chemical
in d u s try p rim a rily as an em ulsifier, stab ilizer, o r th ickening
(gelling) agent (117, 144). The com mercial sources of alginate are
th e b ro w n m a rin e seaw eeds o f th e fam ily Phaeopliyceae,
specifically, those of the genera Ascophyllum, Ecklonia, Fusarium,
Laminaria, and Macrocystis. These alginates form viscous solutions
a t low concentrations, show polyelectrolytic behavior, and form gels
an d films (97). Typically, these alginates are block copolym ers,
linked 1-4, of p-D-m annuronic acid (M) an d its C-5 epim er a-L-
guluronic acid (G). The ratio of M/G varies from one algal species to
th e n ex t an d d ic ta tes th e gelling p ro p ertie s of the po lym er.
Alginates w ith h igh M/G ratios are m ore ex tended and p roduce
elastic, pliable gels due to the sm aller regions of poly-G blocks.
Alginates with low M/G ratios produce strong, b rittle gels, because
of the higher affinity of poly-G for C a^ , and the greater com paction
of the molecule.
Alginate is one of the few eukaryotic polysaccharides th a t
have a po ten tia l p rokaryo tic source. M any species of b ac te ria
p roduce "alginate-like" polysaccharides. This was first discovered
in studies on the "slime" produced by Pseudomonas aeruginosa (13,
75, 76) in in fec tions of cystic fibrosis p a tien ts . Azotobacter
vinlandii (51, 99) was also studied as a potential source of alginate
because of its nonpathogenic nature. At high resp iration ra tes the
m ajority of the carbon used by A. vinlandii goes to resp iration and
the form ation of poly p hydroxybutyrate (PHB, 23). This bacterium
1
2
produces alginate only during encystm ent an d does n o t p roduce
sufficient polym er for industrial production.
Other nonpathogenic pseudom onads, including the fluorescent
pseudom onads (58, 120), and the phytopathogenic pseudom onads,
specifically Pseudomonas syringae (37, 38, 91), produce alginates.
P. syringae produces an alginate conspicuously d ifferent from tha t
of the brow n seaw eeds. As w ith o th e r b ac te ria l alg inates, P.
syringae alginates are generally acetylated a t position C-2 a n d /o r C-
3 of the m annuronic acid residues. Unlike seaw eed alginate, P.
syringae polym ers do n o t contain an extensive block structu re .
The bacterial alginate produces bulky, elastic gels w ith high w ater
re te n tio n because of its lack of ex tensive poly-G blocks, O-
acetylation, and high M/G ratio (47).
The goals of th is study were to determ ine the conditions
w hich influence alg inate p ro d u c tio n from P. syringae a n d to
characterize the properties of the polym er in o rder to determ ine if
the use of this organism is feasible fo r large scale p roduction of
alginate.
REVIEW OF LITERATURE
I. Bacterial Exopolysaccharides: General Characteristics.
Most species of bacteria produce exopolysaccharides (EPS), i.e.,
polysaccharides found outside the cell wall, e ither a ttached in the
form of a capsule o r secreted into the extracellular environm ent as
a slime (135). There have been m any theories proposed to explain
the role of bacterial EPS and all re la te to the survival advantages it
offers to the organism s. Originally, these polysaccharides were
studied because of the ir link to pathogenicity. This was especially
true of the capsules of strains of Streptococcus pneumoniae, Bacillus
anthracis, an d Klebsiella pneum oniae (2). The viru lence of these
organism s is due, in part, to the protection provided by the capsular
polysaccharide surrounding the cell. Within anim al hosts, capsules
in terfere with both phagocytosis and antibody binding (87).
Some b ac te r ia secre te EPS as an aid to colonization by
assis tin g in su rface a tta c h m e n t (16, 50). T he a b ili ty o f
Streptococcus m utans and Streptococcus salivarius cells to adhere to
the surface of teeth is partia lly a function of the EPS p roduced by
these oral bac teria (87). O ther roles of EPS include p reven ting
desiccation, energy storage, concentration and up take of charged
molecules, particu larly m etal ions (11, 17, 46), p ro tec tion against
the effects of u ltrav io let rad ia tion (17), and m ediation of biofilm
and microcolony form ation (18).
B acterial EPS is species specific. Sugars a re common
com ponents in m ost bacterial EPS. D-glucose, D-mannose, and D-
galactose occur frequently , and L-rhamnose and L-fucose a re less
3
4
com m on. The presence of negative charges is a featu re of some
b ac te ria l EPS. U sually these charges a re th e re su lt o f the
incorporation of uronic acids into the polym er. The m ost com m on
negatively charged sugar p resen t in bacteria l EPS is D-glucuronic
acid which is a com ponent of hyaluronan , xan than gum, an d m ost
teichuronic acids. D -m annuronic acid, D-galacturonic acid and L-
guluronic acid are also com ponents of some bacterial polymers. D-
m annuronic acid and L-guluronic acid are associated prim arily with
b ac te ria l a lg inates. D -galactu ron ic ac id is fo u n d in som e
lipopo lysaccharides p ro d u ced by Proteus m irabilis, (77) an d
Xanthomonas campestris (142).
B iosynthesis of EPS n o rm a lly occu rs by e i th e r o f two
m echanism s. EPS m ay be produced at the cytoplasm ic m em brane
using p recursors form ed in tracellu larly , o r they m ay be form ed
fro m specific p re c u rso rs in th e e x tra c e llu la r en v iro n m en t.
G enerally, the firs t type is characteristic of heteropolysaccharide
p ro d u c tio n , w hile th e seco n d is c h a ra c te r is t ic o f c e r ta in
hom opolysaccharides including levans an d dex trans (133). Both
p a th w a y s re q u ire th e p ro d u c tio n o f a c tiv a te d n u c leo tid e
d iphosphate form s of the m onosaccharides and the p recursors for
substituent groups, specifically acetate, pyruvate, and succinate (25,
37). Acetyl CoA was recently confirm ed as the source of acetate in
xan than gum biosynthesis by Xanthomonas campestris (6 6 ). The
p rec u rso r fo r p y ru v a te is p h o sp h o en o lp y ru v a te (71). T hese
substituents introduce negative charges into the polysaccharides. A
high num ber of negative charges, coupled with a high m olecular
weight, p roduce polym ers th a t form highly h y d ra ted gels. While
5
th e ab ility to p ro d u ce EPS is w idesp read , u n d e r la b o ra to ry
conditions this ability is often unstable and lost on repea ted culture
(17).
Bacterial EPS varies enorm ously in structure an d composition,
ran g in g from sim ple hom opolysaccharides to com plex, h ighly
substitu ted and branched heteropolysaccharides (Table 1). These
polysaccharides can be divided into five distinct groups: 1) dextrans
an d levans, o r hom opolysaccharides p roduced by b ac teria using
sucrose as a specific substrate, 2 ) hom opolysaccharides o th e r than
d e x tra n a n d le v a n , i.e., cellu lose, 3) h e te ro p o ly sacch a rid e s
containing m ore than one type of m onosaccharide synthesized from
a specific carbon substrate, 4) heteropolysaccharides form ed from
re p e a t in g u n i t s tru c tu re s , i.e., x a n th a n g u m , a n d 5)
heteropolysaccharides com posed of two types of m onom er w ith no
repeating unit, i.e., alginate (134).
II. Alginate: Overview
1. Compositional Differences, Seaweed vs. Bacterial Alginate
Alginate, the m ajor s truc tu ra l polysaccharide of the brow n
algae (Phaeophyceae), was first discovered by E. C. C. S tanford in
1881 (19). It was n o t un til 30 years la ter th a t uronic acids were
identified as the m ajor com ponents (118). Initially, it was assum ed
th a t p-D -m annuronate was the only m onosaccharide p re sen t in
alginate. Subsequent research established the copolym eric n a tu re
of alginate and the presence of variable am ounts of a second uronic
acid, a-L-guluronate (41).
Table 1. The structures of some bacterial exopolysaccharides.
Species Polysaccharide Structure
Leuconostoc m esenteroides Dextran ( l i t6 glucose)nAcetobacter xylinum Cellulose (16.4 glucose)nAlcaligenes faecalis var. niyxogcnes Curdlan (163 glucose)nStreptococcus m utans Mutan (1“ 3 glucose)nStreptococcus salivarius Levan (26.6 fructose)nAzotobacter vinlandii Alginate 1-4 linked ManA and GulAPseudomonas aeruginosa Alginate 1-4 linked ManA and GulAStreptococci H yaluronan (-3GlcNAcl6.4GlcAl6.)n(Haemolytic group A)Xanthomonas campestris Xanthan (-4Glclh4Glclh4Glclli)n
I1
Aa
Abbreviations: Gal, galactose; Glc, glucose; GlcA, glucuronic acid; GulA, guluronic acid; Man, m annose; ManA, m annuronic acid; GlcNAc, N-acetylglucosamine; OAc, O-acetyl; Pyr, pyruvate.
a A= (-3«lM an(OAc)2hlGlcA4lilM an4-Pyr)
7
W ithin the last 30 years, alginate was found to be produced
by som e p ro k a ry o te s . The o b se rv a tio n th a t Pseudomonas
aeruginosa was able to synthesize an "alginate-like" polysaccharide
was f irs t rep o rted by Linker and Jones (75), a lthough m ucoid
s tra in s o f th is b ac te riu m w ere iso la ted m uch ea rlie r . They
dem onstrated th a t this polym er had com ponents identical to algal
alginate. In a subsequent study, they showed tha t an O-acetylated
a lg inate was the m ajor com ponent of P. aeruginosa slim e (76).
Carlson and Matthews (13) confirm ed the com position of bacterial
alg inates in a study of 13 d iffe ren t m ucoid isolates from cystic
fibrosis p a tien ts . M ore recen tly , the re la ted p seudom onads P.
fluorescens, P. mendocina, P. pu tida (53), and m any pathovars of P.
syringae (37) have been identified as alginate producers. The same
year th a t P. aeruginosa was reported to produce alginate, Gorin and
Spencer (51) showed tha t the polysaccharide produced by strains of
Azotobacter vinlandii was the sam e as the acety lated alginate
produced by pseudom onads. Later it was determ ined th a t alginate
b io syn thesis by A. vinlandii was closely asso c ia ted w ith the
form ation of vegetative cysts (93).
Alginates are unbranched copolymers of p-D-mannuronic acid
an d its C-5 epim er cx-L-guluronic acid, linked 1-4 (Fig. 1). The
actual composition and sequence of the polym er is dependen t upon
the source (73, 96, 130). Alginates from seaweed an d A. vinlandii
generally con tain extensive regions of hom opolym eric blocks of
m annuronic acid and guluronic acid together with some random or
a lte rn a tin g sequences. A lginates d e riv e d fro m s tra in s of
Pseudomonas contain few block hom opolym eric regions. Instead,
p-D-m annuronate
C -( i
ooc
a-L-guluronate
Figure 1 . Structures of the uronic acids of alginates.
9
they a re com posed p red o m in an tly of ran d o m sequences. In
addition, the m annuronic acid residues are highly O-acetylated (2 1 ,
125). The structure, as well as the position of acetylation in these
alginates, has been extensively studied. The O-acetyl groups are
associated exclusively with the C-2 a n d /o r C-3 positions of the D-
m a n n u ro n a te re s id u e s . n u c le a r m ag n e tic re so n a n c e
sp ec tro m etry (NMR) rev ea led th a t som e of th e m a n n u ro n a te
residues were 2,3 di-O-acetylated, although the m ono-O -acetylated
form s were m ore com m on (21, 125, 127). The reason for th e O-
acety la tion in bac teria l alg inates is n o t en tire ly clear. It was
suggested tha t acetylation m ay be p art of a control m echanism tha t
o p e ra te s du rin g b iosyn thesis th a t con tro ls the M/G ra tio by
p rev en tin g ep im eriza tion of th e ca rboxy l g ro u p on th e C-5
m an n u ro n a te carbon (128). A lternatively, it m ay play a ro le in
dictating the solution properties of the polysaccharide (129).
Seaweed and bacteria l alginates d iffer in th e ir M/G ratios.
Most alginates isolated from the brown seaweeds show M/G ratios
in the range of 0.45 (Laminaria hyperborea) to 1.85 (Ascophyllum
nodosum , 117). The m ost com mon industrially available alginate is
extracted from the brow n alga, Macrocystis pyrifera. This polym er
contains 60% 4-linked m annuronate and 40% 4-linked guluronate
residues giving an M/G ratio of 1.5. The alginates p roduced by P.
aeruginosa con tain 80% 4-linked m annuronate an d 20% 4-linked
guluronate residues, giving an M/G ratio of 4.0 (150).
Haworth projections of the (5-D-mannuronate an d the a-L-
gu luronate residues show little d ifference in the two structu res.
Epimerization of the carboxyl group at C-5 does, however produce a
10
m arked change in the th ree dim ensional conform ation of these
m onosaccharides. Since the carboxyl group is th e m ost bulky
s u b s t i tu e n t on th e r in g , th e m ost en e rg e tic a lly fav o rab le
conform ation orien ts this group to the equatorial position. As a
result, p-D-m annuronate exists preferentially in the chair form,
w hereas the a-L-guluronate residues adop t the IC4 conform ation
(Fig. 1). Chains of sugars containing com binations of these two
fo rm s p ro d u c e p o ly m ers w ith d if fe re n t th re e d im en sio n a l
structures. The p-D -m annuronate linkage positions, C -l an d C-4,
are equatorial to the p lane of the sugar ring. These linkages are
axial fo r th e a-L -guluronate residues. X-ray fib er d iffrac tio n
studies of alginates containing high p roportions of m annurona te
residues indicate a flat ribbon-like conform ation in the solid state
(5, 109, 110). This co n fo rm atio n is s im ila r to th e p 1-4
diequatorially linked polym ers such as cellulose. Alginates rich in
polyguluronate, which is 1-4 diaxially linked, ad o p t a buckled 2-
fold chain conform ation (6 , Fig. 2).
2. Properties
The physical com position of alginates dictates the solution and
gelling p ro p e rtie s of th e po lym ers. A lginate is a s tru c tu ra l
com ponent in the brow n seaweeds w here it contributes to bo th the
tensile strength and the flexibility of the algal tissue (64, 143). In
A. vinlandii it serves a s truc tu ra l function for the m etabolically
dorm ant cysts. Evidence exists tha t it is an im portan t com ponent of
bo th the cyst exine and intine, which are m icroscopically d istinct
reg ions ou tside th e cen tra l body of th e cyst (115). A lginate
11
Figure 2. The block struc tu res of alginate. A. The ribbon-like s tru c tu re of poly p -D -m annuronate. B. The b u ck led ch a in conform ation of poly a-L-guluronate. C. A lternating sequences of p- D -m annuronate and a-L-guluronate.
12
production by P. aeruginosa was related to resistance to antibiotics,
phage an d bacteriocins and pro tection against phagocytosis and
antibody attachm ent. This suggests, tha t for P. aeruginosa, alginate
functions as a general protective barrier.
Alginates with low M/G ratios produce strong bu t brittle gels,
w hereas alginates with high M/G ratios form m ore elastic gels (62,
110). This d ifference is due to the a rran g em en t of the block
structures w ithin the polym ers and is particularly evident when the
polysaccharide in teracts with cations. Alginate, being a polyanion,
is a n a tu ra l ion-exchanger w here the selectivity and streng th of
b inding depend on the cation, the conform ational characteristics,
an d the linear charge density of the polym er. Most m ultivalent
cations b ind alginates an d cross-link the polysaccharide to form a
gel m atrix . Some reg ions of the po lysaccharide form stronger
chelation com plexes th an o thers w ith divalent cations, especially
calcium ions (Ca+h). The "egg box model" was proposed to explain
this interaction (82, 83, 8 8 , 109, 110, Fig. 3,). The binding of Ca_l+ is
strong because in ad d itio n to the ionic b inding to the carboxyl
groups, various ring and hydroxyl oxygen atom s are able to chelate
th e c a tio n s . P o ly g u lu ro n a te b in d s Ca4f v e ry s tro n g ly .
Polym annuronate or mixed sequences do no t bind Ca_l+ with as high
an affinity . W hen various block sequences w ere iso lated from
in tact alginate, polyguluronate blocks showed an enhanced binding
of C a ^ in polym ers above 20 residues (61). This in d ica ted a
cooperative m echanism w here binding sites exist in an o rd ered
array, and binding of one ion facilitates binding of a second. Similar
Ca++
S^°-VCa++
Figure 3. The "egg box" model for C a^ induced gelation of poly «- L- guluronate.
14
effects were n o t seen with polym annuronate blocks o r alternating
sequences (6 8 ).
Bacterial alginates tha t contain few polyguluronate blocks (a
h igh M/G ratio ), an d th a t a re O-acetylated, p roduce relatively
bulky, flexible gels w ith high w ater re ten tio n in the p resence of
Ca++( 109). The high num ber of m annuronate residues and O-acetyl
groups reduce the cooperative binding of Ca'l+ and w eaken the gel
network. This fact, in association with the greater positive osmotic
p re s s u re cau sed by th e in c re a se d n u m b e r of d isso c ia ted
counterions, significantly enhances the w ater holding capacity of
bacterial gel m atrices (129). The resulting gel provides cells with
hydrophilic capsules th a t p ro tect them from other microorganisms,
chemicals, antibiotics, and desiccation (116). In P. syringae, alginate
is th o u g h t to p lay an im portan t role in the pathogenicity of the
bacterium , allowing adhesion to the host surface (137).
3. Applications
While alginate production is w idespread among m em bers of
the brow n seaweed (Phaeophyceae), only a few species of brow n
seaweed are used for com mercial production. The principal source
of th e w orld 's supply o f alg inate is the g iant kelp, Macrocystis
pyrifera. O ther seaweeds tha t are used in alginate m anufacture are
Ascophyllum nodosum and species of Laminaria and Ecklonia.
A lg ina te is u sed in foods a n d fo r genera l in d u s tria l
applications because of its unique colloidal behavior and its ability
to thicken, stabilize, em ulsify, suspend, form films, an d p roduce
gels. The p rim ary p ro p erties on which the w idespread use of
15
alginate is based are: 1 ) the fo rm ation of viscous so lu tions a t
relatively low concentrations, 2 ) the polyelectrolytic behavior in
solution, 3) the ability to form gels by chem ical reaction , 4) the
form ation of films on surfaces, and 5) the base exchange properties
(97).
Food p roducts in which alginates are used include frozen
foods, pastry fillings, bakery products, and syrups. Because of their
gelling ab ility , a lg inates a re also used in in s ta n t an d cooked
puddings, and pie fillings. Alginates are excellent em ulsifiers for
salad dressings an d stabilizers for beverages, w hipped toppings,
an d sauces (Table 2). Alginates are also of value in a num ber of
industrial applications, such as the m anufacture of paper, adhesives,
textiles, a ir fresheners, explosives, polishes, antifoam s, ceramics,
and cleaners (19, 97, Table 3).
Since the industria l p roduction of seaweed alginates began,
several com m ercially im portan t derivatives of alginates have been
developed. Commercial derivations include the sodium, potassium,
am monium , calcium, and mixed am monium-calcium salts of alginic
acid, propylene glycol alginate, and alginic acid. The propylene
glycol ester is the only commercial, organic derivative of alginate.
It has im proved acid stability an d resistance to p recip itation by
calcium and o ther m ultivalent m etal ions (19).
4. Commercial M anufacture
Macrocystis pyrifera grows in relatively calm m arine w aters
in large, dense beds. It is a very rapid ly growing p lan t th a t allows
for up to four cuttings p er year. At the time of harvesting, a dense
16
Table 2. Some Food Applications of Alginatesa
Property Product Perform ance
W ater holding Frozen foods
Pastry fillings Syrups
Bakery icings
Maintains texture during freeze-thaw cycles.Produces smooth, soft texture Suspends solids, controls pouring consistency Counteracts stickiness and cracking
Gelling Puddings Firms body and texture Pie and pastry fillings Acts as a cold w ater gel base;
develops soft gel body with broad tem perature tolerance; gives im proved flavor release.
Dessert gels Produces clear, firm, quick-setting gels.
Emulsifying Salad dressings Sauces
Emulsifies and stabilization Emulsifies oils and suspends solids
Stabilizing BeerJuices
syrups and toppings
W hipped toppings
Sauces an d gravies
Maintains beer foam Stabilizes pulp in concentrates and finished drinksSuspends solids; produces uniform bodyStabilizes fat dispersion, and freeze-thaw breakdow n Thickens and stabilizes for a broad range of applications
a Table was adapted from Sandford and Baird (117).
17
Table 3. Some Industrial Applications of Alginatea
Property Product Performance
W ater holding Paper coating Paper sizings
Adhesives
Textile printing
Controls rheology of coatings Improves surface properties, and ink acceptance Controls penetration to im prove adhesion and applicationProduces very fine line prints
Gelling Air freshener
Explosives
Toys
Hydromulching
Firm, stable gels are produced from cold w ater system sElastic gels produced byreaction with boratesNontoxic m aterials m ade forimpressionsHolds mulch to inclinedsurfaces; prom otes seedgermination
Emulsifying Polishes
AntifoamsLatexes
Emulsifies oils and suspends solidsEmulsifies and stabilizes Stabilizes latex emulsions; provides viscosity
Stabilizing CeramicsCleaners
Suspend solids Suspends and stabilizes insoluble solids
a Table was adapted from Sandford and Baird (117)
18
m at of fronds floats on the ocean surface. Harvesting is actually a
m assive prun ing of the kelp beds. U nderw ater blades mow the
kelp approxim ately th ree feet below the w ater surface. The kelp is
then conveyed into the hold of a barge by a moving belt.
The process fo r alg inate ex trac tion is based on an ion-
exchange process. In seaweed, the alginate is p resen t as a mixed
salt of sodium a n d /o r potassium, calcium, and m agnesium (19). The
exact com position of the polym er varies w ith the type of seaweed
bu t this does no t affect processing. The extraction process begins
by grinding the seaweed and washing it with water. A strong alkali
is th en added to the w ashed seaweed an d the m ixture is heated to
ex tract an d dissolve the alginate. The crude alginate solution is
then clarified and precip itated by the addition of calcium chloride.
The calcium alg inate is acid trea ted to p roduce an alginic acid
p recip ita te . Sodium carbonate is then ad d ed to m ake a sodium
alg inate paste w hich is d ried , an d m illed in to sodium alg inate
pow der (Fig. 4).
The extraction costs of seaweed alginate are higher than o ther
industrially available gums due to harvesting and purification costs
(117, Table 4). Because of the unique properties of alginates, and
the high num ber of potential food and industrial applications, there
are no good replacem ents for this polym er. Bacteria have been
studied as potential a lternate sources of alginate. A bacterial source
would give an unlim ited supply of alginate and may reduce costs.
