Clavulanic acid: a review.
ABSTRACT Natural antibiotics are almost universal secondary metabolites, not essential for the growth of the producing organisms generally produced at low growth rates or after growth has ceased. Clavulanic acid (CA), a naturally occurring powerful inhibitor of bacterial beta-lactamases is a major beta-lactam antibiotic produced by organism Streptomyces clavuligerus and is active against a wide spectrum of Gram-positive and Gram-negative bacteria. The review discusses the biosynthetic pathway, fermentative production, downstream processing and applications of CA.
- SourceAvailable from: endoexperience.com[show abstract] [hide abstract]
ABSTRACT: Antibiotics to treat endodontic infections are routinely prescribed based on previously published susceptibility tests. There is increased concern that bacteria have increased resistance to the currently recommended antibiotics. The purpose of this investigation was to perform antibiotic susceptibility tests on a panel of bacteria recently isolated from endodontic infections. The bacteria in this study were aseptically aspirated with a needle from endodontic abscesses, cultivated, and identified at the species level. Each of the 98 species of bacteria was tested for antibiotic susceptibility to a panel of six antibiotics using the Etest. The antibiotics were penicillin V, amoxicillin, amoxicillin + clavulanic acid, clindamycin, metronidazole, and clarithromycin. The percentages of susceptibility for the 98 species were penicillin V: 83/98 (85%), amoxicillin: 89/98 (91%), amoxicillin + clavulanic acid: 98/98 (100%), clindamycin: 94/98 (96%), and metronidazole: 44/98 (45%). Metronidazole had the greatest amount of bacterial resistance; however, if it is used in combination with penicillin V or amoxicillin, susceptibility of the combination with penicillin V or amoxicillin increased to 93% and 99%, respectively. Clarithromycin seems to have efficacy, but it is still considered an antibiotic under investigation because the minimum inhibitory concentration has not been established.Journal of Endodontics 02/2003; 29(1):44-7. · 2.93 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The transmission and rate of filtration of the enzyme yeast alcohol dehydrogenase (YADH) has been studied at capillary pore microfiltration membranes. Photon correlation spectroscopy (PCS) with nanometer resolution showed that the enzyme existed as discreate molecules only for a narrow range of pH and ionic strength. Under such conditions, the transmission of the enzyme was high. However, the rate of filtration still decreased continuously with time. Analyssis of the time dependence of the rate of filtration indicated that this decrease was due to in-pore enzyme deposition at low concentration (“standard blocking model”) and suface depositon at high concentration (“cake filtration model”). Use of atomic force microscopy (AFM) gave unequivocal and quantitative confirmation of these inferences. The work shows the great advantage of using advanced physical characterization techniques, both for the identification of the optimum conditions for filtration (PCS) and for the elucidation of mechanisms giving rise to inefficiencies in the filtration process (AFM). © 1995 John Wiley & Sons, Inc.Biotechnology and Bioengineering 04/1995; 46(1):28 - 35. · 3.65 Impact Factor
- Biotechnology and Bioengineering - BIOTECHNOL BIOENG. 01/1991; 37(5):456-462.
Research review paper
Clavulanic acid: A review
Parag S. Saudagar, Shrikant A. Survase, Rekha S. Singhal⁎
Food Engineering and Technology Department, Institute of Chemical Technology, Nathalal Parikh Marg, Matunga, Mumbai 400 019, India
A B S T R A C TA R T I C L EI N F O
Received 5 October 2007
Received in revised form 25 February 2008
Accepted 9 March 2008
Available online 26 March 2008
Natural antibiotics are almost universal secondary metabolites, not essential for the growth of the producing
organisms generally produced at low growth rates or after growth has ceased. Clavulanic acid (CA), a
naturally occurring powerful inhibitor of bacterial β-lactamases is a major β-lactam antibiotic produced by
organism Streptomyces clavuligerus and is active against a wide spectrum of Gram-positive and Gram-
negative bacteria. The review discusses the biosynthetic pathway, fermentative production, downstream
processing and applications of CA.
© 2008 Elsevier Inc. All rights reserved.
1.Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.Sulbactum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.Tazobactum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.Clavulanic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Clavulanic acid – an introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biosynthesis of clavulanic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Fermentative production of clavulanic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.Development of inoculum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.Strain improvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.Effect of carbon source on clavulanic acid production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.Effect of nitrogen source on production of clavulanic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5. Effect of phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.6.Effect of precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.7. Effect of the operation mode of the bioreactor. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Scale-up studies for clavulanic acid production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stability of clavulanic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Downstream processing of clavulanic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1.Solvent extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2.Membrane technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3.Ion exchange chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pharmacological actions of clavulanic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.1. Clavulanic acid in lower respiratory tract infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.2.Clavulanic acid in urinary tract infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.3.Clavulanic acid in alveolar osteitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.4.Clavulanic acid in endodontic abscesses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.5.Clavulanic acid in pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biotechnology Advances 26 (2008) 335–351
⁎ Corresponding author. Tel.: +91 22 24145616; fax: +91 22 24145614.