C a l c i u mA lka l i + W a t e r C h l o r i d e
W a t e r * H e a t S o l u t i o n
Mi l l ing W a s h i n gW e t o r D r y
S e a w e e dC l a r i f i c a t i o n
D i s s o l u t i o n o f A lg in a te s P r e c i p i t a t i o n
C r u d e A lg in a te S o l u t i o n
W a sh in g s I n s o l u b l e R e s i d u e
C o l o r a n d O d o r S o d i u m
R e m o v a l A c i d C a r b o n a t e
C a l c i u mA l g i n a t e
A c id T r e a t m e n t
A lg i n ic A c i d P r e c i p i t a t e
W a te r a n d
D is so l v e dI m p u r i t i e s
S o d i u m A lg in a te P a s t e
D r y i n g Mil l ingD r y S o d i u m
A lg i n a t e P o w d e r
Figure 4. Flow diagram for the extraction of sodium alginate from seaweed (117).
20
Table 4. Estimates of U. S. Consumption and Price of Polysaccharides.
Polysaccharide% U. S. ConsumDtiona
Food Industrial Retail Price (per 1 0 0 g)b
Agar 0.4 0.6 $ 14.25Alginate 11.5 6.2 $ 14.55Carrageenan 11.5 0.3 $ 15.35Guar gum 19.3 53.8 $ 2 .2 0Gum arabic 29.5 10.8 $ 5.25Gum ghatti 1.3 1.6 $ 12 .2 0Gum tragacanth 1.2 0.3 $ 16.35Karaya gum 1.3 10.8 $ 2.40Locust bean gum 11.5 6.2 $ 2 .1 0Pectin 9.6 0 $ 18.10Xanthan gum 2.9 9.3 $ 11.60
a Percent U. S. consum ption is based on 1980 figures. Percentages represen t the am ount of polysaccharide used for food and industrial applications relative to each o ther (117).b Retail price is based on 1994 figures from Sigma Chemical Co., St Louis, Missouri.
21
III. Alginate Biosynthesis
1. Pathway
In 1966, the biosynthetic ro u te of alginate p roduction was
established for the m arine brow n seaweed Fucus gardneri (73, 74).
Later, a sim ilar pathw ay was proposed for alginate p roduction by
Azotobacter vinlandii (99). In 1981, Piggott e t al (98) proposed a
pathw ay fo r alginate p roduction in Pseudomonas aeruginosa (Fig.
5). This pathw ay was based on the detection of all the enzym es
necessary for b iosynthesis of bac teria l alg inate, except fo r the
epim erase and the O-acetyl transferase.
Fructose 6 -phosphate is considered to be the p recursor in the
alg inate b iosynthetic pathw ay . The p rim a ry ro u te of glucose
ca tabo lism in p seu d o m o n ad s is th ro u g h the Entner-D oudoroff
pathw ay , p roducing g lyceraldehyde 3-phosphate an d pyruvate .
Carlson and Matthews (13) showed tha t the C-6 , bu t no t the C-l of
glucose was incorporated in to alginate. This im plied th a t carbon
atom s 1, 2, an d 3 of glucose were converted to pyruvate by the 2-
keto 3-deoxyphosphogluconate aldolase reac tion an d eventually
were lost to the alginate biosynthetic pathw ay as CO2 an d acetyl
CoA. Carbon atoms 4, 5, and 6 were channeled into alginate through
g lycera ldehyde 3 -phosphate . F ru c to se 6 -p h o sp h a te can be
p roduced either th rough gluconeogenesis o r by condensation of
g lycera ldehyde 3-p h o sp h ate w ith d ihydroxyacetone phosphate.
S u bsequen t stud ies w ith a P. aeruginosa m u ta n t d e fic ien t in
fru c to se 1 ,6 d ip h o sp h a te a ld o lase show ed th a t th e re w as
p referen tia l 14c incorporation from C-6 in to a lg inate com pared
w ith C-l labeled glucose in the wildtype. Incorporation of C-6 and
22
fructose 6-phosphate
Phosphomannose isorerase
Mannose 6-phosphate
Phosohomannonutase
Mannose 1-phosphate
GDP-r.annose pyrcpncspnory 1 ase
GDP-mannose
GDP-nannose denvcrooenasc
GDP-nannuromc acid
Pol viiierase
Epinerase
Acetyl transferase-
ALGINATE
Figure 5. The p ro p o sed b io syn thetic pa thw ay of a lg in a te in A zotobacter vinlandii a n d Pseudomonas aeruginosa. The firs t 4 enzym atic steps have been iden tified in bo th organism s. The po lym erase an d epim erase have been iden tified in A. vin landii only, and the acetyl transferase has not yet been identified in either organism.
23
C-l of glucose were sim ilar in the m u tan t (7, 78). A sum m ary of
the overall ro u tes of in co rp o ra tio n of fruc tose an d glucose in
alginate can be seen in Figure 6 .
F ructose 6 -phosphate can be isom erized to m an n o se 6 -
p h o sp h a te by p hosphom annose isom erase. T ra n s fe r o f th e
phosphate group by phosphom annom utase produces m annose 1-
phosphate from m annose 6 -phosphate. M annose 1-phosphate and
GTP are th en converted in to GDP-mannose, catalyzed by GDP-
m annose pyrophosphorylase. The subsequent oxidation th rough
GDP-mannose dehydrogenase resu lts in th e fo rm atio n of GDP-
m annuronic acid. Polymerization of GDP-mannuronic acid results in
p o lym annurona te w hich is secreted from th e b ac teria l cell and
becom es th e su b s tra te fo r an ex trace llu la r ace ty lase a n d /o r
epim erase in the production of the m ature polym er (47).
The tran sp o rt m echanism of the bacterial alginate across the
cytoplasm ic m em brane is believed to be sim ilar to th a t of some
bacterial cell wall polym ers, w hich use isopreno id lip id carriers
(134). The final step in the biosynthesis of bacterial alginate is the
selective epim erization of the m annuronate residues to guluronate
by an extracellu lar epim erase. The selectivity is th o u g h t to be
d ic ta te d by th e O -ace ty la tion of the m a n n u ro n a te residues.
Acetylation is believed to inh ib it epim erization, thereby dictating
the final com position of the alginates. Acetyl CoA is the probable
source of the acetyl group in bacterial alginates (136), ju st as it is
on th e m a n n o sy l re s id u e s o f x a n th a n gum p ro d u c e d by
Xanthom onas cam pestris (6 6 ). The m echanism of b ac te ria l O
acetylation is no t well defined.
F ructose G 1ucoso >• G l u c o n a t e
2 - K e t o g l u c o n a t eF ru c t o s e 1 - p h o s p h a t e
F r u c t o s e 6 - p h o s p h a t e ■* ► G l u c o s e 6 - p h o s p h a t e
6 - P h o s p h o g l u c o n a t e
A L G I N A T E
F r u c t o s e 1 , 6 - d i p h o s p h a t e
2- Ke t o 3-deoxy p h o s p h o g l u c o n a t e
Di h y d r o x y a c e t o n e ph o s p h a t e
G l y c e r a l d e h y d e 3 - p h o sp h at e
Pyruvate
TCAc y c le
Figure 6 . A com parison of the overall routes of incorporation of fructose an d glucose in alginate produced by Ps. aeruginosa (47).
25
2. Enzymology
Only fo u r of the seven p ro p o sed enzym atic steps in th e
biosynthetic pathw ay of alginate from fructose 6 -phosphate to the
m atu re polym er have been positively iden tified in Pseudomonas
aeruginosa (98). Six of th e steps h av e b een id e n tif ie d in
Azotobacter vinlandii (8 6 ). The first four enzym es in the pathw ay
have been found in bo th organism s an d have been the subject of
m uch investigation . In P. aeruginosa the genes encoding these
enzym es a re lo ca ted a t ap p ro x im a te ly 34 m in u te s o n th e
chrom osom e linkage m ap (89, Fig. 7). W hat is know n abou t these
enzymes is sum m arized below.
P h o sp h o m a n n o se iso m era se : This enzym e catalyses the
reversib le conversion of fructose 6 -p h o sp h a te to m annose 6 -
phosphate . The enzym e is the p ro d u c t of th e algA gene in P.
aeruginosa, and has a molecular weight of approxim ately 56,000 on
th e b a s is o f so d iu m d o d ecy l su lfa te -p o ly a c ry la m id e gel
electrophoresis (SDS-PAGE, 82). O verexpression of the algA gene
p ro d u ces in c reased activ ity of the n ex t two enzym es of the
p a th w a y , i.e., p h o s p h o m a n n o m u ta s e a n d GDP m a n n o se
p y ro p h o sp h o ry la se (48 , 114). In th e o v erexp ressed state,
p h o s p h o m a n n o s e i s o m e r a s e w as s e p a r a t e d from
phosphom annom utase b u t could n o t be se p a ra te d fro m GDP
m annose pyrophosphorylase (114). The inability to separate these
two activities has led to the proposal that, in P. aeruginosa, the algA
gene encodes a single p ro te in having phosphom annose isom erase
and GDP m annose pyrophosphorylase activities.
26
Switching G e n e s " v
7570' algRI srgH
ModulatorG e n e s
a r g B
Posit iveEffectors
algAr~Z------------- \
StructuralG e n e s
Figure 7. The relative location of the alginate (alg) genes on the chromosom e linkage m ap of Pseudomonas aeruginosa. (89).
27
P h o sp h o m a n n o m u ta se : This enzym e converts m annose 6 -
phosphate to m annose 1-phosphate. The enzyme is the p roduct of
th e algC gene, and has a m olecular w eight of 38 ,000 . It was
d e tec ted in the soluble cytoplasm ic fraction of b o th m ucoid an d
nonm ucoid strains of P. aeruginosa, from clinical an d nonclinical
sources (92). Phosphom annom utase has an absolute requ irem en t
for glucose 1,6-diphosphate for its activity (114, 151). Induction of
the algA gene increases phosphom annom utase activity by abou t 10
fold.
GDP m an n o se p y ro p h o sp h o ry la se : This enzym e catalyses
the form ation of GDP m annose from m annose 1-phosphate and GTP.
This enzym e was detected in P. aeruginosa by Piggott e t al (98). It
was suggested th a t the activity of this enzym e m ay rep resen t one
of the two enzym atic activ ities o f the algA gene p roduct. This
enzym e m ay be an example of a bifunctional p ro te in w here the two
activities catalyze noncontiguous steps in a biosynthetic pathway.
GDP m an n o se d e h y d ro g e n a se : This enzym e catalyses the
oxidation of GDP m annose to GDP m annuronic acid (98, 105). The
p roduct of the algD gene, GDP m annose dehydrogenase has been
p u rified an d found to be a hexam er w ith a m olecular w eight of
290,000 (9). Its absence in alginate negative m utants suggests tha t
it is essential for alginate biosynthesis. The algD gene has been
cloned an d sequenced. It is transcrip tionally activated in m ucoid,
b u t n o t in nonm ucoid strains of P. aeruginosa (26, 27). The gene
contains regions th a t have considerable sequence hom ology with
two ou te r m em brane p ro te in genes (ompF and ompC) from E. coli
28
(9). Like ompF and ompC, expression of the algD gene is regulated
by external osm olarity (9).
The final reactions in the biosynthesis of bacterial alginate in
P. aeruginosa are n o t ye t fully understood . From the structu ra l
com position of alginate, it has been p roposed th a t the final steps
include polym erization of the m annuronate residues, O-acetylation
of some of those residues, export of the polym er, and epim erization
of th e carboxyl g roup a t C-5. The assu m p tio n is th a t the
polym erase uses GDP m annuronic acid as its substrate and tha t the
product of this reaction is polym annuronate. The evidence for this
is based on the observation th a t in Azotobacter vinlandii and the
brow n seaw eeds th e re is an ep im erase th a t converts ce rta in
m annuronate residues to gu lu ronate a t the po lym er level. The
polym erase in P. aeruginosa has p roved exceptionally difficult to
m easure and the enzyme has no t been purified. Very low levels of
activity of this enzyme have been m easured in m em brane fractions
p rep ared from cell extracts of m ucoid P. aeruginosa (47), b u t the
characterization of this polym erase is still a t a prelim inary stage.
T he m e c h a n ism b y w h ich g u lu ro n a te re s id u e s a re
incorporated into Pseudomonas alg inate rem ains a m ystery . The
assum ption has been th a t a polym annuronic acid C-5 epim erase
acts a t the polym er level to convert some m annuronate residues to
gu lu ronate residues. The epim erase from A. vinlandii has been
purified to hom ogeneity (126), an d recen tly Piggott e t al (98)
iso la ted the gene encod ing a C-5 ep im erase (algG) fro m P.
aeruginosa. The enzym e requ ired Ca44- for activity, m uch like the
enzyme isolated from A. vinlandii. AlgG m utants were found to be
29
incapable of incorporating guluronic acid residues in to bacterial
alginate.
The epim erase of P. aeruginosa appears to be d ifferen t from
th e enzym e iso la ted from A. vinlandii. The M /G ra tio of P.
aeruginosa alginate is never less than 1 .0 , and this ratio is generally
unaltered by changes in growth conditions. In A. vinlandii the M/G
ratio m ay be less than 1 .0 and the resulting block structu re m ay be
a ltered by changes in CaH+ concentrations in the grow th m edium
(70).
The alginates of P. aeruginosa are h ighly O -acetylated with
th e O -acety l g ro u p s being asso c ia ted exclusively w ith th e
m an n u ro n a te residues. It is though t th a t one of the functions of
acetylation is to p ro tec t the m annuronate residues in the polym er
from ep im erization to gu lu ronate residues (126). Recently, an
alginate m odification gene, algF, was sequenced. This gene codes
for a 28 kd p ro tein which controls the addition of O-acetyl groups to
the m annuronic acid residues. The algF gene was rep o rted to be
n o n essen tia l fo r a lg in a te b io sy n th esis , b u t is re q u ire d fo r
acetylation of the alginate polym er (44).
A m ajor difference betw een the alginates of A.vinlandii, and
P. aeruginosa is the arrangem ent of m annuronate and guluronate
residues w ithin each polym er. The polym er from A. vinlandii is
a r ra n g e d in to h o m o p o ly m eric b locks o f m a n n u ro n a te an d
guluronate, w hereas in P. aeruginosa alginate polyguluronate blocks
are absent. This difference reflects differences in O-acetylation and
ep im eriza tio n ac tiv ities betw een these two b ac te ria . T hese
30
differences m ay indicate alterations in the la tte r stages of alginate
biosynthesis in P. aeruginosa from those proposed in Figure 5.
3. Regulation
In the lungs of cystic fibrosis patien ts Alg (-) stra ins of P.
aeruginosa usually convert to the m ucoid Alg (+) form. In vitro, the
Alg (+) phenotype is unstab le an d Alg (-) revertan ts ap p ear over
time. Genetic m apping experim ents have shown th a t spontaneous
alginate conversion is based on the gene products of the algB, algR,
algS, an d algT genes (42, 45, 79, 90). The p rim ary genetic event
th a t regulates the " o n /o f f switch in alginate production is handled
prim arily by the algS an d algT genes located a t approxim ately 6 8
m inutes on the chrom osom al linkage m ap of P. aeruginosa (89, Fig.
7).
Spontaneous conversion between the m ucoid and nonm ucoid
state is the result of a genetic alteration a t the algS gene locus. The
algS gene is a genetic switch th a t controls the expression of algT
(42). Form ation of the AlgS p ro tein results in the activation of the
algT gene. The gene p roduct of algT acts as a regulatory p ro te in in
the biosynthesis of bacterial alginate in P. aeruginosa by prom oting
the activation of the structu ra l genes involved in the biosynthetic
pathw ay (42, 43).
Along with its ro le in regu la tion of the stru c tu ra l genes in
alginate production, the algT gene p roduct also is involved in the
expression of the algB gene (149). The algB gene is located at
approxim ately 12 m inutes on the chrom osom e m ap (Fig. 7). Its
gene product is no t directly involved in the biosynthetic pathw ay of
31
alginate b u t is ap p a ren tly involved in high level p roduction of
alginate in P. aeruginosa (49). The algB gene p roduct belongs to a
class of p ro te in s th a t contro l gene tran scrip tio n in response to
environm ental stimuli (149).
The la s t gene in v o lv ed in th e re g u la tio n o f alginate
b io sy n th esis is th e algR gene. This reg u la to ry gene controls
transcrip tion o f th e algD gene, which encodes fo r GDP m annose
dehydrogenase, an essential enzyme in the biosynthesis of bacterial
alg inate (98). DNA sequence analysis showed a h igh degree of
hom ology betw een th e algR gene a n d o th e r environm entally
responsive bacterial regulatory genes, including ompR, phoB, ntrC,
an d spoA (28). This ind icates th a t the p roduction of bacteria l
alg inate is affected by environm ental stimuli o r specific chem ical
com pounds presen t in the environm ent.
Recently, the m echanism of alginate p roduction contro l by
algB, algR, an d algT has been exam ined. Com pared w ith Alg (+)
strains, deletion m utations in the algB an d algT genes show ed
highly reduced transcrip tional activ ity in the b iosyn thetic gene
c luster (at 34 m inutes, 49). This ind icated th a t the pathw ay of
alg inate biosynthesis is u n d er contro l no t only by the algR gene
p roduct b u t also by the gene products of algB and algT. W hether
the algT an d algB gene products act directly on any prom oters in
the b iosynthetic gene cluster or on o ther regu la to ry genes is no t
known.
32
IV. Pseudomonas syringae Alginates
Numerous pathovars of P. syringae were rep o rted to produce
bacterial alginates (37). El Banoby and Rudolph (34) repo rted tha t
the EPS from several p lan t pathogenic pseudom onads were capable
of inducing w ater soaked lesions on com patible leaf tissue. This
suggested tha t alginate m ay be necessary for successful colonization
of p la n t h o st tissue. In com m on w ith o th e r bacteria l alg inates,
those produced by P. syringae are highly acetylated. The M/G ratio
is variable am ong strains and am ong d ifferen t p repara tions from
the sam e strain . Fett e t al (38) rep o rted th a t the percen tage of
guluronic acid in P. syringae alginates varied from < 1% to 28%
w hen grown in planta, and th a t the in planta samples h ad a higher
degree of acetylation th an the alginates p roduced in vitro. It was
also rep o rted th a t the P. syringae alginates w ere sm aller, on the
average, th an those from P. aeruginosa (37). The num ber average
m olecu lar weights range from 3.8 x 10^ fo r a lg inates from P.
syringae pv glycinea produced in vitro, to 47.1 x 1 0 ^ for P. syringae
pv papulans alginate produced in vitro, com pared to P. aeruginosa
alginates whose size was repo rted ly in the range of 1 0 ^ (37). It
appears th a t alginate biosynthesis is a com m on p ro p e rty of the
m ajority of pseudom onads in rRNA-DNA hom ology group 1 (29, 40,
94).
V. Polysaccharide Analysis
1. Depolymerization
C h a ra c te riza tio n of an y p o ly sacch a rid e s ta r ts w ith a
com positional analysis. Acid hydrolysis is com mon to m ost m ethods
33
of d e te rm in in g the physical com position of a polysaccharide.
Hydrolysis is usually conducted with dilute m ineral acids, the m ost
com mon being sulfuric acid, hydrochloric acid, or trifluoroacetic acid
a t 100°C for varying lengths of time. Many factors influence the
ra te of h y d ro ly sis o f any po lysaccharide, includ ing ring size,
configuration, conform ation, and polarity of the com ponent sugars
(8).
In studying the com position of alginic acid, m ost researchers
reduce the uronic acids to a n eu tra l po lym er to facilita te acid
h y d ro ly s is w ith o u t th e rm a l d e s tru c tio n o f th e co m p o n e n t
m onosaccharides (13, 37, 38, 91, 150). Acid hydrolysis of neu tra l
polysaccharides has been studied, and the m echanism now accepted
was first suggested by Edward (33) and is depicted in Figure 8 for
the hydrolysis of m ethyl p-D glucopyranoside. The process involves
p ro to n a tio n of the glycosidic oxygen atom to form the conjugate
acid, followed by the form ation of a cyclic carbonium -oxonium ion
which probably exists in the half chair conform ation having C-2, C-
1, O, an d C-5 in a p lane. Reaction with w ater th en gives the
p ro tonated reducing sugar and from it the reducing sugar is form ed.
Alginic acid, being a polyuronide, contains a carboxyl group at
the C-5 position of each com ponent m onosaccharide. This carboxyl
group confers acid resistance to the glycosyluronic acid linkages
(15). M any theories have been advanced as to why. T here is
evidence th a t the enhanced stability m ay be a ttrib u ted to e ith e r
steric factors or to inductive effects (145). Ranby and M archessault
(107, 108) fo rm u la ted an induction-stab ilization theory , w hich
p roposed th a t the glycosidic bond is stabilized by the inductive
Figure 8 . The m echanism of acid hydrolysis of glycosides. The carbonium ion in term ediate (3) is in the half-chair conform ation (8 ).
OJ
35
effect of the po lar carboxyl group. According to this theory, the
presence of an electronegative group, such as a carbonyl or carboxyl
group, can exert inductive influences on the glycosidic oxygen atom.
In a polyuronide, the carboxyl group would oppose the pro tonation
of the glycosidic oxygen atom s by making the electron pairs on the
glycosidic oxygen atoms less basic, resulting in a m ore stable bond.
A possib le fo rm atio n o f six m em bered rings, w here hydrogen
stabilizes the negatively charged carboxyl group and the glycosidic
oxygen atom has also been postu lated (107). A lthough the exact
explanation for the stability of glycosyluronic acid bonds has no t
been pinpointed, suffice it to say tha t these bonds are m ore difficult
to hydrolyze than the neutra l O-glycosidic bonds.
M any fa c to rs in f lu e n c e th e r a te o f h y d ro ly s is of
polysaccharides. The ease of hydrolysis a t a particu lar tem perature
an d acid concentration increases in the o rd e r g lucopyranoside <
fructopyranoside < fructofuranoside (63). This indicates th a t ring
size affects hydrolysis. T here is a d irec t rela tionsh ip betw een the
strain (or free energy) associated with a m olecule and the ra te of
hydrolysis (121). In general, aldofuranosides and aldoheptanosides
a r e h y d ro ly z e d m o re r a p id ly th a n th e co rre sp o n d in g
aldopyranosides. The five and seven m em bered rings are strained
because of the distortion of the te trahedral angle of the ring carbon
atoms. The pyranoid rings can pucker to elim inate strain (35).
The anom eric configuration in pyranosides also plays a role in
the stab ilities of these sugars u n d er acid hydrolysis conditions.
F e a th e r a n d H arris (36) u sed a n o m eric p a irs o f m e th y l
aldopyranosides to study the affects of the anom eric configuration
36
on acid stability. They found th a t an anom er w ith an equatorial
m ethoxy l g roup h y d ro ly zes m ore ra p id ly th a n an an o m e r
containing an axially oriented group. Two explanations have been
p roposed . First, eq u a to ria l bonds are considered to be m ore
accessible th an axial bonds, thus making them m ore available for
p ro to n tran sfe r from a hydron ium ion to the glycosidic oxygen
atom . A lternatively , th e g rea te r reac tiv ity of th e eq u a to ria l
su b stitu en t is due to its h igher free energy caused by a p o la r
in te rac tion betw een the equatorial m ethoxyl group an d the ring
oxygen atom (33).
2. Acid Sensitivity
M onosaccharides are degraded by acid to a g rea ter o r lesser
extent depending on the sugar an d strength of the acid (119). In
add ition to degradation , acids m ay convert sugars in to anh y d ro
derivatives. Spontaneous conversion to a 1,6 anhydroaldopyranose
occurs in acidic solutions of several aldoses an d ketoses having the
ido (100, 102, 146), a ltro (101, 112), an d gulo (131, 132)
configurations. In dilute acid solutions those sugars of the gluco,
m a n n o , a n d g a lac to co n fig u ra tio n s a re a lm o st completely
hydro lyzed to the free aldoses. Reeves (111) first suggested an
explanation for this d ifferen t behavior in term s of conformational
in teractions. He showed th a t (3-D-idose, with all hydroxyl groups
equatorial, has a h igher p ropensity to form 1,6 anhydrides than
does p-D-glucose whose hydroxyl groups are all o rien ted axially.
This system represents a good example of conform ational control of
37
an equilibrium. The position of the equilibrium is dependen t on the
steric arrangem ent of the groups no t taking part in the reaction.
The solutions of any reducing sugar contain an equilibrium
m ixture of the IC4 and 4 c i forms of the a and (3 anom er s. Only the
IC4 form of the (3 anom er can directly form the anhydride w ithout
change in configuration and conformation. 1,6 A nhydride form ation
is believed to occur in two steps: 1 ) conversion of o ther form s of the
sugar into the IC4 form of the |3-D anom er, and 2) form ation of the
anhydride . P ra tt an d R ichtm yer (104) show ed th a t an axial
hydroxyl group at C-3 is the m ost im portan t axial constituen t in
determ ination of 1,6 anhydride form ation. An axial hydroxyl group
a t C-3 can in te rac t with an anhydride bridge an d destabilize the
anhydride. The four hexoses having axial hydroxyl groups at C-3
(D-glucose, D -m annose, D -galactose, an d D -talose) fo rm less
anhydride than those hexoses whose C-3 hydroxyl group is orien ted
equatorially (D-allose, D-altrose, D-gulose, and D-idose). The overall
ability for 1,6 anhydride form ation then is:
ido > altro, gulo > talo > alio > galacto > m anno > gluco
Some debate still exists abou t the placem ent of talose com pared to
allose in the above scheme. Some researchers claim talose is more
p rone to 1,6 anhydride form ation. O thers claim allose form s 1,6
anhydrides m ore readily (103, 141).
38
VI. Goals of This Study
This s tu d y was designed to op tim ize th e cond itions fo r
p roduction of bacteria l alginate from Pseudom onas syringae pv
phaseolicola, ATCC 19304 to determ ine the feasibility of large scale
p roduction , an d to characte rize th e physical p ro p ertie s of th e
p roduct, rela ting those p ro p erties to the resu lting so lu tion an d
gelling properties of the polymer.
MATERIALS AND METHODS
I. Organisms, Growth Conditions, and M aintenance
Pseudom onas syringae subsp. phaseolicola ATCC 19304 was
obtained from the American Type Culture Collection, Rockville, MD.
It was selected fo r th is research because of its lack of hum an
pathogenicity an d its po ten tia l for producing a highly acety lated
bacterial alginate (39).
Cultures were m ain ta ined at 4°C on Dworkin Foster (DF) agar
(32) supp lem en ted w ith 2% (w/v) gluconic acid. This m edium
co n ta in ed (in gram s p e r lite r of deionized w ater): KH2PO4 , 4.0;
Na2 HTC>4 , 6.0; NaCl, 0.4; KNO3 , 9.1; (NH4 )2 S0 4 , 0.9; MgS0 4 • 7 H2 O,
0.2; gluconic acid , 20; an d agar, 15. The gluconic ac id was
aseptically added to the salts m edium after separate sterilization in
an autoclave a t 121°C, a t 15 lb s . / in ^ of p ressu re for 15 m inutes.
The pH of the agar was betw een 6.9 an d 7.0 p rio r to sterilization.
Plates w ere inocu lated w ith 0.1 ml of a standard ized 48 h o u r DF
b ro th cu ltu re of P. syringae. This culture showed an absorbance
between 1.9 and 2.0 a t 660 nanom eters. The inoculum was spread
using a flame sterilized, ben t glass rod. After growth a t 30°C for 48
hours, the p lates w ere sto red a t 4°C. Cultures w ere transferred
every fou rth week.
All b ro th sta rter cultures used in this work were p rep a red by
inoculating P. syringae ATCC 19304 from agar plates in to 100 ml of
DF b ro th in 250 ml Erlenm eyer flasks. Cultures were incubated at
30°C for 48 hours a t 180 rpm on a NBS Model G25-KC ro tary shaker
(New Brunswick Scientific Co. Inc., Edison, NJ). C ultures w ere
39
40
s ta n d a rd iz e d w ith s te rile w a te r to a n a b s o rb a n c e a t 660
n an o m ete rs betw een 1.9 a n d 2.0 on a G ilford R esponse II
spectrophotom eter (Gilford Instrum ent Lab., Oberlin, OH) p rio r to
use.
II. Analytical M ethods
1. Cell Mass D eterm ination
Dry cell weight for calculation of specific yield of alginate or
p ercen t acety lation was m easured directly . Broth cu ltu res w ere
cen trifuged at 17,000 x g for 15 m inutes in a Sorval Superspeed
M odel RC-5B centrifuge (Du Pont Co., W ilmington, DE) to rem ove
bacterial cells. Cells from agar m edia were resuspended in 100 ml
of deionized water. The suspension was cen trifuged a t 27,000 x g
for 60 m inutes to pellet the cells from the highly viscous solution.
Once the cells were separated, the pellets were trea ted equally. The
pelle ts w ere w ashed twice in 10 m l o f d e ion ized w ater. The
su p ern a tan t was d iscarded and the pellet was resu sp en d ed in an
equal volum e of deionized water. The cell suspension was p ou red
into a pre-dried, tared , alum inum weighing dish and was d ried in a
drying oven a t 100°C to a constant weight.
2. Total Carbohydrate Quantitation
The to ta l ca rb o h y d ra te p re se n t in P. syringae EPS was
determ ined by the phenol-sulfuric acid assay (31). The protocol
was as follows:
41
1) To 2 ml of carbohydrate solution containing 10 to 100 |.ig of
carbohydrate, 0.05 ml of 80% (v/v) aqueous phenol was
added.
2) The solution was shaken in a Vortex m ixer for a count of 5.
Then, 5 ml of concentrated sulfuric acid was added and the
solution mixed again for another count of 5.
3) The tubes were incubated at room tem perature for 30
m inutes to allow the color to develop. The absorbance was
m easured at 485 nanom eters on a Gilford Response II
spectrophotom eter (Gilford Instrum ent Lab., Oberlin, OH).
This value was com pared to a standard curve of sodium
alginate from Macrocystis pyrifera (Sigma Chemical Co., St
Louis, MO) for determ ination of the total carbohydrate
p resen t in the solution.
3. Alginate Q uantitation
Alginate concen tra tions were d e te rm in ed by two d iffe ren t
m ethods. These m ethods included the uronic acid assay described
by Blum enkrantz and Asboe-Hansen (10), and the carbazole assay
of K nutson an d Jeanes (69). Each m ethod has its benefits. The
uronic acid assay is rap id , bu t is only accurate up to 150 itig/ml of
uronic acid. The carbazole assay takes m uch longer to perform , bu t
it is accurate up to 1 0 0 0 ng/ml.
The uronic acid assay was used predom inately w ith alginates
produced in b ro th cultures. This assay allowed d irect testing of the
superna tan t w ithout color in terference from the rem aining salts in
solution. The protocol was as follows:
42
1) To 0.2 ml of sample containing from 0.5 to 30 f.ig uronic
acid, 1.2 ml of 12.5 mM tetraborate in concentrated sulfuric
acid was added.
2) The tubes were chilled in an ice bath for 10 minutes.
3) The m ixture was shaken in a Vortex mixer, and the tubes
heated in a w ater ba th at 100°C for 5 minutes.
4) After cooling in a water-ice bath, 20 |xl of 0.15% (w/v)
m eta-hydroxydiphenyl in 0.5% (w/v) sodium hydroxide was
added to the above mixture.
5) The tubes were shaken for a count of 5 and the absorbance
read a t 520 nm as described previously. This value was
com pared to a standard curve of sodium alginate from
Macrocystis pyrifera to obtain the concentration of uronic
acid.
The carbazole assay was used fo r h igh concentrations of
purified alginate. Experim ental e rro r dim inished w ith th is assay
because of few er d ilu tion steps. The protocol fo r the carbazole
assay was as follows:
1) 0.5 ml of a purified alginate solution containing 0 to 500 jxg
of alginate was equilibrated in a water-ice bath for 10
minutes.
2) Three ml of cold concentrated sulfuric acid was added to
this solution. The m ixture was re-equilibrated in a water-ice
bath.
3) The solution was then mixed with a Vortex mixer for a
count of 4 and heated at 55°C for 20 m inutes in a w ater bath.
43
4) The solution was then re-equilibrated in a water-ice bath
for 10 m inutes, an d 0 .2 ml of a 0 .2% (w/v) carbazole solution
(Eastman Kodak, Rochester NY) in ethanol was added.
5) The sample was mixed with a Vortex m ixer for a count of
10 and allowed to incubate at room tem perature for 3 hours
for color development.
6 ) The sample absorbance was read at 530 nm as described
previously. This value was com pared to a standard curve of
sodium alginate from Macrocystis pyrifera to determ ine
bacterial alginate concentrations.
4. Acetyl Q uantitation
The p e rc e n t ace ty la tio n was d e te rm in ed by th e m e th o d
described by McComb an d McCready (80). A s ta n d a rd curve for
percen t acetylation was p rep ared with glucose pen taaceta te (Sigma
Chemical Co., St. Louis, MO). P rior to assay, all sam ples w ere
desalted by dialysis for 48 hours against deionized w ater a t room
tem perature. The protocol was as follows:
1) One volum e of 9.4% (w/v) sodium hydroxide was added to
one volume of 3.75% (w/v) hydroxylam ine solution.
2) To 2 ml of the above mixture, 0.5 ml of the sample solution
was added with agitation.
3) After 5 m inutes, 0.5 ml of acid m ethanol was added with
agitation, then 1.3 ml of the ferric perchlorate solution was
added.
44
4) After 5 m inutes, the precipitated hydroxam ic acid and
ferric complex was rem oved by m icrocentrifugation for 3
m inutes in a Sorval Microspin 24s M icrocentrifuge (Du Pont
Co., Wilmington, DE).
5) Color intensity was determ ined by m easuring absorbance
a t 520 nm as described above. This value was com pared to
the standard curve of glucose pentaacetate to determ ine the
percent acetylation.
The reagents for acetyl quantitation were p repared as follows:
1) Acid M ethanol Solution
Chilled reagent grade absolute m ethanol was added to 35.2 ml
o f chilled 70% perchloric acid to m ake a 500 ml solution. This
solution was used as the acidic m ethanol solution.
2) Ferric Perchlorate Solution
Ferric ch lo rid e (1.93 g) was d isso lved in 5 m l o f 70%
perchloric acid and evaporated alm ost to d ryness. It was th en
d ilu ted to 100 ml with w ater for use as the stock ferric perchlorate.
Then 8.3 ml of 70% perchloric acid was ad d ed to 60 ml of stock
ferric perch lorate solution. This solution was cooled in an ice bath
an d m ade to 500 ml with chilled reagent grade absolute m ethanol.
3) Glucose Pentaacetate Standard Solution
Pure crysta lline p-D-glucose p en taace ta te (108.9 mg) was
dissolved by heating w ith gentle agitation in abou t 5 ml of ethy l
alcohol, and m ade to 50 ml with deionized w ater. 2, 4, 5, and 7 ml
of this solution were then taken and m ade to 50 ml with deionized
w ater. These solutions rep resen t 120, 240, 300, an d 420 |ng/ml of
acetyl.
45
III. Optimization of Bacterial Alginate Production
1. Media Composition:
A. Carbon Source
P. syringae ATCC 19304 was tested for its ability to p roduce
an acety la ted bacteria l alg inate w hen grown on d ifferen t carbon
sources. The carbon sources tested were fructose, glucose, sucrose,
glycerol, and gluconic acid. Each separate solution of test carbon
source was autoclaved and then aseptically added to the DF bro th .
The concen tra tion of each stock solution was 20% (w /v o r v /v ).
Each carbon source was tested a t a final concentration of 2% (w/v or
v /v ). After inoculation of DF b ro th from sta rter cultures, (3%, v /v ),
P. syringae was incubated for 48 hours w ith shaking as described
previously . At 48 hours, the cu ltu re b ro th was cen trifuged to
rem ove bacterial cells, an d the cell mass determ ined as described
previously. The cell free b ro th was analyzed by the phenol-sulfuric
assay (to ta l c a rb o h y d ra te p ro d u c tio n ), th e u ro n ic ac id assay
(alginate production), and the acetyl assay (degree of acetylation).
B. Nitrogen Source
P. syringae was tested fo r its ab ility to p roduce acetylated
bac teria l a lg inate on d iffe ren t n itrogen sources. The n itro g en
sources tested were am m onia, [(NH4 )2 9 0 4 ], n itrate, (KNO3 ), n itrite,
(KNO2 ), and urea. Each DF b ro th was m ade by incorporating only
the test com pound as a potential n itrogen source for the organism .
Each n itro g en source was p laced in to th e m ed iu m a t in itia l
concentrations of 2 mM, 5 mM, 9 mM, 12 mM, an d 15 mM (w /v).
Gluconic acid was used as the carbon source a t a final concentration
46
of 2% (w/v). After inoculation, (3%, v /v), from standardized sta rter
cu ltures, P.syringae was incubated for 48 hou rs w ith shaking as
described previously. At the ap p ro p ria te tim e the cu ltu re b ro th
was cen trifu g ed to rem ove b ac te ria l cells, an d th e cell m ass
d eterm ined as described previously. The b ro th was analyzed by
the uronic acid assay for alginate production.
2. pH and Tem perature
The effect of pH on bacterial cell yield and alginate production
was investigated in DF broth . The initial pH's were 6.0, 6.2, 6.4, 6 .6 ,
6 .8 , 7.0, and 7.2. Cell yield and alginate production were m easured,
as described previously, after incubation a t 30°C fo r 48 h o u rs a t
180 rp m in a NBS M odel G25-KC ro ta ry shaker (New Brunswick
Scientific Co. Inc., Edison, NJ). In each case, phosphate buffer (0.03
M) was used to m aintain the pH of the culture broth. Gluconic acid,
a t a final concentration o f 2% (w/v), was the carbon source.
The effect of tem peratu re on bacterial cell yield an d alginate
p roduction was investigated in DF broth . The tem pera tu res tested
w ere 25°C, 28°C, 29°C, 30°C, 31°C, and 32°C. Both cell y ield and
alginate production were m easured, as described previously. Each
culture was grown under the same conditions as the pH cultures.
3. Agar vs. Broth Culture
Cell y ie lds an d a lg ina te p ro d u c tio n w ere m easu red in 5
d ifferent media, both on agar and in b ro th culture. All m edia were
m ade up in deion ized w ater. The m ed ia inc luded DF m edia,
n u tr ien t m edia (Difco Lab. Detroit, MI), m edia com posed of 3 g/L
47
beef extract (Difco Lab. Detroit, MI), m edia containing 5 g/L peptone
(Difco Lab. Detroit, MI), and m edia containing a m ixture of 3 g/L
beef extract and 5 g/L peptone. All m edia were supplem ented with
2% gluconic acid (w/v). The gluconic acid was sterilized separately
by autoclaving as a stock solution of 2 0 % (w /v), an d added to the
cu ltu re m edia asep tically a fte r sterilization . Agar m ed ia w ere
p repared with 15 g/L agar. The pH of each test m edia was betw een
6.9 an d 7.0 p r io r to s te riliza tion . All in o cu la w ere fro m a
standard ized 48 h o u r s ta rte r cu ltu re of P. syringae grow n in DF
bro th a t 30°C with shaking as described previously. Liquid cultures
were inoculated from the standardized 48 h o u r s ta rter cu lture to a
fined concentration of 2% (v/v). Solid cultures were inoculated with
0.1 ml of the same standard ized s ta rte r cu ltu re p e r 100 x 15 m m
petri dish containing 25 ml of m edium . The inoculum was spread
using a flam e sterilized, b en t glass rod . All test cu ltu res w ere
allowed to grow for 48 hou rs a t 30°C. The liquid cultures w ere
incubated with shaking as described previously. At the appropriate
tim e, the cell yield and alginate p roduction of each cu lture were
m e asu red as d esc rib ed p rev iously . A lginate p ro d u c tio n was
m easured by the uronic acid assay. Total alginate production on
solid m edia was m easured by scraping the bacteria l growth from
half of a pe tri dish (allowing 2 m easurem ents p e r p e tri dish) and
resuspending th a t growth in 10 ml of deionized w ater. The cells
were then rem oved by centrifugation and the cell mass determ ined,
as described previously. Alginate p roduction was assayed by the
uronic acid assay. The to ta l a lg inate p ro d u c tio n fo r the solid
cultures was reported in (.ig/cm2.
48
IV. Batch Ferm entations
Batch ferm entations were conducted in 1 liter volum es in a
NBS 2.5 lite r Bioflo II b a tch /co n tin u o u s cu ltu re fe rm en te r (New
Brunswick Scientific Inc., Edison, NJ) in DF b ro th supplem ented with
2% (w/v) gluconic acid. T em perature was m aintained a t 30°C, and
the pH was m a in ta in ed a t 7.0 by titra tio n w ith 3 M sod ium
hydroxide. Air, filtered through a sterile W hatm an H epavent filter
(W hatman Inc., Clifton NJ) was supplied through a sparger a t a ra te
o f 1 s tan d ard liter p e r m inute (SLPM), an d ag itation was a t 100
rpm . Samples were periodically w ithdraw n aseptically, th roughout
the bacterial growth cycle. Growth was m easured by absorbance at
660 nanom eters as described previously. Alginate was m easured in
the cell free supernatan t by the uronic acid assay.
V. Purification of Bacterial Alginate
The bacterial alginates used for characterization studies w ere
obtained from either DF agar or N utrient agar plates supplem ented
with 2% (w/v) gluconic acid. Bacterial growth was scraped off the
agar plates using a ben t glass rod an d resuspended in 150 ml of
d e io n ized w ater. The sam ples w ere v o rtex ed u n til evenly
suspended and then the bacterial alginate was separa ted from the
cells by centrifugation as described previously. Three volum es of
isopropanol were added to one volum e of clarified su perna tan t to
p recip ita te the polysaccharide. This solution was m ixed fo r 15
m inutes an d the p rec ip ita ted alginate was rem oved by w inding
a ro u n d a glass rod . The p rec ip ita te was th en d ried in acetone
followed by a ir drying.
49
Purity was determ ined by the carbazole assay using sodium
alg in a te from M acrocystis pyrifera a s th e s ta n d a rd , an d by
w avelength scans betw een 200 nm and 300 nm in increm ents of
0.5 n m on a G ilford R esponse II sp ec tro p h o to m e te r (Gilford
Instrum ent Lab., Oberlin, OH). Absence of detectable peaks a t 260
nm an d 280 nm indicated an absence of nucleic acid an d p ro te in
respectively. A large peak a t 230 nm ind icated the presence of
large am ounts of carbohydrate. This carbohydrate was determ ined
to be alginate by the carbazole assay. The purity of the bacterial
alginate produced from P. syringae ATCC 19304 was m ore than 98%
(w/v).
VI. Deacetylation of Bacterial Alginate
The alginates from P. syringae ATCC 19304 were deacety lated
fo r com parison w ith seaw eed alg inate an d acety lated bacteria l
a lg inate . The com parisons w ere to d e te rm in e th e effects of
acetylation on the solution and gelling p roperties of the polym er.
Purified, acety lated bacteria l alginate was dissolved in deionized
w ater a t a concentration of 1 m g/m l. Three volumes of this solution
w ere m ixed w ith one volum e of 1 N sodium hydrox ide solution.
After incubation for 20 m inutes at room tem peratu re , w ith gentle
ag ita tion , one volum e o f 1 N hydroch lo ric acid was ad d e d to
n eu tra lize th e so lu tion (final pH was ab o u t 7.0) an d stop the
reac tio n . The d eace ty la ted bac teria l a lg inate was ex tensively
dialyzed against deionized water. The effectiveness of the process
was d e te rm in ed from co n cen tra tio n s of acety l g roups in the
preparation.
50
VII. Alginate Size and Q uantity Determ inations
M olecular w eights w ere d e te rm in ed by gel p e rm e a tio n
chrom atography (GPC) from alginate solutions [100 (ng/ml (w/v)] in
deion ized w ater. A lginate sizes and polydispersity indices were
d e te rm in e d by m e a su re m e n t of m u ltian g le lig h t scattering
in tensities using a DAWN-Photometer (W yatt Technology, Santa
Barbara, CA). The DAWN GPC detec to r m easures the scattering
intensities of a sample at 15 different angles and transm its the data
to a com puter for digital conversion and subsequen t processing
u n d e r con tro l of the ASTRA™ (or ASTRA 202) softw are (W yatt
Technology, Santa Barbara, CA). Acetone an d cyclohexane were
used for instrum ent calibration. Concentrations were obtained from
a W aters Model 410 D ifferential Refractom eter (M illipore Corp.,
Milford, MA). Alginate sizes were based on the GPC calculation and
the following external standards: T10, T40, and T500 (Pharm acia
Co., Uppsala, Sweden) dextrans. Sample injection volumes were 100
jil and the GPC column was an U ltrahydrogel Linear colum n (Waters,
Millipore Corp., Milford, MA). The running buffer was 0.1 M NaNC>3 ,
and the tem perature was 45 °G
VIII. Sugar Sensitivity to Acid
1. Thin Layer Chrom atography
Standards of D-mannose and L-gulose (Sigma Chemical Co., St.
Louis, MO) were p repared a t a concentration of 6 .6 6 m g/m l (w/v)
in deionized water. One m l of acid (1 N HC1 or 1 N H2SO4 ) and 1 ml
of s tan d ard sugar solution were m ixed and the solution hea ted a t
100°C for 0.5, 1, 2, 3, an d 4 hours. Each so lu tion was then
51
neutralized with 1 ml of 1 N NaOH giving a final sugar concentration
of 1.67 m g/m l (w/v) and a final pH of between 6.0 and 7.0.
T h in la y e r c h ro m a to g ra p h y was p e r fo rm e d o n th e
h y d ro ly sa tes on 0.25 m m p la tes co a ted w ith silica gel 150A
(W hatman Co., Maidstone, England), o r kieselgel 60 F254 (E. Merck
Co., D arm stadt, Germ any). The hydro lyzed sugar solutions w ere
spotted a t a concentration of 50 fig sugar/spot. The running solvent
was n -p ropanol an d w ater in a ra tio of 85:15 (v /v ). The p lates
were developed by spraying with 20% (v/v) H2SO4 in m ethanol and
charring a t 100°C for 15-20 m inutes.
2. Ion Chrom atography
Standards of D-mannose and L-gulose (Sigma Chemical Co., St.
Louis, MO) were p rep ared a t a concentration of 1 m g /m l (w/v) in
deionized w ater. One ml of sugar solution was mixed with 1 ml of
acid (1 N HC1 o r 1 N H2SO4 ). Each solution was hydrolyzed at 100°C
fo r 0.5, 1, 2, 3, and 4 hours. After hydrolysis each solution was
neutralized with 1 ml of 1 N NaOH giving a final sugar concentration
of 0.33 m g/m l (w/v) and a final pH between 6.0 and 7.0.
Q uantitative determ inations of the acid sensitivities of each
sugar w ere m ade with ion chrom atography. Ion chrom atography
was perfo rm ed on each hydrolyzed standard , using a Dionex ion
chrom atography system (Dionex Corp., Sunnyvale, CA) fitted with a
Carbopac PA1 colum n (4 x 250 mm). A 100 mM solution of NaOH
was used as e luen t an d pum ped a t 0.5 m l/m in u te by a g rad ien t
pum p. A 50 |il sample was injected and the signal was detected by
a pulse am perom etric detector. Integration was accom plished by a
52
Dionex 4400 in teg ra to r. The rela tive peak areas w ere used to
q u a n tita te th e p e rc e n t d eg rad a tio n o f each sugar u n d e r the
experim ental conditions employed.
IX. Identification of the Acid Hydrolysis Product of L-Gulose
1. Thin Layer Chrom atography
Thin layer chrom atography was used to help identify the acid
hydrolysis p ro d u c t of L-gulose. L-Gulose (Sigma Chemical Co., St.
Louis, MO) was acid hydrolyzed and separa ted on a TLC p la te as
described previously. At the end of a run , the TLC plate was air
dried . The silica gel in the region corresponding to the Rf value of
the spo t of in te re s t was scraped from th e p la te an d e lu ted in
deionized w ater for 10 m inutes. The solution was m icrocentrifuged
in a Sorval Microspin 24S microcentrifuge (Du Pont Co., Wilmington,
DE) for th ree m inutes to pellet the silica gel. The su p ern a tan t was
freeze d ried in a Flexi-Dry freeze d ry apparatus (FTS Systems, Stone
Ridge, NY).
The freeze d r ie d sam ple was resu sp en d ed in 0.2 ml of
deionized w ater and again spotted on a 0.25 m m TLC p la te coated
w ith kieselgel 60 F254 (E. Merck Co., D arm stadt, G erm any) as
described previously. The sample was ru n in n-propanol and w ater
in a ra tio of 85:15 (v /v ) w ith sam ples o f 1,6 an h y d ro (3-D-
m a n n o p y ra n o se , a n d 1,6 anhydro -p -D -g lucopyranose (Sigma
Chemical Co., St. Louis, MO) an d the m igration of the unknown
com pared to the known standards.
53
2. Stability of 1,6 Anhydro p-L-Gulopyranose
The stability of 1,6 anhydro |3-L-gulopyranose was m easured
by m olecu lar m odeling on SYBYL m olecular m odeling software
version 6.2 (Tripos Assoc. Inc., St. Louis, MO). The m olecule was
draw n using the above software and then m inim ized to its lowest
energy level a t pH 7.0 with a dielectric constant of 78.8 (equivalent
of water). Once the molecule was a t its lowest energy levels for the
above conditions, it was solvated in w ater by com puter and heated
to 400°K for a total of 100,000 fem toseconds (10"11 seconds). This
allowed the solvent to a tta in equilibrium . A distance depen d en t
dielectric constan t was used to avoid the conditions of a vacuum .
Energy m easurem ents were m ade a t 250 fem tosecond intervals.
X. Alginate Reductions
Alginates were chemically reduced p rio r to acid hydrolysis of
the polym ers. The m ethod used for the reduction of the uronic
acids in alginates to the corresponding neu tra l sugars was th a t of
Taylor e t al (140). An aqueous solution of alginate containing 100
m icroequivalents of carboxylic acid in 10 ml of deionized w ater was
ad justed to pH 4.75 with 0.1 M NaOH. One millimole of 1-ethyl-3-
(3-d im ethylam inopropyl)carbodiim ide was added to the alginate
solution to convert the uronides to esters. The pH of the reaction
m ixture was m aintained a t 4.75 by titra tion with 0.1 M HC1. The
reaction was allowed to continue until hydrogen ion uptake ceased
(45-60 m inutes). Then, 25 ml of a 3 M NaBH4 solution was added
dropwise over a 1 hour period to reduce the uronides to the m ore
readily hydrolyzable neu tra l polymers. The pH was m ain tained at
54
7.0 by titra tion with 4 M HC1. 1-Propanol was added dropwise, as
necessary to m inim ize foam ing. The reac tion m ixture was th en
m ade slightly acidic to destroy any rem aining sodium borohydride,
an d the solution was dialyzed exhaustively against deionized water.
Each reduced alginate sam ple was then con cen tra ted in a Buchi
Model R110 ro ta ry evaporator (Buchi Lab., Flawil, Switzerland) and
precip itated by addition of th ree volumes of isopropanol and dried
by washing in acetone.
XI. Alginate Compositions
Composition m easurem ents of the alginates from Macrocystis
pyrifera (Sigma Chemical Co., St. Louis, MO), and P. syringae ATCC
19304 were m ade by ion chrom atography of the acid hydrolyzed,
reduced polymers. Each alginate sam ple was p rep ared in the same
m an n er as the D-m annose, and L-gulose sugar stan d ard s for ion
chrom atography described previously. The reduced sam ples were
m ixed a t a concen tration of 1 m g/m l an d 1 m l of solution mixed
w ith 1 ml of acid (1 N HC1 or 1 N H2 9 0 4 ). The sam ples w ere th en
hydrolyzed at 100°Cfor 0.5, 1, 2, 3, and 4 hours. After hydrolysis,
5 0 [xl o f e a c h sam ple was in je c te d in to th e D ionex io n
chrom atography system described above. The resulting peak areas
w ere th en co rre la ted an d ex trap o la ted back to tim e zero to
determ ine the percen tage of m annose an d gulose p resen t in the
reduced polym er. This com position was then directly correlated to
the com position of both Macrocystis pyrifera and P. syringae ATCC
19304 alginates.
55
XII. Properties of Bacterial Alginates and Effects of Acetylation
1. Viscosity
The viscosities of Macrocystis pyrifera alginate and acetylated
an d deacetylated P. syringae ATCC 19304 alginate were m easured
as a fu n c tio n of te m p e ra tu re , co n cen tra tio n , a n d d eg ree of
acetylation. In each case, viscosities w ere d e te rm in ed by the
m eth o d of A llison a n d M atthew s (1) using a sim ple U -shaped
Ostwald capillary viscom eter designed for small volumes. The time
taken for a sam ple to fall a fixed distance und er gravity (N), divided
b y th e tim e taken by w ater to fall th a t same distance (No) was
expressed as a m easure of com parative viscosity (Visc.COm = N/N0).
The effect of tem peratu re on alginate solution viscosity was
m easured at alginate concentrations of 400 tig/m l (w /v) over a
te m p e ra tu re ran g e of 30°C to 85°C. The effects of a lg inate
concentration on viscosity was m easured a t 50°C a t concentrations
ranging from 50 fxg/ml (w/v) to 1000 f.ig/ml (w /v). The effect of
a c e ty la tio n on v iscosity was m e asu re d a t 50°C a t a lg in a te
concentrations of 50 ng/m l (w/v), 100 ng/m l (w /v), an d 200 ng/m l
(w/v) and the values averaged. Deacetylation of bacterial alginate
was as described previously with m inor variations. A 400 ng/m l
sam ple of h ighly acety lated bacterial alginate was deacety lated to
various degrees by varying the concentration of NaOH used in the
reac tio n an d by altering the reac tion tim e. Sodium hydrox ide
concentrations ranged from 0.25 M-1.0 M while reaction times w ere
betw een 5 m inutes and 20 m inutes. A cetylated seaweed alginate
was ob ta ined from Jin W. Lee (72) and p artia lly deace ty la ted as
described previously.
56
2. W ater Holding Capacity
W ater holding capacities of alginate gels w ere m easured by
determ ining the am ount of w ater lost from the gels upon drying.
A lginate gel beads w ere m ade from a 0.6% (w /v) so lu tion of
seaweed, acetylated or deacetylated bacterial alginate. Using a 5 ml
p ipet tip, 4 ml of the alginate solution was d rip p ed slowly in to 30
ml of CaCl2 , FeCl3 , o r PbCl2 solution. Each m etal solution was tested
at concentrations of 0.05 M, 0.1 M, 0.25 M, o r 0.5 M. The beads
w ere allowed to form in each m etal solution for 15 m inutes after
w hich th e y w ere w ashed th o ro u g h ly in d e io n ized w ate r (5
m inutes). A fter a ir d ry ing fo r 5 m inutes, 10 beads from each
sam ple were p laced in to p red ried , ta red alum inum weigh dishes
an d weighed. The weighing dishes were th en p laced in a drying
oven a t 100°C and the sam ple d ried un til a co n stan t w eight was
reached . The dishes were then rew eighed an d the w ater holding
capacity of each bead calculated as g w ater/ g d ry alginate gel.
3. Surface Tension
The relative surface tensions of beads were m easured using
bead d iam eter, the sm aller the bead d iam eter th e h ig h e r the
surface tension on the bead. Gel beads w ere m ade as described
prev iously in 0.5 M CaCl2 . U pon form ation , 1 /3 of each bead
sam ple was w ashed in deionized w ater for 5 m inutes an d allowed
to d ry fo r 5 m inutes p r io r to d iam eter m easurem ent w ith a dial
caliper (L. S tarre tte Co., Athol, MA). The second po rtion of each
sample was incubated a t 4°C for 24 hours in deionized w ater prior
to m easurem ent, and the final th ird of each sam ple was incubated
57
a t 4°C fo r 24 h o u rs in th e 0.5 M CaCl2 so lu tion p r io r to
m easurem ent. W ater holding capacity was d irectly re la ted to the
relative surface tension on each bead. A lower surface tension on
the bead resu lted in a la rger b ead d iam eter an d h ig h e r w ater
holding capacity of the gel.
4. Precipitation by Metal Ions
The p rec ip ita tio n of seaw eed alg inate an d acety la ted and
d ea ce ty la ted b ac te ria l a lg ina te by m eta l ions was com pared .
P u rified a lg in a te s w ere d isso lv ed in d e io n ize d w a te r a t a
concentration of 400 M.g/ml (w /v). M etal salts w ere dissolved in
deionized w ater to p rep are for the solutions with concentrations of
0 to 25 o r 100 mM. The m etal ions tested were: Csl+, Rbl+, Mg2+,
Ca^+, Sr2+, Mn2+, Fe^+, Co2+, Cu2+, Zn2+, Pb2+, an d U ^ . All m etal
salts were ob tained from Sigma Chemical Co., St. Louis, MO, except
fo r u ran y l ace ta te (Eastm an Kodak Co., R ochester, NY). Four
volum es of seaweed alginate solution or acetylated or deacetylated
bacteria l alg inate solution were m ixed w ith one volum e of each
m etal solution, respectively. The m ixtures were incubated for 12
hou rs a t room tem p era tu re an d cen trifuged (17,000 x g fo r 30
m inutes) in a Sorval Superspeed Model RC-5B centrifuge (Du Pont
Co., W ilmington, DE). The su p ern a tan ts were sep ara ted an d the
co n cen tra tio n of a lg inate rem ain ing in each su p e rn a ta n t was
m easu red by th e u ron ic acid assay. The am ounts of a lg inate
separated as a gel were calculated by difference and those values
w ere used to determ ine the rela tive precip ita tion of the alginate
solutions by the m etal ions.
RESULTS
I. Production of Bacterial Alginate
1. Media Compositions and Conditions
In b ro th cu lture , alg inate p roduction by P. syringae ATCC
19304 fo llow ed th e g row th cu rve of th e o rgan ism (Fig. 9).
Maximum cell mass (dry weight) an d alginate yields were obtained
48 hours post inoculation. The type of carbon source used affected
the alginate yield of P. syringae ATCC 19304, as well as the degree
of acetylation within the polym er (Table 5). P. syringae grew well
on glucose, sucrose, glycerol, and gluconic acid, bu t d id no t grow on
fructose. Sucrose grown cells yielded an EPS, only 77% percen t of
which was uronic acid. Gluconic acid grown cells yielded the m ost
alg inate (approxim ately 2 0 0 i-ig/mg cell d ry w eight), w ith th e
h ighest degree of acetylation (approxim ately 100%). Gluconic acid
was the carbon source of choice due to the increased alginate yield
of gluconic acid grown P. syringae.
P. syringae ATCC 19304 u tilized am m onia as a n itro g en
source, b u t was unable to utilize nitrate, n itrite, or urea. There was
an inverse co rre la tio n betw een a lg ina te y ie ld a n d th e in itia l
concen tra tion of am m onia in the m edia. As in itia l am m onium
co n cen tra tio n increased from 2 mM to 15 mM th e cell m ass
increased 2.4 fold, from 0.66 mg cell d ry w eight/m l to 1.58 mg cell
d ry w eight/m l. At the same time, alginate yield decreased 2.4 fold,
from 1200 j.ig/ml to 500 ^g/m l (Fig. 10). This ind ica ted th a t at
h ig h e r in itia l am m onium concen tra tions the ca rbon norm ally
d es tin ed fo r alg inate p roduction was used by the cells for cell
58
Cell
mas
s (m
g dr
y w
eigh
t/
ml
brot
h)
59
10002
- 8 0 0
- 6 0 0
1
- 4 0 0
- 2 0 0
01006040 800 20
do*3ddadod3ddtuod
oH
Tim e (hr)
Figure 9. The re la tionsh ip betw een cell mass (♦), and alginate (uronic acid) accum ulation (D) by P. syringae ATCC 19304 with time in shake flask culture.
tyig
/ml
brot
h)
Table 5. Effects of Carbon Source on Alginate Yield from P. syringae ATCC 19304
CarbonSourcea>b
Cell Yield (m g/m l)c
Yield Total EPS Oig/ml)d
% Alginatee Yield Alginate (ng/m g cell)f
% AcetylationS
Glucose 1.25 175 1 0 0 140 107 (±23)
Sucrose 1.23 211 77 132 4 (± 1)
Glycerol 0.96 134 1 0 0 140 84 (± 17)
Gluconic acid 1.04 213 100 205 97 (± 30)
a Cultured in DF bro th supplem ented with 2% carbon source, and grown for 48 hours a t 30°C with shaking a t 180 rpm .b Fructose was also tested bu t there was no growth. c Cell yield was m easured as mg cell d ry w eight/m l broth. d Yield of total EPS was m easured as ng EPS/ml broth. e The percen t of to tal EPS th a t was alginate.f Yield of alginate was m easured as [ig alginate/m g cell d ry weight.8 The fimolar ratio (%) of acetyl to uronic acid.
61
3©*&
asAtxo'H
b©awwrt2
©L>
1400
- 1200
- 1000
- 80 0
- 6 0 00.8 -
4 0 00.612 15i 9
d©S
P
I sn3 -% a00 ar t 5 "
3©H
In itia l [Am m onium ] (mM)
Figure 10. The effect of the initial am m onium concentration on cell mass (♦), and total alginate (uronic acid) accum ulation (□), by P. syTingae ATCC 19304 at 48 hours.
62
division. As initial am m onium concentrations increased from 2 to 9
mM, the specific yield of alginate decreased from 2100 fxg/mg cell
d ry w eight to 500 ^ig/mg cell d ry weight. In itia l am m onium
concentrations above 9 mM resulted in a consistently lower specific
alginate yield (Fig. 11).
Initial pH of the DF b ro th and tem peratu re bo th affected the
cell mass and alginate yield of P. syringae ATCC 19304. M aximum
growth of P. syringae ATCC 19304 occurred in DF b ro th with initial
pH's betw een 6.4 and 7.2. Alginate yield was greatest in DF b ro th
with initial pH's betw een 6.8 and 7.0. Above pH 7.0 the alginate
y ield declined (Fig. 12). The optim um tem p era tu re fo r alg inate
p roduction was 30°C. T here was little change in cell m ass w ith
tem p era tu res betw een 25°C to 32°C. A lginate p ro d u c tio n was
extrem ely tem pera tu re dependen t, with a sharp optim um at 30°C
(Fig. 13).
2. Agar vs. Broth Culture
The ability of P. syringae ATCC 19304 to grow an d produce
a lg in a te was co m p ared on ag ar an d in b ro th cu ltu re . Upon
successive tran sfe rs in b ro th cu ltu re , a lg inate y ie ld dec reased
dram atically. Initially, alginate yield reached a maxim um of 550 |ng
a lg inate / mg cell d ry weight a t 27 hours. After one tran sfe r back
into b ro th culture, the yield decreased to only 50 (.ig a lg in a te / mg
cell d ry weight. After a second transfer, there was less th an 10 j.ig
alg inate/ mg cell d ry weight. On agar m edia alginate production by
P. syringae ATCC 19304 was consisten t upon tran sfe r from one
Spec
ific
algi
nate
ac
cum
ulat
ion
tyig
/mg
cell
dry
wei
ght)
63
3000
2000
1000
0
In itia l [A m m onium ] (mM)
t '-----------1----------- 1------------'-----------1------------'-----------r
2 5 y 12 15
Figure 11. The effect of the initial am m onium concentration on specific yields of alginate (uronic acid) by P. syringae ATCC 19304 at 48 hours.
Cell
mas
s (m
g dr
y w
eigh
t/m
l br
oth)
64
5 0 0
1 .6 -
4 0 0
- 3 0 0
0.8 -
- 2 0 0
0.6
0 .4 1006.0 6.2 6.4 6.6 6.8 7.0
d3to
h* 2 « &s a.23>CO
3oH
In itia l pH
Figure 12. The effect of initial pH on cell m ass (♦), and alginate(uronic acid) accum ulation (0), by P. syringae ATCC 19304 a t 48hours.
65
f l©PXi
as•pjd&o
pt?&Qa0)CO
a©©
2.75
2 .50
2.25
2.00
1.75
1 . 5 0 -
1.25 -
1.0 0 -
0 .75 -
0 .50 -
0 .25 -
0.00
500
- 4 0 0
300
d©3«iqpag
a sd ©© ps s
d &oS 5 'rt
5©H
24—1"26
—r-28
—r~30
—r~32
20034
Tem p. (°C)
Figure 13. The effect of tem perature on cell mass (♦), and alginate(uronic acid) accum ulation (0), by P. syringae ATCC 19304 at 48hours.
66
culture to another. The organism averaged approxim ately 1870 jug
a lg inate / mg cell d ry weight over 5 successive transfers (Table 6).
Total and specific alginate yields were determ ined in various
m edia. P. syringae was tested in b ro th and on an agar surface. DF
salts, n u trien t m edia, peptone m edia, and beef ex tract m edia were
tested fo r th e ir ability to support bacteria l growth and prom ote
alginate production by P. syringae ATCC 19304 (Table 6). In both
b ro th an d on agar, n u tr ie n t m edia sup p o rted the h ighest to tal
alginate yield in a 48 hour period. The specific alginate y ield was
1.5 to 2 fold g reater in beef extract m edia than in any of the o ther
m edia tested . In all cases, grow th on agar m edia increased the
specific yield of bacterial alginate by abou t 3 fold. In DF b ro th the
specific yield was approxim ately 500 M-g/mg cell d ry w eight an d
was constan t from 24 hours to 96 hours. This ind icated th a t the
to tal yield of alginate increased at a constant ra te over this period.
On agar m edia specific alginate yields reached approxim ately 1850
ng/m g cell d ry weight betw een 24 and 72 hours an d then began to
decrease after 72 hours.
Ferric ion affected alginate production . By increasing the
initial concentrations of ferric ion from 0 mM to 1 mM, the specific
alginate yield by P. syringae ATCC 19304 decreased by 87% (Fig.
14). This indicated tha t iron starvation m ay have a role as a trigger
for alginate production.
Table 6. Alginate Yield by P. syringae ATCC 19304 on Different Media.
Yield in Liquid Media Yield on Solid Media
Mediaa >b Specific (ng/m g cell)c
Total(fig/ml)d
Specific (fig/mg cell)c
Total (fig/cm2 )e
DF Salts 550 480 1870 630
Beef Extract, Peptone
580 620 2030 980
Nutrient 590 640 2040 960
Peptone 680 540 2060 700
Beef Extract 1320 450 3000 550
a Cultures were grown a t 30°C for 48 hours. Liquid cu ltu res w ere shaken a t 180 rpm . All cultures were supplem ented with 2% (w/v) gluconic acid.t> Beef extract was used a t a 3 g/L concentration and Peptone was used a t a 5 g/L concentration. Beef extract, Peptone, and N utrient m edia were from Difco Labs., Detroit, ML c Specific production was m easured as ng alginate/m g cell d ry weight. d Total production in liquid culture was m easured as fig alg inate/m l broth. e Total production on solid culture was m easured as fig alginate/cm 2 of agar surface.
Spec
ific
algi
nate
ac
cum
ulat
ion
(pg/
mg
cell
dry
wei
ght)
68
2000
1000 -
0.2 0.4 0.8 1.0 1.20.G0.0
In itia l [Fe+++] (mM)
Figure 14. Effect of in itial ferric ion (Fe^+) concentration on thespecific yields of alginate (uronic acid) by P. syringae ATCC 19304at 48 hours.
69
II. Characterization of Bacterial Alginate
1. Recovery
Alginate p roduced by P. syringae ATCC 19304 was recovered
from the surface of DF agar plates supplem ented w ith 2% (w/v)
gluconic acid. Several d iffe ren t alcohols w ere tested fo r th e ir
ability to precip itate bacterial alginate. Recoveries were com pared.
All of the alcohols tested: m ethanol, ethanol, n-propanol, and iso
p ropano l, p rec ip ita ted 100% of th e alg inate a t a fin a l a lcohol
concentration o f 50% (v /v ). In o rd e r to de term ine the effect of
acetylation on alcohol precip itation , equal am ounts of acety lated
a n d d e a c e ty la te d a lg in a te w ere p re c ip ita te d w ith v a r io u s
concentrations of iso-propanol. Acetylation d id no t affect p ro d u ct
recovery.
2. M olecular Weight
The w eight average m olecu lar w eight (Mw ) a n d n u m b e r
av e rag e m o lecu la r w eigh t (Mn) w ere d e te rm in e d fo r b o th
acety lated and deacety lated bacterial alginates by gel perm eation
ch ro m ato g rap h y (GPC). The Mw an d th e Mn of th e seaw eed
alginate were approxim ately 65% sm aller than the native bacterial
alginate. D eacetylation w ith sodium hydroxide d id n o t a lte r the
m olecular weights appreciably. Upon deacetylation, the Mw of the
bacterial alginate decreased by 6%, and the Mn decreased by 11%.
In each case, th e po lyd ispersity was approx im ately 3.00. This
ind ica ted th a t th e re was a wide range of m olecular sizes in each
alginate sam ple (Table 7).
70
Table 7. M olecular Weights of Alginates
Alginate sample Mna (x 104 ) Mw b (x 104 ) Mw /M n c
Macrocystis 1.4(± 1.5 x 103)
4.7(± 2.6 x 103)
3.36
A cetylated P. syringae
4.3(± 8.2 x 103)
12.7 (±7.2 x 103)
2.95
Deacetylated P. syringae
3.8(± 7.7 x 103)
11.9(± 8.9 x 103)
3.13
a Num ber average m olecular weight, b Weight average m olecular weight. c Polydispersity
71
3. Composition
The d e te rm in a tio n of alg inate com position req u ired the
reduction of the uronic acids to their corresponding neu tra l sugars
p rio r to acid hydrolysis of the polym er. This reduction facilitated
the acid hydrolysis of the glycosidic bonds by conversion of the acid
resistan t glycosyluronic acid bonds to the m ore acid labile glycosyl
bonds. The acid sensitivities of D -m annose an d L-gulose (Sigma
Chem ical Co., St. Louis, MO) w ere s tu d ied using th in la y e r
chrom atography (TLC) and ion chrom atography. TLC indicated that
D -m annose and L-gulose have m arked d ifferences in th e ir acid
sensitivity. Acid hydrolyzed (HC1) D-mannose resulted in only one
spot on TLC (Rf=.40). The intensity of this spot rem ained constant
betw een 0 an d 4 h o u rs hyd ro lysis tim e (Fig. 15). L-gulose
produced a second spot (Rf=.56) after m ore than 1 hou r hydrolysis.
The in tensity of this spot increased with hydrolysis tim e. At the
sam e tim e, the in tensity of the gulose spo t (Rf=.37) decreased
p ropo rtiona lly (Fig. 16). Both D-m annose an d L-gulose reac ted
similarly in HC1 and H2 SO4 (Table 8 ).
The relative acid sensitivity of each sugar was m easured by
Dionex ion chrom atography. D-Mannose was acid stable. After 4
hours of HC1 o r H2 SO4 hydrolysis, 98% of the original sugar was
recovered. L-gulose was relatively stable for 1 hour, after which it
began to rapidly breakdown. After 4 hours hydrolysis, only 22% of
the original sugar was recovered (Fig. 17).
The acid breakdow n p roduct of L-gulose was no t definitively
identified. TLC of this com pound showed a m igration (Rf=.58) equal
to 1,6 anhydro p-D-mannopyranose (Rf=.58), and very close to 1,6
72
.4. .... *: ■ MW.",., ?A--' - 4 . ) > M»r-5)-St>i*>•
I
1 2 3 4 5 6 7
Figure 15. Thin layer chrom atograph of HC1 hydrolyzed D-mannose. Lane 1= unhydrolyzed D-mannose in H20, Lane 2= unhydrolyzed D- m annose in HC1, Lanes 3-7= hydrolyzed D-mannose in HC1 for 0 .5 ,1 , 2 ,3 , and 4 hours respectively at 100°C
73
1 2 3 4 5 6 7
Figure 16. Thin layer chrom atograph of HC1 hydro lyzed L-gulose. Lane 1= unhydro lyzed L-gulose in H2 O, Lane 2= unhydro lyzed L- gulose in HC1, Lanes 3-7= hydrolyzed L-gulose in HC1 for 0.5, 1, 2, 3, an d 4 hours respectively a t 100°G
74
Table 8 . Thin Layer Chrom atography (Rf values )a
Sample Spot A Spot B
D-mannose in HC1 .40 (± .03) N/AC
L-gulose in HC1 .37 (± .03) .56 (± .03)
D-mannose in EI2SO4 .37 (± .04) N/A
L-gulose in H2 SO4 .34 (± .03) .50 (± .04)
a Each Rf value was calculated by the distance of the spot from the origin divided by the total distance traveled by the running solvent, b Each sample was hydrolyzed in an equal volum e of 1 N acid and hydrolyzed for 4 hours a t 100°G c N/A= Not Applicable
75
HWkHo>oooPiurtooi/j
120
100
80
GO
40
203 41 7
H y dro lysis Tim e (h r)
Figure 17. The stability of m onom eric D-mannose an d L-gulose in HC1 and H2904 under hydrolysis conditions at 100°C, as determ ined by ion chrom atography. L-gulose in HC1 (□), D-m annose in HC1 (♦), L-gulose in H29D4 (■), D-m annose in H2SO4 (0 ).
76
anhydro p-D-glucopyranose (Rf=.60, Fig. 18). This indicated tha t the
m olecule m ay be a 1,6 anhydride . M olecular m odeling of 1,6
anhydro p-L-gulopyranose (Fig. 19) showed tha t this molecule has a
low total energy (approxim ately -1400 kcal/m ol) and thus is stable
(Table 9). This lended em phasis to the possibility of 1,6 anhydride
form ation upon acid hydrolysis of L-gulose.
D estruction of the reduced sugars in the alginates on acid
hydrolysis paralleled the results seen w ith the D-m annose an d L-
gulose m onom ers. A fter co rrection fo r gulose d es tru c tio n by
extrapolation back to tim e 0, a composition of 60% m annuronic acid
and 40% guluronic acid was obtained for Macrocystis alginate, and
82% m annuronic acid and 18% guluronic acid for P. syringae ATCC
19304 alg inate (Fig. 20). This co rre la ted well to the rep o rted
com position fo r a lg in a te p ro d u ced by P. aeruginosa o f 80%
m annuron ic acid an d 20% guluronic acid, an d fo r Macrocystis
alginate of 60% m annuronic acid and 40% guluronic acid (150).
III. Functional Properties
1. Viscosity
The viscosity of a solution depends on the m olecular weight
an d the rig id ity of the solute, as well as env ironm ental factors,
especially tem perature. The concentration, percent acetylation, and
tem peratu re all con tribu ted to variations in the flow properties of
alginates. At an alginate concentration of 0.1% (w/v) the viscosity
of acetylated bacterial alginate was 2.7 fold greater th an seaweed
alginate. Deacetylated bacterial alginate was 2.0 fold m ore viscous
than seaweed alginate. As alginate concentrations increased from
77
§ •
Figure 18. T hin lay e r ch rom atograph of th e acid hydro lyzed p roduct of L-gulose. Lane 1= unhydrolyzed L-gulose in H2O, Lane 2= standard 1,6 anhydro p-D-mannopyranose, Lane 3= standard 1,6 anhydro p-D-glucopyranose, Lane 4= acid hydrolysis p ro d u ct of L- gulose.
Figure 19. Com puter derived image of 1,6 anhydro p-L-gulopyranose. The image is depicted cross stereo view.
Table 9. The Energetics and Stability of 1,6 Anhydro p-L-Gulopyranosea
Sample # Time(Femtosec)b
PotentialEnergyc
KineticEnergy
TotalEnergy
T em perature(°K)
2 250 -2307.73 861.11 -1446.62 387.77
3 500 -2432.09 886.92 -1545.17 399.39
4 750 -2475.61 855.84 -1619.77 385.40
5 1000 -2512.66 878.87 -1633.79 395.77
7 1500 -2542.10 870.16 -1671.94 391.85
14 3250 -2554.33 865.87 -1688.46 389.92
31 7500 -2512.04 820.92 -1691.11 369.67
33 8000 -2569.74 875.05 -1694.70 394.05
a Values w ere ob ta ined using SYBYL m olecular m odeling software (Tripos Assoc. Inc., St. Louis, MO).b 1 fem tosecond equals 10" 15 seconds. c All energy values are m easured in kcal/m ol.
80
50
■OImo►oo4>P4to©53OM
4 0 -
3 0 -
20 -
10 -
I4
H ydro lysis T im e (h r)
Figure 20. Gulose recovered, expressed as a p ercen t of the to tal sugar p re se n t in the HC1 hydro lyzed reduced alg inates, from M acrocystis pyrifera (□), a n d P. syringae ATCC 19304 (♦), as determ ined by ion chrom atography.
81
0 jig/m l to 1000 (.ig/ml (w /v), the com parative viscosity (N/No) of
each solution increased non linearly . In each case, th is increase
exhibited non-new tonian flow dynam ics (Fig. 21). Seaweed alginate
deviated least from new tonian flow. A cetylated bacteria l alginate
deviated most. The difference in the viscosities of acety lated and
deacety lated alginates ind icated th a t acety lation linearly affected
th e flow dynam ics of a lg inate so lu tions. An average of the
c o m p a ra tiv e v isc o s ity o f seaw eed a lg in a te m e a s u re d a t
c o n c e n tra tio n s of 50, 100, a n d 200 f-ig/ml (w /v) in c reased
approxim ately 8% per a 50% increase in acetylation. An average of
the com parative viscosity of bacteria l a lg inate m easu red a t th e
sam e co n cen tra tio n s , in c reased 16% p e r a 50% in c re a se in
acety la tion (Fig. 22). This d ifference was p ro b ab ly due to the
in c re a se d average m o lecu lar w eight o f th e bac teria l alg inate
polym er, an d its m ore extended structure due to high am ounts of D-
m annuronic acid.
T em perature also affected the viscosity of alginate solutions.
Each alg inate so lu tion re sp o n d e d sim ilarly to th e affects of
te m p e ra tu re . The v iscosity o f each a lg in a te so lu tio n a t a
co n cen tra tio n of 400 f.ig/ml (w /v) was co n s tan t from 30°C to
approxim ately 52°C. At 52°C the viscosities o f each a lg inate
so lu tion decreased d ram atica lly . Between 52°C a n d 85°C th e
viscosities of the seaweed alginate sam ple decreased 3.4%, on the
average, p e r °C. The viscosities of the acety la ted bac teria l an d
deacetylated bacterial samples decreased 9.2% p er °C, and 6.5% per
°C respectively . At 85°C the com parative viscosities of each
alginate solution were approxim ately equal (Fig. 23).
Com
para
tive
Vis
cosi
ty
(N/N
o)
82
10
8
6
4
2
00 200 4 0 0 600 800 1000 1200
A lg ina te C o n cen tra tio n (^ig/m l)
Figure 21. Com parative viscosity, (N/N0), of Macrocystis alginate (□), a c e ty la te d P. syringae a lg ina te (I), an d d e a c e ty la te d P. syringae alginate (♦), as a function of alginate concentration, (w/v).
Com
para
tive
Vis
cosi
ty
(N/N
o)
83
2.0
1 . 6 -
1 .4 -
0 50 100 150
A cety la tion (%)
Figure 22. The effects of acetylation on the com parative viscosity, (N/Nq), of Macrocystis alginate (D), and P. syringae alginate (♦).
Com
para
tive
Vis
cosi
ty
(N/N
o)
84
6
4
3
2
130 32 38 42 4 4 50 52 58 0 4 08 7 4 78 80 84
T em p e ra tu re (°C)
Figure 23. The effects of tem perature on the com parative viscosity,(N/No), of Macrocystis a lg inate (0), acetylated P. syringae alginate(I), and deacetylated P. syringae alginate (♦).
85
2. Physical Effects
The affects of acetylation on gelation, w ater holding capacity,
surface tension, and gel porosity were determ ined. Calcium induced
gelation was altered by bo th acetylation, and calcium concentration.
The gels p ro d u c e d fro m ac e ty la ted b ac te ria l a lg in a te h e ld
approxim ately 100 g w ate r/g d ry alg inate gel com pared to the
deacetylated bacterial alginate gel which held approxim ately 3 2 g
w ate r/ g d ry alginate gel (Table 10). As the concentration of CaCl2
increased from 0.05 M to 0.50 M, the w ater holding capacity of each
Ca-alginate gel decreased linearly. The w ater holding capacities of
bacterial alginate gels m ade with 0.50 M CaCl2 were approxim ately
54% th a t of gels m ade with 0.05 M CaCl2. In contrast, Ca-alginate
gels m ade w ith seaweed alginate showed a 41% decrease in w ater
holding capacity over the same calcium ion increase.
The w ater holding capacities of bo th Fe-alginate (ferric), and
P b-alg inate gels w ere d e te rm in e d fo r seaw eed a lg in a te an d
acety lated and deacety lated bacterial alginates. The resu lts were
com pared to those obtained from Ca-alginate gels. In each case, Fe-
alg inate gels held less w ater th an th e ir Ca-alginate counterparts,
b u t m ore than Pb-alginate gels. The w ater holding capacity of Fe-
a lg inate (0.05 M FeCl3 ) gels m ade w ith seaw eed a lg inate was
approxim ately 82% th a t of Ca-alginate gels. Pb-alginate (0.05 M
PbCl2) gels held w ater a t 67% th a t of Ca-alginate gels (Fig. 24). As
the concen tra tion of m etal (Fe^+ o r Pb2+) increased , the w ater
holding capacity of the resulting gels decreased. By increasing the
m etal concentration from 0.05 M to 0.50 M in increm ents of 0.05 M,
the w ater holding capacity in Fe-alginate gels decreased by an
86
Table 10. Effects of Calcium Concentration on the W ater HoldingCapacitiesa of Alginate Gels.
Alginate sample 0.05 M
Calcium Concentration^
0.10 M 0.25 M 0.50 M
Macrocystis 65 47 36 27(±3) (±4) (±4) (±3)
A cetylated 100 85 69 56P. syringae (±6) (±5) (±5) (±5)
Deacetylated 32 26 24 17P. syringae (±3) (± 2) (±2) (± 1)
a W ater holding capacities are calculated as g w ater/g d ry alginate gel.b Calcium was derived from CaCl2 (Sigma Chemical Co., St. Louis, MO).
Wat
er
Hol
ding
{%
of C
alci
um)
87
90
80 -
70 -
60 ■■
50 -
40.25 M.05 M .1 M .5 M
M etal c o n c e n tra tio n
Figure 24. P ercen t d iffe ren ce in w ater ho ld ing cap ac ity ofMacrocystis alginate gels m ade with ferric iron (D), and lead (♦), ascom pared to calcium alginate gels.
88
average of 2.2% per 0.05 M increase, and in Pb-alginate gels by an
average of 3.7% p er 0.05 M increase. The w ater holding capacities
of Fe-alginate gels and Pb-alginate gels m ade with acety lated and
deacety la ted bacterial alginate were sim ilar to those of seaweed
alginate. The w ater holding capacity of Fe-alginate (0.05 M FeCl3)
gels m ade w ith acety la ted an d deace ty la ted bac teria l a lg inate
averaged approxim ately 79% tha t of the corresponding bacterial Ca-
alginate gels. The w ater holding capacity of Pb-alginate (0.05 M
PbCl2) gels m ade under the same conditions averaged only 46% that
of the corresponding bacterial Ca-alginate gels (Fig. 25, 26). As in
seaw eed a lg in a te gels, th e w ater h o ld in g cap ac ity fo r both
acetylated and deacetylated bacterial alginate gels decreased as the
m etal concentration increased. The w ater holding capacities of Fe-
alg inate gels m ade w ith acety la ted an d d eace ty la ted bac teria l
alginate decreased by an average of 2.0% p e r 0.05 M increase in
iron concentration. The w ater holding capacity of Pb-alginate gels
decreased by an average of 3.0% p e r 0.05 M increase in lead
concentration (Fig. 25, 26).
The bead size is a function of the surface tension of the gel
th a t m akes up the bead, the sm aller the bead the higher the surface
tension . A cetylated bacteria l alginate form s gel beads in 0.5 M
CaCl2 th a t averaged 4.04 millimeters in d iam eter (Table 11). These
beads are 37% larger th an equivalent beads m ade with seaweed
alg inate , a n d 45% la rg e r th a n beads m ade w ith d eace ty la ted
bacterial alginate.
Incubation of the gel beads in deionized w ater or 0.5 M CaCl2
a t 4°C fo r 24 hours a lte red the size of the gel beads. Beads
Wat
er
Hol
ding
(%
of C
alci
um)
89
90
80 -
70 -
60 -
50 -
4 0 -
30.05 M 1 M .25 M .5 M
M etal c o n c e n tra tio n
Figure 25. P ercen t d iffe ren ce in w ater ho ld ing cap ac ity ofacetylated P. syringae alginate gels m ade with ferric iron (D), andlead (♦), as com pared to calcium alginate gels.
Wat
er
Hol
ding
{%
of C
alci
um)
90
80
7 0 -
6 0 -
50 -
4 0 -
3 0.05 M ] M .25 M .5 M
M etal c o n c e n tra tio n
Figure 26. P ercen t d iffe ren ce in w ater ho ld ing cap ac ity ofdeacety lated P. syringae alg inate gels m ade w ith ferric iro n (□),and lead (♦), as com pared to calcium alginate gels.
91
Table 11. Effects of Acetylation on Bead Surface Tensiona
Bead Diameters (mm.)
Time in W aterb Tim einCaCl?c
Alginate sample 5 m inutes 24 hours 24 hours
Macrocystis 2.95 2.69 2.64(± 0.05) (± 0.04) (± 0.06)
Acetylated 4.04 2.82 2.26P. syringae (± 0.06) (± 0.04) (± 0.05)
Deacetylated 2.79 1.96 1.68P. syringae (± 0.05) (± 0.04) (±0.05)
a The rela tive surface tension was m easured by bead d iam eter (mm.), the sm aller the bead the h igher the surface tension on the bead.b W ater values were m easured after 15 m inutes in 0.5 M CaCl2 and incubation in water a t 4°C for the relevent times. c CaCl2 values were m easured after incubation in 0.5 M CaCl2 at 4°C for 24 hours.
92
in cu b a ted in deion ized w ater m ade from ace ty la ted b ac te ria l
alginate h ad diam eters averaging 2.82 m illim eters, a decrease of
42% from the initial size. These beads were still 5% larger th an the
seaweed alginate gel beads and 44% larger th an the deacety la ted
bacterial alginate gel beads. This indicated th a t the relative surface
tension of acetylated gels was less th an deacety lated alginate gels.
Extended incubation (24 hours) of Ca-alginate gel beads in 0.5 M
CaCl2 p roduced d ifferen t results. The Ca-alginate gel beads m ade
from seaweed alginate were the largest with an average d iam eter
o f 2 .64 m illim eters. These beads w ere 17% la rg e r th a n the
acetylated alginate beads and 57% larger than the beads m ade from
d eace ty la ted bac teria l alg inate. This in d ica ted th a t acetylation
d ecreased the surface tension on the Ca-alginate gel beads, an d
extended exposure to calcium decreased the size of the beads and
increased surface tension on the beads.
The re la tiv e p o ro sity of the C a-alginate a n d Fe-alginate
(ferric) gel beads were determ ined from the ra te of w ater loss w ith
time (Fig. 27). A large absolute slope indicated a m ore rap id w ater
loss resulting from larger relative pore sizes. Ca-alginate gels, m ade
from acety lated bacteria l alginate, showed the m ost rap id loss of
w ater (absolute slope= 6.8 x 10 '2). The ra te of w ater loss in the
gels m ade from deacetylated bacterial alginate (absolute slope= 5.2
x 10" 2) was 24% slower th an the acetylated bacterial polym er and
13% faster than seaweed alginate (absolute slope= 4.6 x 10"2). The
ra tes of w ater loss of Fe-alginate gels were also d e te rm in ed and
com pared as a percentage of Ca-alginate gels. Both the gels m ade
from seaw eed a lg inate an d deace ty la ted bac teria l a lg inate lost
93
■N
fed Qa •©isOas« ,£rt£ M
Im&
to
5
4
30 10 20 30
Time (m in.) a t 50 C
Figure 27. Rate of w ater loss of calcium alginate gels m ade fromMacrocystis alginate, I slope l= 4.6 x 10"2 (□), acety lated P. syringaealginate, I slope 1= 6.8 x 10"~ (♦), and deace ty la ted P. syringaealginate, I slope 1= 5.2 x 10"2 (I), as a function of incubation time at 50°C.
94
w a te r ap p ro x im a te ly 2 fo ld fa s te r th a n th e ir C a-a lg in a te
counterparts, indicating a m ore open structure. The gels produced
by the acety lated bacteria l alginate show ed a ra te of w ater loss
approxim ately equal to th a t of Ca-alginate gel beads indicating
com parable pore sizes between these gels (Fig. 28).
3. Cation Precipitation by Alginates
P re c ip ita tio n o f ac e ty la te d a n d d e a c e ty la te d b ac te r ia l
alginates, m easured as gelation, by cations was com pared to th a t of
seaw eed alginate. Twelve m etal ions w ere screened an d th en
classified into 3 groups, depending on the ir ability to p recip ita te
acetylated a n d /o r deacetylated bacterial alginate. The groups were:
1) those cations having the ability to p recip itate half the available
bacterial alginate at a concentration of less than 20 mM m etal, (U6+,
Cu2+, Pb2+,Ca2+, Sr^+, and Fe3+), 2) those cations having the ability
to precipitate half the available bacterial alginate at a concentration
of g reater than 20 mM m etal, (Zn2+, Co^+, and Mn^+), and 3) those
cations unable to precip itate half the available bacterial alginate at
a concentration up to 100 mM metal, (Mg2+, Csl+, and Rbl+).
Of all the cations tested, uranium , copper, lead and ferric ions
precip itated both acetylated and deacetylated bacterial alginate a t
low concentrations. They were able to precipitate m ore than 90% of
the alginate a t m etal concentrations less th an 5 mM. A cetylation
d id n o t significantly affect the ability of these ions to p recip ita te
bac teria l alg inate. U ranium p rec ip ita ted g rea te r th a n 90% of
deacetylated bacterial alginate a t a m etal concen tra tion of 1 mM
an d g rea te r th an 90% of the acety la ted bac teria l a lg inate a t a
95
UiBa § rt dQ °
S 3tt or °s Wi WHS3 4> 12 bo2 do rt2 8 J S SfldEt
110
100 -
9 0 -
80 -
70 -
GO10 20 300
Tim e (m in.)
Figure 28. Comparison of the w ater loss of ferric iron alginate gels of Macrocystis alginate (D), acetylated P. syringae alginate (♦ h a n d deacetylated P. syringae alginate (I), com pared to calcium alginate gels as a function of incubation time at 50°G
96
concentration of 5 mM (Fig. 29). Cupric ion reacted m uch the same
way, precipitating 98% of the deacetylated bacterial alginate a t 2.5
mM copper, and greater than 90% of acetylated bacterial alginate a t
a m etal concentration of 5 mM (Fig. 30). Lead induced gelation of
bacterial alginates was least affected by acetylation. G reater than
90% of b o th ace ty la ted a n d d eace ty la ted b ac te ria l alginates
precipitated in the presence of 1 mM lead chloride (Fig. 31). Ferric
ions p rec ip ita te d b o th ace ty la ted an d d eace ty la ted b ac te ria l
alginate. Acetylation increased the precipitability of the bacterial
po lym er by ferric ion. A pproxim ately 90% of the ace ty la ted
b ac teria l po lym er p rec ip ita ted w ith 1 mM ferric ch loride. In
con trast, 90% of the deacety la ted bacteria l alginate p recip ita ted
with 2 mM ferric chloride (Fig. 32).
A high affinity of calcium ions for polyguluronate residues is
the basis for the cu rren t gelling theory (the "egg box model") for
seaw eed alginates. As expected, alm ost 100% of the seaw eed
alg inate was p rec ip ita ted by 5 mM calcium ch lo ride (Fig. 33).
Calcium p rec ip ita tio n was less efficient for b ac teria l alg inates,
p robab ly due to the absence of extensive polyguluronate blocks.
The am o u n t of bac teria l a lg inate p rec ip ita ted by calcium ions
increased as the calcium concentration increased. A pproxim ately
95% of the deacetylated bacterial alginate and 65% of the acetylated
bacterial alginate precipitated with 60 mM calcium chloride.
As m ight be expected from its chemical sim ilarity to calcium,
stron tium was also an effective precip itan t of bo th acetylated and
deacetylated bacterial alginate. As with calcium, strontium showed
a g re a te r ab ility to p rec ip ita te d eace ty la ted th a n ace ty la ted
97
■0$Bao0kH&3rtabjo<
100
80 -
60 -
4 0 -
20 -
103 84 (> 7 90 1 2
U ran iu m ch lo rid e (mM)
Figure 29. Precipitation of Macrocysns alg inate (D), acety la ted P. syringae alginate (♦), an d deacetylated P. syringae a lg inate (I) by uran ium ions. Relative precipitation is expressed as the % alginate precipitated by U&+- ions.
98
3 Bao
£$rtaao<
120
100 -
80 -
60 -
4 0 -
20 -
0 +0 2 64 8 10
C upric ch lo rid e (mM)
Figure 30. Precipitation of Macrocysns alginate (D), acety la ted P. syringae alginate (♦), and deacetylated P. syringae a lg inate (I) by copper ions. Relative p recip itation is expressed as the % alginate precipitated by Cu2+ ions.
99
’O$B‘8.ooWn&B«aGJO<
120
100 -
80 -
GO -
4 0 -
20 -
0 ■*0 1 2 4
Lead ch lo rid e (mM)
Figure 31. Precipitation of Macrocystis alg inate (D), acety la ted P. syringae alginate (♦), and deacetylated P. syringae a lg inate (I) by lead ions. Relative p rec ip ita tion is expressed as th e % alg inate precip itated by Pb^+ ions.
100
■oB$Gk-o<bu
O h
O4->(0a&0VN
<
100
80 -
6 0 -
4 0 -
20 -
7 4 6 8 10
Ferric ch lo rid e (mM)
Figure 32. Precipitation of Macrocystis alginate (□), acety lated P. syringae alginate (♦), and deacetylated P. syringae a lg inate (I) by ferric ions. Relative p recip ita tion is expressed as the % alginate precip itated by F e ^ ions.
101
*0B&Q.oOb*a,Bcadbo<M
100
80 -
60 -
4 0 -
20 -
0 •#0 20 6040 80
C alcium ch lo rid e (mM)
Figure 33. Precipitation of Macrocystis alg inate (D), acety lated P. syringae alginate (♦), and deacetylated P. syringae a lg inate (I) by calcium ions. Relative precip itation is expressed as the % alginate precipitated by Ca^+ ions.
102
bacterial alginate. S trontium precipitated 100% of the deacetylated
bacterial alginate a t a final concentration of 20 mM. Approxim ately
78% of the acetylated bacterial alginate was p rec ip ita ted w ith 60
mM strontium chloride (Fig. 34).
The second set of cations were those tha t precip itated half the
available bacterial alginate a t a concentration of g rea te r th an 20
mM m etah . Zinc ions fell in to this category. Zinc p rec ip ita ted
d e a c e ty la te d b a c te r ia l a lg in a te b e t te r th a n its a c e ty la te d
counterpart. The precipitation of both acetylated and deacetylated
bacterial alginate d id no t begin until zinc concentrations were above
10 mM. The concen tra tion of p rec ip ita ted b ac teria l a lg inate
increased as the concen tration of zinc increased. A pproxim ately
55% of the acetylated polym er precipitated in 60 mM zinc chloride,
an d 95% of the deace ty la ted polym er p rec ip ita ted a t th e sam e
m etal concentration (Fig. 35). It appears th a t acetylation decreased
the precipitability of alginate by zinc ions.
M anganese an d cobalt ions show ed a lim ited affin ity for
deacety la ted bacteria l alginate an d no affinity fo r the acetylated
po lym er. M anganese p rec ip ita ted 100% of th e d eace ty la ted
bacterial polym er a t a concen tration of 75 mM (Fig. 36). Cobalt
p rec ip ita ted approx im ately 87% of the d eace ty la ted b ac te ria l
polym er a t th a t concen tra tion (Fig. 37). A cetylation com pletely
in h ib ite d th e ab ility of m anganese an d co b a lt to precipitate
bacterial alginate. N either acety lated n o r deacety lated bacteria l
alg inate p rec ip ita ted w ith cesium, rub id ium , o r m agnesium ions
(Fig. 38 ,39).
103
120
100T>$S 80a,o<L>
3rta 40&o<M 20
00 20 40 GO 80
S tro n tiu m ch lo rid e (mM)
Figure 34. Precipitation of Macrocystis alg inate (D), acety la ted P. syTingae alginate (♦), an d deacetylated P. syringae a lg inate (I) by strontium ions. Relative precipitation is expressed as the % alginate precip itated by Sr^+ ions.
104
•o$JS•pN&•HoOlM&
coo&o
pN<
120
100 -
8 0 -
60 -
4 0 -
20 -
200 40 60 80
Zinc ch lo rid e (mM)
Figure 35. Precipitation of Macrocystis alg inate (0), acety la ted P. syiingae alginate (♦), and deacetylated P. syTingae a lg inate (I) by zinc ions. Relative p rec ip ita tio n is expressed as the % alginate precipitated by Zn^+ ions.
105
x?B BoOu&Brta•̂ 4ao<
120
100 -
80 -
6 0 -
4 0 -
20 -
-200 50 100 150 200 25 0
M anganese ch lo rid e (mM)
Figure 36. Precipitation of Macrocystis alginate (0), ace ty la ted P. syringae alginate (♦), and deacetylated P. syringae a lg inate (I) by m anganese ions. Relative p rec ip ita tion is exp ressed as the % alginate precipitated by Mn^+ ions.
106
•0$Bf t•vMo4kn&&rtaeuo<
120
100 -
80 -
GO -
4 0 -
20 -
-200 50 100 150 200
C obalt ch lo rid e (mM)
Figure 37. Precipitation of Macrocystis alginate (□), acety la ted P. syringae alginate (♦), an d deacetylated P. syringae a lg inate (I) by cobalt ions. Relative p recip itation is expressed as the % alginate precipitated by Co2+ ions.
107
■eis5a»HokH6
i s
a&0fN<
3
1
10 20 40 60 80
Cesium ch lo rid e (mM)
Figure 38. Precipitation of Macrocystis alg inate (0), acety lated P. syiringae alginate (♦), and deacetylated P. syTingae alg inate (i) by cesium ions. Relative precip itation is expressed as the % alginate precipitated by Csl+ ions.
108
■o8 8aoOa8rtaCUD
<M
100
90
80
70
60
50
40
30
20
0 10 3020 40 50 60 70
M agnesium ch lo rid e (mM)
Figure 39. Precipitation of Macrocystis a lg inate (□), acety la ted P. syringae alginate (♦), and deacetylated P. syringae a lg inate (I) by m agnesium ions. Relative p rec ip ita tion is expressed as the % alginate precipitated by Mg 2+ ions.
109
The relative ability of these m etal ions to precip itate alginates
w ere com pared to the P i /2 values fo r seaw eed a lg in a te an d
reco rded as the fold difference from those values (Table 12). The
P l /2 value is defined as the concen tra tion of m etal ions (mM)
requ ired to precipitate 50% of a polym er from a 400 iig/m l (w/v)
solution. The re la tive o rd e r of p rec ip ita tion of ace ty la ted an d
deace ty la ted bac teria l alg inate by these ions, as d e te rm in ed by
P l /2 , were as follows:
A cetylated bacterial alginate: Pb2+=Fe3+=u6'1- > Cu2+ > Sr2+ > Ca2+ >
Zn^+ > Mg 2+ =Co 2+ =Cs 1+=Mn 2+ =Rb 1+
Deacetylated bacterial alginate: Pb2+=Fe3+=U(5+ > Cu2+ > Sr2+ > Ca2+ >
Zn2+ > Mn2+ > Cq2+ > Mg 2+ =Cs 1+=Rb 1+
110
Table 12. The Precipitation of Macrocystis Alginate and Acetylated and Deacetylated P. syringae Alginate by Metal Ions
Fold Increase from P i / 2 a of Macrocystis Alginate^
Ions Periodic Macrocystis Acetylated Deacetylated Group Alginate P. syringae P. syringae
(P l/2 )c Alginate Alginate
Csl+Rbl+
I A No affinity No affinity
No affinity No affinity
No affinity No affinity
Mg 2+ Ca2+Sr2+
II A No affinity 3.1 1.8
No affinity 3.4 3.7
No affinity 1.7 1.1
Mn2+ VII A 30.0 No affinity 0
pe3fCb2+
VIII A 1.89.6
-0.7 No affinity
-0.63.7
Cu2+ I B 0.5 1.6 2.0
Zn2+ II B 7.0 4.3 2.3
Pb2+ IV B 0.5 0 0.2
U&f Actinidem etal
0.9 -0.2 -0.4
a P l /2 is the co n cen tra tio n of m etal ions (mM) req u ired to p rec ip ita te 50% (w /v) a lg inate from 400 [.ig/ml (w /v) alg inate solutions.b V alues exp ressed a re th e fo ld d iffe ren ce fro m P l/2 o f Macrocystis alg inate (positive values = less ability to precipitate, Negative values = g reater ability to precipitate, zero values = equal ability to precipitate).c " No affinity" signifies th a t the ion d id no t precipitate 50% of the alginate sample up to 100 mM ion concentration.
DISCUSSION
Alginate biosynthesis is a common characteristic of a m ajority
of pseudom onads in rRNA-DNA hom ology group I (94). This group
in c lu d e s a ll th e f lu o re s c e n t a n d a few n o n f lu o re s c e n t
pseudom onads. Until recently, only certain strains of P. aeruginosa
isolated alm ost exclusively from cystic fibrosis patients, and strains
of A. vinlandii were known to produce O-acetylated alginate as EPS
(67). The fluorescent pseudom onads, P. fluorescens , P. mendocina,
an d P. pu tida , w ere subsequently found to p roduce alginate, bu t
o n ly u n d e r co n d itio n s o f stress (bac terioc in , b ac te rio p h ag e ,
an tib io tic , 53, 58). W ithin the p a s t seven years, m any p la n t
pathogenic fluorescen t pseudom onads have been recogn ized as
alginate producers u n d er the appropriate conditions. This strongly
in d ica tes th a t th e ab ility to syn thesize a lg inate m ay have an
im portan t evolutionary role.
L ittle is know n ab o u t th e syn thesis of a lg in a te by th e
p h y to p a th o g en ic p seu d o m o n ad s. P seu d o m o n a s syringae p v
phaseolicola ATCC 19304 produces high am ounts of O -acetylated
alginate in vitro. The am ount of alginate, as well as the degree of
acetylation of the polym er, varied with carbon source. P. syringae
ATCC 19304 grown on sucrose produced a mixed population of EPS.
A lthough alginate was the p redom inan t EPS produced, P. syringae
ATCC 19304 also produced levan (C-2 -» C-6 fructan backbone) on
sucrose (57). Levan production indicates th a t the bacterium also
has the ability to synthesize levansucrase. Sucrose was no t utilized
as the carbon source in this research since the presence of levan in
111
112
the exopolym er extracts m ade purification of the alginate m ore
difficult.
Gluconic acid was chosen as the carbon source in this work
because cells grown on gluconic acid produced approxim ately 21%
m ore alginate th an cells grown on glucose. It is well estab lished
th a t the p rim ary rou te of glucose catabolism in pseudom onads is
th rough the Entner-D oudoroff pathway, which produces pyruvate
an d glyceraldehyde 3-phosphate (52). Glucose conversion to 6-
ph o sphog luconate is re q u ire d p rio r to en try in to th e E ntner-
Doudoroff pathway. This conversion can be accom plished in one of
two ways. Glucose can be phosphorylated to glucose 6-phosphate
by a hexokinase and subsequently oxidized to 6-phosphogluconate
by a glucose 6-phosphate dehydrogenase (148), o r glucose can be
converted to gluconate a t the surface of the cell by an NAD(P)
d ep en d en t glucose dehydrogenase followed by gluconolactonase.
G luconate is th en oxidized to 2-ketogluconate by a m em brane
bound gluconate dehydrogenase during transport of the sugar into
the cell. Once inside the cell the 2-ketogluconate is phosphorylated
to 6-phosphogluconate (47). Most pseudom onads, i.e., P. aeruginosa,
P. fluorescens, an d P. putida oxidize glucose to gluconate using the
second m echanism prior to transport of the sugar into the cell (52).
The increased production of alginate by gluconate grown cells over
glucose grow n cells p ro b ab ly resu lts from a decreased energy
req u irem en t fo r the enzym atic oxidation of glucose to gluconate
p rio r to transport into the cell.
In P. aeruginosa, the C-6 of g luconate in co rp o ra te s into
alginate (13). Carbon atom s 1, 2, and 3 of gluconate are converted
113
to pyruvate through the 2-keto 3-deoxyphosphogluconate aldolase
reaction an d are eventually lost as CO2 and acetyl CoA. Carbon
a to m s 4, 5, a n d 6 a re ch a n n e le d in to a lg in a te th ro u g h
g lyceraldehyde 3-phosphate. G lyceraldehyde 3 -phosphate can
condense w ith d ihydroxyacetone phosphate to p roduce fructose
1,6-diphosphate and ultim ately fructose 6-phosphate, w hich is the
starting m aterial for alginate biosynthesis (7, 78). Besides being a
source of fructose 6-phosphate, gluconic acid can also be oxidized to
acetyl CoA (7). Acetyl CoA is reported ly the source of the O-acetyl
groups on the m annosyl residues of xan than gum (66), and is the
probable source of acetyl in bacterial alginates (136).
P. syringae ATCC 19304 utilized am m onia as a n itrogen source
fo r grow th. It was unab le to use n itra te , n itr ite , o r u rea. All
pseudom onads p ro d u ce energy by resp ira tion . In som e cases,
n itra te can be used as an a lte rn a te e lec tron accep to r allowing
grow th to occur anaerobically . Those pseudom onads th a t can
conduct n itra te resp ira tio n can reduce n itra te beyond the toxic
n itrite stage to m olecular n itrogen by "denitrification." P. syringae
was unab le to utilize n itra te as a n itrogen source and therefore
lacks the cellular m akeup to conduct n itra te respiration. This m ay
be due to the absence of cytochrom e C in the electron tran sp o rt
chain of P. syringae (94). In p lan ts , free am m onium ions are
p re se n t a t v ery low levels. A m m onium is toxic to the p lan ts,
because it inhibits the production of ATP in the m itochondrial and
photosynthetic electron tran sp o rt systems. Most n itrogen presen t
in plants is found associated w ith organic com pounds. In plants,
am m onium is converted in to organic com pounds prim arily through
114
three d ifferent reactions: 1) form ation of glutamic acid by reaction
w ith a-ketoglutarate, 2) form ation of g lutam ine by reac tion w ith
glutamic acid, and 3) form ation of carbam yl phosphate in arginine
b io syn thesis an d p y rim id in e b iosyn thesis . P. syringae is a
proteolytic bacterium (94). This bacterium probably provides itself
w ith am m onia through deam ination of the com ponent am ino acids
allowing the survival of the bacterium on the host leaf surface.
Polysaccharide production by m icroorganism s is enhanced in
n itrogen lim ited, high carbon conten t m edia (147). The changing
c a rb o n /n itro g e n ra tio ex h ib ited th e sam e effect on a lg ina te
p ro d u c tio n in P. syringae th a t has b een r e p o r te d fo r o th e r
p seu d o m o n ad s. H ig h er y ie ld s w ere o b ta in e d w ith h ig h
carbon /n itrogen ratios (low n itrogen content, high gluconate). It
has been proposed tha t lim itation of essential nu trien ts, in this case
n itro g e n , in h ib its g row th a n d d ire c ts th e co u rse o f to ta l
po lysaccharide b iosynthesis from cell w all m a te ria l (LPS a n d
peptidoglycan) to extracellular polysaccharide synthesis (133). It
was fu rth e r postu lated th a t during active growth the same pool of
isoprenoid lipid carriers involved in polysaccharide synthesis are
u tilized by cell wall an d LPS precursors (133). These conditions
d irec t a lg inate b iosynthesis tow ard p ro d u c tio n as a secondary
m etab o lite . In the case o f P. syringae ATCC 19304, a lg inate
biosynthesis d id no t fully fit this hypothesis. A lginate p roduction
by P. syringae is necessary for successful colonization of p lan t host
tissue (113). A lthough alginate p roduction d id inc rease as the
ca rb o n /n itro g en ratio increased , a lg in a te was p ro d u ced by P.
syringae coincidental with cellular reproduction.
115
Bacteria secrete EPS for m any different reasons. Bacterial EPS
m ay aid colonization (16, 50), prevent desiccation, store energy, or
help concentrate and take up charged molecules, particu larly m etal
ions (11, 17, 46). In cystic fibrosis patien ts P. aeruginosa secretes
alg inate to help colonize the lungs an d to p ro tec t itself against
phagocytosis. P. syringae produces alginate to aid in colonization
and parasitization of leaf surfaces (30, 138).
The ab ility of P. syringae to p roduce alg inate decreased
ra p id ly u p o n successive tra n sfe rs in liqu id m edia. A lginate
p ro d u ctio n rem ain ed stable upon tran sfe r on solid m edia. The
pathogenicity of P. syringae is dependen t upon its ability to produce
alginate (113). Colonization of a leaf surface by P. syringae causes
halo blight (leaf spots) o f the leaf. Alginate m ust be p re se n t to
p roduce leaf spots (34). These leaf spots are p robab ly due to an
in te rru p tio n of th e p h o to sy n th e tic pathw ay in th e leaf. All
pseudom onads respire, and thus m ust have a source of ferric iron
fo r synthesis of cytochrom es. On the leaf surface, the sources of
ferric iron are lim ited to ferredoxin and the cytochrom e b f complex.
The b reak d o w n of fe rred o x in o r the cy tochrom e b f com plex
in te rrup ts photosynthesis by stopping electron tran sp o rt an d aids
in leaf spotting. A cetylated P. syringae a lg inates have a h igh
affinity for ferric iron. By increasing the ferric iron concentration in
the growth m edia alginate production decreased. The parasitization
o f P. syringae ATCC 19304 m ay be due to th e ab ility of the
acetylated alginate to scavenge the ferric iron from ferredoxin or
the cytochrom e bf complex. By scavenging the iron, photosynthesis
116
w ould be in te rru p te d a n d leaf spots w ould occur due to the
inability of the p lant to make energy for chlorophyll biosynthesis.
The n a tiv e a lg in a tes of P. syringae ATCC 19304 w ere
polydisperse. This in d ica ted th a t th e re was a h igh deg ree of
variability in size of the alginate p roduced by the biological system.
The Mw for the native bac teria l polym er was 1.3 x 1 0 $. Upon
deacetylation, the Mw dropped approxim ately 6 % to 1.2 x 105. This
d rop was due to the loss of the acetyl groups at C-2 a n d /o r C-3 of
the m annuron ic acid residues w ithin the polym er. By monitoring
the difference in the acid stability of D-mannose an d L-gulose from
the acid hydro lyzed , red u ced alginates, m ore accu ra te a lg inate
com positions were obtained for the bacterial polym er. The alginate
p roduced by P. syringae ATCC 19304 had a final com position of 82%
m annuronic acid and 18% guluronic acid. This com position was in
con trast w ith the previously rep o rted com position of g rea ter th an
95% m annuronic acid an d less th an 5% guluronic acid w here the
relative acid sensitivity of each sugar was no t considered (38).
Both the solution an d gelling properties of bacterial alginate
w ere a lte red by the degree of acety lation on the polym er. The
v isc o s ity o f th e a c e ty la te d p o ly m e r was h ig h e r th a n th e
d e a c e ty la te d b a c te r ia l o r seaw eed a lg in a te s a t e q u iv a le n t
concentrations. The native bacterial alginates were acety lated with
an average of 1.2 acety l un its p e r u ron ic acid resid u e . This
p ro d u ced a 6 p e rcen t increase in Mw an d a 26% increase in
viscosity a t a concen tra tion of 1 m g/m l. A cetylation rep o rted ly
increases the viscosity of alginate solutions (129). These increases
m ay be d u e to th e increase in to ta l m olecu lar w eight of the
117
polym er, o r a lte red conform ations of the po lym er in aqueous
solution. Alginates high in m annuronic acid exhibit a flat ribbon
like conform ation. High am ounts of gu lu ronate p roduce a m ore
puckered structu re . It is possible th a t the in troduction of acetyl
groups shifts the conform ational energy w ithout increasing the
accessible surface of the molecules, eventually producing a flexible
polym er with an increased num ber of conform ations. Because the
acety la ted po lym er show ed a 4 fold increase in v iscosity over
m olecular weight, it is probable tha t both factors play a role.
A cetylation a lte red the affin ity of th e po lym er fo r m any
cations, including calcium. Except for ferric an d to som e ex ten t
copper ions, the ability of m ultivalent cations, particularly calcium,
to induce gelation of th e alginate was reduced in the acety lated
polym er. In parallel, there was a m arked decrease in the strength
of the gels. The effects of acetylation on the gelling p roperties of
bacteria l alginates were seen in the w ater holding capacities, the
surface tensions, and the relative porosities of the Ca-alginate gels.
Acetylation profoundly affected the rigidity of the Ca-alginate
gels and the ir ability to hold w ater. Generally, the volum e of an
ionic gel is d ep e n d en t upon a positive osm otic p ressu re . The
osmotic pressure of the gel is due m ainly to the positive en tropy of
the mixing of counterions with water, which is counterbalanced at
equilib rium by a negative p ressu re due to th e elasticity of the
netw ork (139). For Ca-alginate gels, which are en thalp ic ra th e r
th a n en tro p ic (3), the elastic ity d ep en d s on the n u m b er an d
strength of the crosslinks. Since the in troduction of acetyl groups
im pairs the cooperative b inding of calcium ions, the num b er of
118
dissociated counterions p er polymer chain increases with increasing
degree of acetylation. The high num ber of dissociated counterions
enhances the positive osm otic p ressu re . At th e sam e tim e, it
weakens the forces holding the network together. Reduction in the
cooperative binding of calcium ions reduces bo th the streng th and
the num ber of crosslinks in the network. Fewer crosslinks lessen
the surface tension on the gel and as a resu lt allows h igher w ater
holding capacities w ithin the gel network. This was observed w ith
th e P. syringae alginate. A cetylated bacterial alg inate gels h ad a
m uch low er surface tension , an d as a re su lt a w ate r ho ld in g
cap ac ity th a t was ap p ro x im ate ly 68 p e rc e n t h ig h e r th a n its
deace ty la ted co u n terp art. Fe-alginate gels an d Pb-alginate gels
showed the same general properties, however neither the ferric no r
lead alginate gels held as m uch water as the Ca-alginate gels. These
differences are attribu tab le to the relative affinities of each cation
to the alginate. Ferric an d lead ions h ad a h igher affin ity th an
calcium ions for both acetylated and deacetylated alginates.
Because of its h igher viscosity and lower affin ity for m any
polyvalent cations, acetylated bacterial alginate m ay be a possible
substitu te for seaweed alginate in m any applications. More viscous
so lu tions can be m ade w ith low er co n cen tra tio n s of bacterial
a lg in a te th a n w ith seaw eed a lg inate , red u c in g th e p o ly m er
requ irem en ts in a given application. By varying the degree of
acetylation, it should be possible to produce alginates with designer
properties.
As a polyelectrolyte, alginate has the potential to concentrate
toxic, heavy a n d /o r valuable m etals from the environm ent. The
119
rem o v al of m etals by m icroorganism s rep o rted ly d ep en d s on
p hysicochem ica l ad so rp tio n to ce llu la r co m p o n en ts such as
polysaccharides and p ro te ins (84). Because of the h igh negative
c h a rg e on th e ce ll w alls o f b a c te r ia , fu n g i, a n d a lgae,
m icroorganism s have been used to concentrate m etal ions including
uran ium , copper, m anganese, cadm ium , m olybdenum , gold, and
m ercu ry (20, 56, 65). The polyanionic n a tu re of alg inates also
allows the binding and concentration of heavy m etals through m etal
induced gelation of the polym ers. The form ation of alginate gels in
the presence of polyvalent cations is a ltered by acetylation of the
alginate, as well as the native conform ation of the polym er. The
physical binding of calcium ions to guluronate residues in seaweed
alginate is due to charge charge in teractions betw een the positive
charges from calcium an d the negative charges from the carboxyl
groups. The size of calcium ions is such th a t they fit in to the space
form ed by the polyguluronate stretches of seaweed alginates (83).
Randomly organized bacterial alginate also gels in the presence of
m any polyvalent cations. This casts some dou b t on the principal
tenan t of the "egg-box" theory; tha t only polyguluronate residues in
a lg inate p lay a key ro le in gelation . The ab ility of bac te ria l
alginates, w ithout polyguluronate blocks, to gel in the presence of
calcium indicates a reevaluation of this theory is necessary.
M ultivalent cations in te rac t d iffe ren tly w ith alg inates (59,
60). Each ca tion exam ined show ed varia tions in its ab ility to
p recip ita te seaweed and bacterial alginate. With the exception of
fe r r ic a n d u ra n iu m ions, p o ly v a len t ca tio n s m ore re a d ily
p rec ip ita ted seaweed alginate over its bacteria l co u n te rp art. In
120
m ost instances, deacetylation of the bacterial polym er enhanced its
ca tion p rec ip itab ility . This ab ility to ad ju s t th e affin ities of
alginates for specific polyvalent cations increases the feasibility of
using th is polym er to rem ove toxic m etals, i.e., lead o r u ran ium
from drinking water.
Acid h y d ro ly s is is u sed to d ep o ly m erize po ly m ers for
com positional analysis. Ideally, hydro lysis goes to com pletion
w ithout loss of the com ponents. Practically, however, some sugar
loss occurs an d m ust be accounted fo r to accurate ly determ ine
polysaccharide compositions. Com positional analysis of alginates
re q u ire s th e red u c tio n of th e u ro n ic ac id res id u es to th e ir
co rresp o n d in g n e u tra l sugars p rio r to acid hydro lysis. This
reduction facilitates the acid hydrolysis of the glycosidic bonds by
converting the acid resistan t glycosyluronic acid bonds to the m ore
acid labile glycosyl bonds. Reduction of alginate polym ers liberates
D-mannose and its C-5 epim er L-gulose after acid hydrolysis. Both
D-mannose and L-gulose react differently u n d er acid conditions. D-
M annose is re la tive ly acid stab le. L-gulose is m ark ed ly acid
sen sitiv e . In general, aldose con ta in ing p o ly saccharides a re
com pletely hydrolyzed w ith m inim al sugar loss by 1 M H2SD4 a t
100°C for 5 hours (119).
At equ ilib rium D -m annose resid u es favor th e ch a ir
confo rm ation while the L-gulose residues ad o p t th e IC4 ch a ir
conform ation because th e bu lky g ro u p a t C-5 o rien ts to an
eq u a to ria l position . T hese sugars a re n o t locked in to these
conform ations. In so lu tion these m onom ers exist in a dynam ic
equilibrium . This equilibrium allows alternation betw een both ^C \
121
an d IC4 conform ations, as well as the boat, half-chair, an d open
conform ations. The open chain conform ation allows anom erization
around C-l producing both the a an d p anom er. This equilibrium
allows num erous conform ational possibilities for each m onom er
including the possible form ation of 1,6 anhydrohexopyranoses in
acid solutions.
Sugars of the gulo., ido., an d altro . configurations undergo
spontaneous conversion to 1,6 anhydrides in acids. Sugars of the
gluco., m anno., an d galacto. configurations produce very little 1,6
anhydride a t equilibrium and are alm ost com pletely hydrolyzed to
the free aldoses under acid conditions (4, 81). This is because of the
o rien ta tion of the hydroxyl groups a t C-2, C-3, an d C-4. Axially
o rien ted hydroxyl groups, particu larly a t C-3, destabilize anhydride
fo rm a tio n . E quato ria lly o rien ted hyd roxy l groups fav o r the
form ation of 1,6 anhydrides. The equilibrium is d ep en d en t on the
steric arrangem en t of hydroxyl groups which do n o t take p a r t in
th e re a c tio n . Once a 1,6 a n h y d rid e b o n d is fo rm ed , th e
conform ational equilibrium is shifted so the sugar no longer exists
in a dynam ic state. The form ation of a second ring w ith in the
molecule locks it into th a t particular conformation.
D -M annose, w hose fre e e n e rg y fav o rs th e 4 q l c h a ir
conform ation, does no t readily form the 1,6 anhydride bond when it
is in the I-C4 chair conform ation and the hydroxyl group attached to
th e an o m eric ca rb o n is in th e p p o sitio n . A lth o u g h th is
conform ation brings C-6 and the anom eric hydroxyl group into axial
positions around the ring and into proximity, this conform ation also
brings the hydroxyl groups a t C-2 and C-3 into axial positions which
122
destabilize an d inh ib it anhydride form ation. The axial hydroxyl
group a t C-3 in teracts w ith the potential anhydride bond blocking
its form ation. In the p conform ation D-mannose, D-glucose, and D-
galactose all have axial hydroxyl groups a t C-3 and therefore do no t
readily form 1,6 anhydrides.
T h e f re e e n e rg y o f L -gulose fav o rs th e 1Gj. c h a ir
conform ation. At dynam ic equilibrium it can also exist in the 4 c i
conform ation , w hich places the bulky group a t C-5 in an axial
orientation. If the hydroxyl group a t the anom eric carbon is in the
p position the C-6 and the anom eric hydroxyl group come into close
enough proxim ity to allow 1,6 anhydride form ation. An exception
is th a t the anhydride bond form s below the plane of the ring w ith
L-gulose ra th e r than above it. This orientation places the hydroxyl
groups a t C-3, and C-4 in equatorial positions a round the ring and
the hydroxyl group a t C-2 axial. In this position the free energy
favors 1,6 anhydride form ation due to the absence of a destabilizing
axial hydroxyl group a t C-3.
U pon ac id h y d ro ly s is , L-gulose lo ck ed in to a n e w
conform ation . This was seen by the increase in in ten sity of a
second spot on TLC over time. Although the acid hydrolysis product
o f L-gulose was n o t defin itive ly id en tified , the m olecule was
ten tatively iden tified as a 1,6 anhydride because it h ad the same
m igration as 1,6 anhydro p-D-m annopyranose, and 1,6 anhydro p-
D-glucopyranose on TLC. Com puterized m olecular m odeling of 1,6
anhydro p-L-gulopyranose using SYBYL software ind icated th a t the
m olecule has a low total energy, favorable for its p roduction and
stability.
123
Existing m ethods fo r de term ination of the com position of alginates recom m end hydrolysis in 1 M H2 SO4 for 90 m inutes at
100°C (38). Since L-gulose is m ore susceptible to conform ational
m odification u n d er these conditions than is D -m annose, lack of
accounting for gulose destruction has resulted in inaccurate reports
of composition.
Analysis of acid hydrolyzed reduced alginates paralleled those
o b ta in ed w ith the D -m annose and L-gulose m onom ers. W hen
correcting fo r gulose destruction, by extrapolation back to tim e 0 ,
the results of the seaweed alginate analysis correlated exactly with
the pub lished results of 60% m annuronic acid and 40% guluronic
acid as determ ined by the reductive cleavage m ethod of Zeller and
G ray (150). The a lg inates p roduced by th e phy topa thogen ic
p seu d o m o n ad s ap p a re n tly have a h ig h e r co n cen tra tio n of L-
guluronic acid than h ad been previously reported . The rep o rted
com position of the P. syringae ATCC 19304 alginate is g reater than
95% D -m annuronic acid an d less than 5% L-guluronic acid (38).
A fter co rrec tin g fo r th e re la tiv e acid sen sitiv itie s o f each
corresponding neutral sugar, the actual com position of the bacterial
a lg inate was 82% D -m annuronic acid and 18% L-guluronic acid.
This ra tio co rre la tes well to the re p o rte d com position of P.
aeruginosa alginate of 80% D-m annuronic acid and 20% L-guluronic
acid (150).
P. syringae ATCC 19304 produced large am ounts of acetylated
alginate w hen grown on gluconic acid. While p roduction of this
po lym er decreased dram atica lly on subculture in liquid m edia,
p roduction was stable on solid m edia. Maximum p roduction was
124
d ep e n d en t on the pH, tem pera tu re , an d a high carbon/nitrogen
ra tio w ithin the growth m edia. The Mw of the bacteria l polym er
was approxim ately 1.7 tim es larger th an the seaweed coun terpart
with a composition of 82% m annuronic acid and 18% guluronic acid,
and an average of 1.2 acetyl groups p er m onom er. These physical
characteristics allowed for m uch higher solution viscosities a t equal
concentrations, an d ca tion in d u ced gels w ith in c reased w ater
holding capacity. By controlling the degree of acety lation it was
possib le to con tro l th e so lu tion an d gelling p ro p e rtie s of the
polym er to some extent. The polyanionic n a tu re of the polym er
allows for the binding of m any toxic and heavy metals. Ultimately,
it should be possible to create inexpensive designer alginates whose
p ro p ertie s are co n tro lled by the degree of ace ty la tion on the
polymer.
REFERENCES
1. A lliso n , D. G., a n d M. J. M atth ew s. 1992. Effect of p o ly sacch arid e in te ra c tio n on an tib io tic su sep tib ility of Pseudomonas aeruginosa. J. Appl. Bacteriol. 7 3 :484-488.
2. A n d e rs o n , P., a n d D. H. S m ith . 1977. Iso la tio n of po ly sacch arid e from cu ltu re su p e rn a ta n t of Haemophilus influenza type B. Inf. and Imm. 15:472-477.
3. A n d re se n , I. L., a n d O. S m id sro d . 1977. T em pera tu re d e p e n d en c e on th e elastic p ro p e r tie s o f a lg in a te gels. Carbohydr. Res. 58:271-279.
4. A ngyal, S. J., a n d K. Dawes. 1968. Conform ational analysis in carbohydrate chem istry II. Equilibrium betw een reducing sugars an d th e ir glycosidic an h y d rid e s . Aust. J. Chem. 21:2747-2760.
5. A tk in s , E. D., a n d I. A. N ieduszynsk i. 1973. S tructuralcom ponents of alginic acid I. The crystalline structu re of polyp-D -m annuron ic acid . Results of X-ray d iffra c tio n an d polarized infrared studies. Biopolymer 12:1865-1878.
6 . A tk in s , E. D., a n d I. A. N ied u szy n sk i. 1973. S tructuralcom ponents of alginic acid II. The crystalline structu re of polya-L-guluronic acid. Results of X-ray diffraction and polarized in frared studies. Biopolymer 12:1879-1887.
7. B anerjee , P. V., V anags, R. I., C h a k rab a rty , A. M., an d P. K. M a itra . 1985. Fructose 1 ,6 -b isphosphate a ldo lase activity is essential for synthesis of alginate from glucose by Pseudomonas aeruginosa. J. Bacteriol. 1 6 1 :45 8-460.
8 . BeM iller, J. N. 1967. Acid catalyzed hydrolysis of glycosides. Adv. in Carbohydr. Chem. and Biochem. 22:25-108.
125
126
9. B erry , A., D eV ault, J. D., R o y ch o u d h u ry , S., Z ielinski, N. A., May, T. B., W ynne, E. C., R othm el, R. K., F ialho, A. M., H ussein , M., K rylov, V., a n d A. M. C h ak rab a rty . 1988. Pseudom onas aeruginosa in fection in cystic fibrosis: m o lecu lar ap p ro ach es to a m edical problem . Chimicaoggi 5:13-19.
10. B lu m en k ran tz , N., a n d G. A sb o e-H an sen . 1973. New m ethod for quantita tive determ ination of uronic acids. Anal. Biochem. 54:484-489.
11. B uckm ire, F. L. A. 1984. Selective accum ulation of ions in cellu lar com ponents of ind iv idual cells w ith in colonies of Pseudomonas aeruginosa. Microbios Letters 22:151-162.
12. C ap o n , B. 1963. In tra m o le c u la r ca ta ly sis in glycoside hydrolysis. Tetrahed. Letters 14:911-913.
13. C arlson , D. M„, a n d L. W. M atth ew s. 1966. Polyuronic ac id s p ro d u c e d b y Pseudom onas aeruginosa. Biochem . 5:2817-2822.
14. C h itn is , C. E., a n d D. E. O h m an . 1990. C loning of P seudom onas aeruginosa algG, w hich co n tro ls a lg in a te structure. J. Bacteriol. 172:2894-2900.
15. C o n rad , H. E., B am berg , J. R., Epley, J. D., a n d T. J. K ind t. 1966. The s tru c tu re o f aerobacter aerogenes A3 polysaccharide II. Sequence analysis and hydrolysis studies. Biochem. 5:2808-2817.
16. C orpe, W. A. 1980. M icrobial surface com ponents involved in adsorption of m icroorganism s onto surfaces. In Britton, G., and K. C. M arshall (eds.), A dsorption of m icroorganism s onto surfaces, p. 106-135. Wiley Interscience, New York.
17. C o sterto n , J. W., G eesey, G. G., a n d K. J. C heng. 1978. How bacteria stick. Sci. Amer. 238:86-95 .
18. C osterton , J. W., Cheng, K. L., Geesey, G. G., Ladd, T. I., N ickel, J. C., D asg u p ta , M., a n d T. J. M arrie . 1987. Bacterial biofilms in n a tu re and disease. Ann. Rev. Microbiol. 41:435-464.
127
19. C o ttre l l , I. W., a n d P. K ovacs. 1980. A lg inates. In Davidson, R. L. (ed.), Handbook of w ater soluble gum s an d resins, p. 1-43. McGraw-Hill, New York.
20. D arn a ll, D. W., G reene , B., H enzl, M. T., H osea, J. M., M cPherson , R. A., S n ed d o n , J., a n d M. D. A le x a n d e r . 1986. Selective recovery of gold an d o th e r m etal ions from algal biomass. Environ. Sci. Technol. 20:206-208.
21. D av idson , J. W., S u th e r la n d , I. W., a n d C. I. Law son.1977. Localization of O-acetylated groups. J. Gen. Microbiol. 98:603-606.
22. D avies, D. G., C h a k ra b a r ty , A. M., a n d G. G. G eesey. 1993. Exopolysaccharide production in biofilms: substra tum ac tiv a tio n of a lg in a te gene ex p ress io n by Pseudomonas aeruginosa. Appl. Environ. Microbiol. 59:1181-1186.
23. D aw es, E. A., a n d P. J. S en io r . 1973. The ro le and regu la tion of energy reserve polym ers in m icroorganism s. Adv. Microbial Physiol. 10:135-266.
24. D elb en , F., C esaro , A., P ao le tti, S., a n d V. C resen z i. 1982. M onom er com position of ace ty l c o n te n t as m ain d e te rm in a n ts o f th e io n iza tio n b e h a v io r o f a lg in a tes . Carbohydr. Res. 10Q:C-46-C-50.
25. D e n t in i , M ., C re s c e n z i, V., a n d D. B las i. 1984. Conform ational p roperties of xan than deriva tives in d ilu te aqueous solution. Int. J. Biol. Macromol. 6:93-98.
26. D ere tic , V., Gill, J. F., a n d A. M. C h a k ra b a r ty . 1987. G ene algD cod ing fo r GDP m an n o se d eh y d ro g e n a se is tra n sc r ip tio n a lly a c t iv a te d in m u c o id Pseudomonas aeruginosa. J. Bacteriol. 169:351-358.
27. D ere tic , V., G ill, J. F., a n d A. M. C h a k ra b a r ty . 1987. Alginate biosynthesis: a m odel system for gene regulation and function in pseudom onads. Biotechnology 5 :469-477.
128
28. D eretic , V., D ikshit, R., K onyecsni, W. M., C h a k ra b a rty , A. M., a n d T. K. M iskra . 1989. The algR gene, w hich regulates m ucoidy in Pseudomonas aeruginosa, belongs to a class o f en v iro n m e n ta lly resp o n siv e genes. J. Bacteriol. 171:1278-1283.
29. DeVos, P., G oor, M., G illes, M., a n d J. DeLey. 1985. R ib o so m al r ib o n u c le ic a c id c is t ro n s im ila r i t ie s of phytopathogenic Pseudomonas species. Int. J. Syst. Bacteriol. 35:169-184.
30. D rigues, P., D em ery -L affo rg u e , D., T rig a le t, A., D upin , P., S am ain , D., a n d J. A sse lin ea u . 1985. Com parative studies of lipopolysaccharide an d exopolysaccharide from a v iru len t strain of Pseudomonas solanacearum an d from three avirulent m utants. J. Bacteriol. 162:504-509.
31. D ubois, M., Gilles, K. A., H am ilton , J. K., R ebers, P. A., a n d F. S m ith . 1956. Colorimetric m ethod fo r determ ination of sugars and rela ted substances. Anal. Chem. 28:350-356.
32. D w orkin , M., a n d J. W. F oste r. 1958. Experim ents w ith some m icroorganism s which utilize e thane an d hydrogen. J. Bacteriol. 75:592-603.
33. Edw ard, J. T. 1955. Stability of glycosides to acid hydrolysis. Chemistry and Industry (London) p. 1102-1104.
34. El Banoby, F. E., a n d K. R udolph. 1979. Induction of w ater soaking in p lan t leaves by extrcellu lar polysaccharides from phytopathogenic pseudom onads and xanthom onads. Physiol. Plant Path. 15:341-349.
35. Eliel, E. L., A llin g er, N. L., A ngyal, S. S., a n d G. A. M o rriso n . 1965. C on fo rm atio n a l A nalysis, p . 351-432 . Interscience Publishers, New York.
36. F ea th er, M. S., a n d J. F. H a rris . 1965. The acid catalyzed hydrolysis of glycopyranosides. J. Org. Chem. 30:153-157.
37. F e tt, W. F., O sm an, S. F., F ish m an , M. L., a n d T. S. S ieb le s III. 1986. Alginate p roduction by p lan t pathogenic pseudom onads. Appl. Environ. Microbiol. 5 2:466-473.
129
38. F e tt, W. F., a n d M. F. D unn . 1989. Exopolysaccharides p r o d u c e d b y p h y to p a th o g en ic P seudom onas syringae pathovars in infected leaves of suseptible hosts. Plant Physiol. 89:5-9 .
39. F e tt, W. F., O sm an , S. F., a n d M. F. D u n n . 1989. C haracterization of exopolysaccharides p roduced by p la n t a s so c ia ted f lu o re sc e n t p se u d o m o n ad s . A ppl. E nv iron . Microbiol. 55:579-583.
40. F ialho, A. M., Z ielinski, N. A., Fett, W. F., C h a k ra b a rty ,A. M., a n d A. B erry . 1990. D istribution of alginate gene sequences in th e Pseudomonas rRNA hom ology g roup I- Azom onas-Azotobacter lineage of superfam ily B prokaryotes. Appl. Environ. Microbiol. 56:436-443.
41. F ischer, F. G., a n d H. D orfel. 1955. Die polyuronsauren der b raunalgen (kohlenhydrate d e r algen I). Z. Physiol. Chem. Hoppe-Seylers 302:186-203.
42. F lynn, J. L., a n d D. E. O hm an. 1988. Cloning of genes from m ucoid Pseudomonas aeruginosa w hich con tro l spontaneous conversion to the alginate production phenotype. J. Bacteriol. 170:1452-1460.
43. F ly n n , J. L., a n d D. E. O h m an . 1988. Use o f gene rep lacem en t cosm id vector fo r cloning alg inate conversion genes from m ucoid and nonm ucoid Pseudomonas aeruginosa s tra in s : algS co n tro ls ex p ress io n o f algT. J. B acterio l. 170:3228-3236.
44. F ran k lin , M. J., a n d D. S. O hm an. 1993. Identification of algF in the alginate biosynthetic gene cluster of Pseudomonas aeruginosa w hich is re q u ire d fo r a lg inate acety la tion . J. Bacteriol. 175:5057-5065.
45. Fyfe, J. A., a n d J. R. W. G ovan. 1980. Alginate synthesis in m u co id Pseudom onas aeruginosa : a ch ro m o so m al locus involved in control. J. Gen. Microbiol. 119:443-450.
46. Fyfe, J. A., a n d J. R. W. G ovan. 1983. Synthesis, regulation an d biological function of b ac te ria l alg inate . Progress in industrial m icrobiology 18:45-85.
130
47. G acesa, P., a n d N. J. R ussell. 1990. The s tru c tu re and properties of alginate. In Gacesa, P., and N. J. Russell (eds.), Pseudomonas infection and alginates, p. 29-49. Chapm an and Hall, London.
48. Gill, J. F., D ere tic , V., a n d A. M. C h a k ra b a r ty . 1986. O v erp ro d u c tio n an d assay o f Pseudom onas aeruginosa phosphom annose isomerase. J. Bacteriol. 1 6 7 :611-615.
49. G o ld b erg , J. B., a n d D. E. O hm an . 1984. Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate. J. Bacteriol. 158:1115-1121.
50. G ordon, A. S., an d F. J. M iller. 1984. Electrolyte effects on a tta c h m e n t of an e s tu a r in e b ac te riu m . Appl. E nviron . Microbiol. 4 7 :495-499.
51. G orin , P. A. T., a n d J. F. T. S pencer. 1966. Exocellular alginic acid from Azotobacter vinlandii. Can. J. Chem. 44:993- 998.
52. G o ttsc h a lk , G. 1979. B acteria l M etabolism , p. 93-97. Springer-Verlag, New York.
53. G ovan, J. R. W., Fyfe, J. A. M., an d T. R. Ja rm a n . 1981. Iso lation of a lg ina te p roducing m u tan ts o f Pseudomonas fluorescens, P seudom onas p u tid a , a n d Pseudomonas mendocina. J. Gen. Microbiol. 1 2 5 :217-220.
54. G rasdalen , H., Larsen, B., a n d O. Sm idsrod . 1979. A P. M. R. study of the com position an d sequence of u ronate residues in alginates. Carbohydr. Res. 68:23-31.
55. G ra sd a le n , H. 1983. High field ^H-NMR spectroscopy of alg inate: sequen tia l s tru c tu re an d linkage conform ations. Carbohydr. Res. 118:255-260.
56. G reene, B., H enzl, M., H osea, J. M., a n d D. W. D arnall. 1986. Elimination of b iocarbonate interference in the binding of U(VI) in m ill w aters to freeze d ried Chlorella vugaris. Biotechnol. Bioeng. 28:764-767.
131
57. Gross, M., a n d K. R udolph. 1984. Partial characterization of slimes p roduced by Pseudomonas phaseolicola in vitro and in planta (Phaseolus vulgaris). Proc. of the 2nd Working Group on Pseudomonas syringae Pathovars. p. 66-67.
58. H acking, A. J., T ay lo r, I. W. F., Ja rm an , T. R., a n d J. R. W. G ovan . 1983. A lginate b iosyn thesis by Pseudomonas mendocina. J. Gen. Microbiol. 125:217-220.
59. H aug, A. 1961. The affin ity of some d iv a len t m etals to d iffe ren t types of alginates. Acta. Chem. Scand. 15:1794- 1795.
60. H aug, A., a n d O. S m idsrod . 1965. The effect of d ivalen t m etals on the properties of alginate solution. II. Com parison of different m etal ions. Acta. Chem. Scand. 19:341-351.
61. Haug, A., Larsen, B., a n d O. S m id sro d . 1966. A study of the constitution of alginic acid by partial acid hydrolysis. Acta. Chem. Scand. 20:183-190.
62. H aug, A., a n d B. L arsen . 1971. Biosynthesis of alginate; p a rt II. Polym annuronic acid C-5 epim erase from Azotobacter vinlandii. Carbohydr. Res. 17:297-308.
63. H eid t, L. J., a n d C. B. Purves. 1944. Therm al ra tes and activation energies for the aqueous acid hydrolysis of a - and p-methyl, phenyl, and benzyl-D-glucopyranosides, a- an d p- m ethyl an d p-benzyl-D -fructopyranosides, and a-m ethyl-D- fructofuranosides. J. Am. Chem. Soc. 66:1385-1389.
64. H e lle b u s t, J. A., a n d A. H aug. 1972. Photosynthesis, translocation, and alginic acid synthesis in Laminaria digitata and Laminaria hyperborea. Can. J. Chem. 5 0:169-176.
65. H o rik o sh i, T., N akajim a, A., a n d T. S ak ag u ch i. 1979. Uptake of u ran iu m by Chlorella regularis. Agric. Biol. Chem. 43:617-623.
6 6 . le lp i, L., Couso, R., a n d M. D an k e rt. 1981. Lipid-linked in term ediates in the biosynthesis of xan than gum. FEBS Lett. 130:253-256.
132
67. Ja rm a n , T. R. 1979. Bacterial alginate synthesis. In Berkely, R. C. W., Gooday, G. W., and D. C. Ellwood (eds.), M icrobial po lysaccharides a n d polysaccharases. p. 35-50. A cadem ic Press, New York.
6 8 . K ohn, R. 1975. Ion b inding on po lyuronates-alg inate and pectin. Pure Appl. Chem. 42:371-397.
69. K nutson , C. A., a n d A. Jeanes. 1968. A new m odification of th e carbazo le assay: application to heteropolysaccharides. Anal. Biochem. 24:470-481.
70. L arsen , B., a n d A. H aug. 1971. Biosynthesis of alginate: p a r t I. Com position an d structu re of alg inate p ro d u ced by Azotobacter vinlandii. Carbohydr. Res. 32:217-225.
71. Lee J. W. (L o u is ian a S ta te U n iv e rs ity ) . 1993. Personal communication.
72. L ee , J . W. (L o u is ia n a S ta te U n iv e r s i ty ) . 1993. A cetylation of seaw eed alg inate by Pseudom onas syringae. Ph.D. Dissertation, p. 43.
73. Lin, T. Y., a n d W. Z. H assid . 1966. Isolation of guanosine diphosphate uronic acids from a m arine alga, Fucus gardneri silva. J. Biol. Chem. 241:3283-3293.
74. Lin, T. Y., a n d W. Z. H assid. 1966. Pathway of alginic acidsynthesis in the m arine brown alga, Fucus gardneri silva. J.Biol. Chem. 241:5284-5297.
75. L in k e r , A., a n d R. S. Jo n es. 1964. A p o ly sacch arid e resem bling alginic acid from a Pseudomonas m icroorganism . Nature 204:187-188.
76. L inker, A., a n d R. S. Jones. 1966. A new po lysaccharideresem bling alginic acid isolated from pseudom onads. J. Biol.Chem. 241:3845-3851.
77. L u d e ritz , O., G m ein er, J., K ickhofen , H., M ay er, H., W estpha l, O., a n d R. W. W heat. 1968. Identification of D- m annosam ine and quinovosam ine in Salmonella an d re la ted bacteria. J. Bacteriol. 9 5 :490-494.
133
78. L ynn, A. R., a n d J. R. S okatch . 1984. Incorpora tion of iso tope from specifically labelled glucose in to a lg inate of P seudom onas aeruginosa a n d A zo tobac ter vinlandii. J. Bacteriol. 158:1161-1162.
79. M acGeorge, J., K orolik, V., M organ, A. F., A she, V., a n dB. H o llo w ay . 1986. T ra n sfe r o f a chrom osom al locus responsible for m ucoid colony m orphology in Pseudomonas aeruginosa is o la te d fro m cy stic f ib ro s is p a t ie n ts to Pseudomonas aerusinosa PAO. J. Med. Microbiol. 2 1 :331-336.
80. McComb, E. A., a n d R. M. M cCready. 1957. D eterm ination o f acety l in pec tin an d acety lated carbohydrate polym ers. Anal. Chem. 29:819-821.
81. Mills, J. A. 1955. Cyclic derivatives of carbohydrates. Adv. in Carbohydr. Chem. 1 0 :49-51.
82. M orris, E. R., Rees, D. A., a n d D. Thom . 1978. Chiroptical and stoichiometric evidence of a specific p rim ary dim erization process in alginate gelation. Carbohydr. Res. 66:145-154.
83. M orris, E. A., a n d D. A. Rees. 1980. Competitive inhibition of in te rchain in teractions in polysaccharide system s. J. Mol. Biol. 138:363-374.
84. N ak a jim a , A., H rik o sh i, T., a n d T. S ak ag u ch i. 1982. Recovery of u ran ium by im m obilized m icroorganism s. Eur. J. Appl. Microbiol. Biotechnol. 16:88-91.
85. N a rb ad , A., H ew lins, M. J. E., G acesa, P., a n d N. J. R u sse ll. 1990. The use of l^C-NMR spectroscopy to m onitor a lg inate b iosyn thesis in m ucoid Pseudom onas aeruginosa. Biochem J. 267:579-584.
8 6 . N a rb a d , A., G acesa , P., a n d N. J. R u sse ll. 1990. Biosynthesis of alginate. In Gacesa, P., and N. J. Russell (eds.), Pseudomonas infection and alginates, p. 181-205. C hapm an and Hall, London.
87. N eid h a rd t, F. C. 1990. The bacterial cell. In Sherris J. C. (ed.), Medical Microbiology: An Introduction to Infectious Disease, p. 11-48. Elsevier, New York.
134
8 8 . N ilsson , S. 1992. A therm odynam ic analysis of alginate gel form ation in the presence of in e rt electrolyte. Biopolymers 32:1311-1315.
89. O 'hoy, K., a n d V. K rishnap illa i. 1987. Recalibration of the Pseudomonas aeruginosa strain PAO chromosom e m ap in tim e units using high frequency of recom bination donors. Genetics 115:611-618.
90. O hm an , D. E., a n d A. M. C h a k ra b a r ty . 1981. G enetic m apping of chrom osom al determ inants for the p roduction of the exopolysaccharide alginate in a Pseudomonas aeruginosa cystic fibrosis isolate. Inf. and Imm. 33:142-148.
91. O sm an , S. F., F ett, W. F., a n d M. L. F ish m an . 1986. E xopo lysaccharides of th e p h y to p a th o g en Pseudomonas syringae pv. glycinea. J. Bacteriol. 166:66-71.
92. P a d g e t t , P. J . , a n d P. V. P h ib b s . 1 9 8 6 . P hosphom annom utase activ ity in w ild type a n d a lg in a te producing strains of Pseudomonas aeruginosa. Curr. Microbiol. 14:187-192.
93. Page, W. J., a n d H. L. Sadoff. 1975. Relationship betw een calcium and uronic acids in the encystm ent of Azotobacter vinlandii. J. Bacteriol. 122:145-151.
94. P alle ron i, N. J. 1984. Family I. Pseudom onadaceae. In Kreig, N. R., an d J. G. Holt (eds.), Bergeys m anual o f system atic bacteriology. Vol. 1. p. 141-219. The Williams and Wilkens Co. Baltimore, M aryland.
95. P a rso n s , A. B., a n d P. R. D ugan . 1971. P roduction of extracellular polysaccharide m atrix by Zooglea ramigera. Appl. Microbiol. 21:657-661.
96. Penm an, A., an d G. R. S a n d e rso n . 1972. A m ethod for the determ ination of u ronic acid in alginates. C arbohydr. Res. 25:273-282.
97. Percival, E., a n d R. H. McDowell, (eds.). 1967. Alginic acid. In C h e m is try a n d E n zy m o lo g y o f M a rin e Algal Polysaccharides, p. 99-126. Academic Press, London.
135
98. P ig g o tt, N. H., S u th e r la n d , I. W., a n d T. R. Ja rm a n .1981. Enzymes involved in the biosynthesis of alg inate by Pseudomonas aeruginosa. Eur. J. Appl. Microbiol. Biotechnol. 13:179-183.
99. P in d ar, D. F., a n d C. Bucke. 1975. Biosynthesis of alginic acid by Azotobacter vinlandii. Biochem. J. 15 2 :617-62 2.
100. P ra tt, J. W., R ichtm yer, N. K., a n d C. S. H udson. 1952. D- idoheptulose and 2,7 anhydro p-D- idoheptulopyranose. J. Am. Chem. Soc. 74:2210-2214.
101. P ra tt, J. W., R ich tm yer, N. K., a n d C. S. H udson . 1952. Proof of the structure of sedoheptulosan as 2,7 anhydro p-D- altroheptulopyranose. J. Am. Chem. Soc. 74:2200-2205.
102. P ra tt, J. W., R ich tm yer, N. K., a n d C. S. H udson . 1953. 1,6 A nhydro D-glycero p-D-ido-heptopyranose. J. Am. Chem. Soc. 75:4503-4507.
103. P ra tt, J. W., a n d N. K. R ic h tm y e r. 1954. P roduction of glycosans by the action of acid on sugars w ith alio and talo configurations. Abstract Papers, Am. Chem. Soc. 126:220-230.
104. P ra tt, J. W., a n d N. K. R ich tm y er. 1957. Crystalline 3- deoxy-a-D -ribohexose. P rep ara tio n an d p ro p ertie s o f 1,6 anhydro 3-deoxy-p-D-arabino-hexopyranose, 1,6 anhydro 3- deoxy-p-D-ribo-hexopyranose and re la ted com pounds. J. Am. Chem. Soc. 79:2597-2600.
105. P u g a sh e tti , B. K., V adas, L., P r ih a r , H., a n d D. S. F e in g o ld . 1983 . G D P-m annose d e h y d ro g e n a s e a n d biosynthesis of alginate-like polysaccharide in a m ucoid strain of Pseudomonas aeruginosa. J. Bacteriol. 153:1107-1110.
106. R am phal, R., a n d G. B. P ie r. 1985. Role of Pseudomonas aeruginosa mucoid exopolysaccharide in adherence to tracheal cells. Inf. and Imm. 47:1-4.
107. R anby, B. G., a n d R. H. M a rc h e ssa u lt. 1959. Inductive effects in the hydrolysis of cellulose chains. J. Polym er Sci. 36:561-563.
136
108. R anby , B. G. 1961. Weak links in polysaccharide chains as rela ted to modified groups. J. Polymer Sci. 53:131-140.
109. R ees, D. A. 1977. Shapely po lysaccharides. Biochem. J. 126:257-273.
110. Rees, D. A., M orris, E. R., Thom , D., a n d J. K. M adden .1982. Shapes and in te ractions of ca rb o h y d ra te chains. In Aspinall,G. (ed.), The Polysaccharides I. p. 195-281. Academic Press, New York.
111. R eeves, R. E. 1950. The shape of pyranoside rings. J. Am. Chem. Soc. 72:1499-1506.
112. R ich tm y er, N. K., a n d C. S. H u d so n . 1940. The ring structure of D-altrosan. J. Am. Chem. Soc. 6 2 :961-964.
113. R u d o lp h , K. 1978 . A h o s t sp ec ific p r in c ip le fro m Pseudomonas phaseolicola inducing w ater soaking in leaves. Phytopathol. Z. 93:218-226.
114. S a 'C o rre ia , I., D arz ins , A., W ang, S. K., B erry , A., an d A. M. C h a k rab a rty . 1987. Alginate biosynthesis enzym es in m u c o id a n d n o n m u c o id P seudom onas aeruginosa: O v e r p r o d u c t io n o f p h o s p h o m a n n o s e isomerase, phosphom annom utase, and GDP m annose pyrophosphorylase by overexpression of phosphom annose isom erase (p m i) gene. J. Bacteriol. 169:3224-3231.
115. S a d o ff , H. L. 1975. E ncystm en t a n d g e rm in a tio n in Azotobacter vinlandii. Bacteriol. Rev. 39:516-539.
116. S a k a g u c h i, T ., T su ji, T ., a n d a. N a k a jim a . 1979. Accum ulation of cadm ium by green m icroalgae. Eur. J. Appl. Biotechnol. 8:207-215.
117. S an d fo rd , P. A., a n d J. B aird . 1983. Industrial utilization of polysaccharides. In Aspinall, G. (ed.), The polysaccharides Vol. 2. p. 411-485. Academic Press, New York.
118. Schoeffel, E., a n d K. P. Link. 1933. Isolation of a and p-D- m annuronic acid. J. Biol. Chem. 100:397-405.
137
119. S e lv e n d ran , R. R., M arch, J. F., a n d S. G. Ring. 1979. Determ ination of aldoses and uronic acid conten t of vegetable fiber. Ann. Biochem. 96:282-292.
120. Sengha, S. S., A nderson , A. J., H acking, A. J., a n d E. A. D aw es. 1989. The p roduction of alg inate by Pseudomonas mendocina in batch and continuous culture. J. Gen. Microbiol. 135:795-804.
121. S h afizad eh , F. 1958. Form ation and cleavage of the oxygen ring in sugars. Adv. in Carbohydr. Chem. 1 3 :9-61.
122. Shatw ell, K. P., a n d I. W. S u th e rlan d . 1991. Influence of the acetyl substituent on the interaction of xanthan w ith p lan t p o ly sa c c h a rid e s-I . X an than-L ocust b e a n gum systems. Carbohydr. Polymers 14:29-51.
123. Shatw ell, K. P., a n d I. W. S u th e rlan d . 1991. Influence of the acetyl substituent on the interaction of xanthan with p lan t polysaccharides-II. X anthan-G uar gum systems. C arbohydr. Polymers 14:115-130.
124. S hatw ell, K. P., a n d I. W. S u th e rla n d . 1991. Influence of the acetyl substituent on the interaction of xanthan w ith p lan t polysaccharides-lll. Xanthan-Konjac gum systems. Carbohydr. Polymers 14:131-147.
125. S herbrock-C ox, V., Russell, N. J., a n d P. G acesa. 1984. The purification and chemical characterization of the alginate p resen t in extracellular m aterial p roduced by m ucoid strains of Pseudomonas aeruginosa. Carbohydr. Res. 13 5:147-154.
126. Skjak-Braek, G., L arsen , B., a n d H. G rasd a len . 1985. The ro le of O-acetyl groups in the biosynthesis of alg inate by Azotobacter vinlandii. Carbohydr. Res. 145:169-174.
127. Skjak-B raek, G., G ra sd a len , H., a n d B. L arsen . 1986. M onomer sequence and acetylation pa tte rn in some bacterial alginates. Carbohydr. Res. 154:239-250.
128. S k jak -B raek , G .,S m id sro d , O., a n d B. L a rse n . 1986. Tailoring of alginates by enzymatic m odification in vitro. Int. J. Biol. Macromol. 8:330-336.
138
129. Skjak-B raek, G., Z ane tti, F., a n d S. P ao le tti. 1989. Effect of acety la tion on som e solution an d gelling p ro p ertie s of alginates. Carbohydr. Res. 185:131-138.
130. S k jak -B raek , G. 1992. A lginates: B iosynthesis a n d som e structu re function rela tionsh ips re leven t to biom edical and b io technological app lications. Biochem . P lan t Polysacch. 20:27-33.
131. S tew art, L. C., R ichtm yer, N. K., a n d C. S. H udson . 1952. L-guloheptulose and 2,7 anhydro (5-L-guloheptulopyranose. J. Am. Chem. Soc. 74:2206-2210.
132. S tew art, L. C., a n d N. K. R ichtm yer. 1955. Transform ation of D-gulose to 1,6 anhydro p-D-Guiopyranose in acid solution. J. Am. Chem. Soc. 77:1021-1024.
133. S u th e r la n d , I. W. 1977. Bacterial exopolysaccharides- the ir n a tu re an d p roduction . In Su therland , I. W. (ed.), Surface carb o h y d ra tes of the prokaryotic cell. p. 27-96. Academ ic Press, London.
134. S u th e r la n d , I. W. 1982. B iosyn thesis o f m ic ro b ia l exopolysaccharides. Adv. Micro. Physiol. 23:79-142.
135. S u th e r la n d , I. W. 1989. Bacterial exopolysaccharides- the ir na tu re and production. Antibiot. Chemother. 4 2 :50-55.
136. S u th e r la n d , I. W. 1990. B io techno logy o f microbial exopolysaccharides. Cambridge University Press, Cambridge.
137. S u th e r la n d , I. W. 1993. B iosynthesis of ex trace llu la r polysaccharides (exopolysaccharides). In W histler, R. L., and J. N. BeMiller (eds.), Industrial gums: polysaccharides and their derivatives, p. 69-85. Academic Press, San Diego.
138. S u tto n , J. C., a n d P. H. W illiam s. 1970. Com parison of extracellular polysaccharides of Xanthomonas campestris from culture and from infected cabbage leaves. Can. J. Bot. 48:645- 651.
139
139. T anaka, T. 1987. In Nicolino, C. (ed.), Structure and Dynamics of Biopolymers, p. 237-257. NATO ASI series, Boston.
140. T ay lo r, R. L., S h ively , J. E., a n d H. E. C o n rad . 1976. Stoichiom etric reduction of uronic acid carboxyl groups in polysaccharides. M ethods Carbohydr. Chem. Vol VII. p. 149- 151.
141. T h o m p so n , A., A nno , K., W o lfro m , M. L., a n d M. In a to m e . 1954. Acid reversion p roducts from D-glucose. J. Am. Chem. Soc. 76:1309-1311.
142. V olk , W. A. 1968. Iso lation of D -galacturonic ac id 1- phosphate from hydrolysates of cell wall lipopolysaccharide extracted from Xanthomonas campestris. J. Bacteriol. 95:782- 786.
143. V re e la n d , V. 1970. Localization of a cell wall polysaccharide in a brow n alga w ith labelled antibody. J. of Histochem. and Cytochem. 18:371-373.
144. W ells, J. 1977. Extracellu lar m icrobial polysaccharides, a critica l overview . In Sandford , B., a n d A. Laskin (eds.), Extracellular Microbial Polysaccharides, p. 299-313. American Chemical Society, W ashington D. C.
145. W h is tle r, R. L., a n d G. N. R ich ard s. 1958. Uronic acid fragm ents from slash pine (Pinus elliotti) and th e ir behavior in alkaline solution. J. Am. Chem. Soc. 80:4888-4891.
146. W iggins, L. F. 1949. Derivatives of 1,6 anhydro p-D-idose. J. Chem. Soc. 2:1590-1592.
147. W ilk inson , J. F. 1958. The extracellu lar polysaccharides of bacteria. Bacteriol. Rev. 22:46-73.
148. W olff, J. A., M acG regor, C. H., E isenberg , R. C., a n d P. V. P h ib b s. 1991. Isolation and characterization of catabolite rep ressio n con tro l m u tan ts of Pseudom onas aeruginosa. J. Bacteriol. 173:4700-4706.
140
149. W ozniak, D. J., a n d D. E. O hm an. 1993. Involvem ent of the alginate algT gene and integration host factor in the regulation o f th e Pseudom onas aeruginosa algB gene. J. B acteriol. 175:4145-4153.
150. Zeller, S. G., a n d G. R. G ray. 1992. Analysis of Macrocystis pyrifera an d Pseudom onas aeruginosa alginic acid by th e reductive cleavage m ethod. Carbohydr. Res. 226:313-326 .
151. Z ielinsk i, N. A., C h a k ra b a r ty , A. M., a n d A. B erry . 1991. C haracterization an d reg u la tio n of th e Pseudomonas aeruginosa algC gene encoding phosphom annom utase. J. Biol. Chem. 266:9754-9763.
VITA
Richard David Ashby was b o m in Kansas City, Kansas on May
6, 1962. After graduating from Frem ont High School in Sunnyvale,
California in 1980, he a ttended Brigham Young U niversity w here he
obtained his Bachelor of Science degree in Microbiology in 1987. In
August 1988, he began his g raduate tra in ing a t Louisiana State
University in Baton Rouge, Louisiana.
141
DOCTORAL EXAMINATION AND DISSERTATION REPORT
candidate: Richard David Ashby
Major Field: M ic r o b io lo g y
Title of Dissertation: j h e P ro d u c t io n and C h a r a c t e r i z a t i o n o f A l g i n a t eProduced by Pseudomonas s y r i n q a e
Appro?
f % K._Major Professor and Chaxrman
Dean of the Grac^iate School
EXAMINING COMMITTEE:
y g.-yr , . M■ v/
^.u f H ' L Jkl
j2/^ P u ,
Date of Examination:
June 2 1 , 1994