E-mail address: firstname.lastname@example.org (R.S. Singhal).
0734-9750/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/biotechadv
Toxicity associated with clavulanic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Clavulanic acid in diarrhea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Naturally produced antibiotics are almost universal secondary
metabolites. These are generally produced at lowgrowth rates or after
growth has ceased, nonessential for growth of the producing
organisms in pure culture, and typically possessing unusual structures
not found in primary metabolites of central metabolism. Although
antibiotic biosynthesis is not essential to the producing organism in
pure culture, these compounds are critical to the producing organisms
in their natural environment, both for survival and competitive ad-
vantage (Demain, 1989). Secondary metabolites are structurally
diverse and unusual, generally produced in mixtures with other
members of the same chemical family. In batch cultures some pro-
cesses leading to the production of antibiotics are sequential; i.e., they
exhibit a distinct growth phase (trophophase) followed by production
phase (idiophase). In other processes, trophophase and idiophase
overlap. Timing depends on nutritional environment presented to the
culture and/or on the growth rate. Delay in the antibiotic production
until after trophophase is useful to the producing organism, since the
organism is sensitive during growth to its own antibiotic. Resistance
develops during idiophase. Resistance mechanisms include enzymatic
modification of the antibiotic, alteration of the target, and decreased
uptake of the excreted antibiotic. Mechanisms that control the ini-
tiation of antibiotic synthesis include repression and inhibition of
antibiotic synthetases. In cases of the branched pathways leading to
the primary metabolites and an antibiotic, the primary metabolite
interferes with antibiotic formation by inhibiting an early step of the
common pathway, thus preventing accumulation of antibiotic
Specific mechanisms regulating the onset of antibiotic synthesis
include carbon catabolite repression, nitrogen metabolite regulation,
phosphate regulation, and induction. Cessation of the antibiotic bio-
synthesis occurs via decay of antibiotic synthetases, feedback inhi-
bition, and repression of these enzymes.
Routine strain improvement programs have successfully yielded
high producing mutants of antibiotic producers. Attempts at inten-
tional deregulation of such cultures have begun only recently, but
have been successful. As the emergence of antibiotic resistant
organisms threatens to return treatment of infectious diseases to
pre-antibiotic era, strategies aimed at overcoming acquired resistance
are of increasing interest. One of the best examples of the wide clinical
application of this strategy is the development of β-lactamase
inhibitors for use in combinations with conventional β-lactam
antibiotics. The β-lactamase inhibitors that have gained considerable
importance in the world market include sulbactum, tazobactum and
Sulbactam, a derivative of the basic penicillin nucleus, is a β-lac-
tamase inhibitor with a chemical designation (2S, 5R)-3, 3-dimethyl-
7-oxo-4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylate 4,4-dioxide.
It is used to increase the antibacterial spectrum of penicillins and
cephalosporins against penicillinase producing and β-lactamase-pro-
ducing organisms such as Staphylococcus aureus and Moraxella cata-
rrhalis that are resistant to ampicillin alone (Singh, 2004).
Sulbactam inhibit β-lactamase irreversibly. However, it is not able to
interact with the ampC cephalosporinase. Thus, it confers little
protection against bacteria such as Pseudomonas aeruginosa, Citrobac-
ter,Enterobacter, andSerratia, whichoften express this gene. It is awhite
to off-white crystalline powder (assay: 97% to 103%), having an optical
rotation of +242o∼+253o,a molecular weightof 233.24 and a molecular
formula as C8H11NO5S.
In the United States, sulbactam is combined to form cefoperazone/
sulbactam (Sulperazone) and ampicillin/sulbactam (Sultamicillin).
Sultamicillin is a white to off-white crystalline powder; soluble in
aqueous diluents to yield reconstitution of ampicillin and sulbactam.
Tazobactam is a compound that inhibits the action of bacterial β-
lactamases. It is added to the extended spectrum β-lactam antibiotic
piperacillin to produce Tazocin®. Tazocin® is marketed by Wyeth, USA
and has an annual sale of close to a billion dollars in the US. It broadens
the spectrum of piperacillin by making it effective against organisms
that express beta-lactamase and would normally degrade piperacillin.
molecular weight of 300.28 and a molecular formula as C10H12N4O5S1.
1.3. Clavulanic acid
The antibacterial market is currently best described as saturated,
highly segmented and increasingly flooded with generics. Furthermore,
bacterial drug-resistance is threatening currently marketed drugs,
although this represents an opportunity for the more audacious drug
maker. Glaxo SmithKline's (GSK) former community blockbuster
‘Augmentin’ is a classical example of the impact of generic incursion
following patent expiry. Since the first amoxicillin/CA generics entered
theUS market in 2002, Augmentin's USsales plummeted from a peakof
$1.6 billion in 2001 to only $107 million in 2004, representing 93% sales
GSK launched two follow-up products; Augmentin ES-600 and
Augmentin XR. However, although these two products partially offset
the loss in Augmentin revenues, dampening the drop in sales from 93%
to 68% by generating combined sales worth $505 million in 2004, GSK
still registered a significant loss in antibacterial revenues.
CA has limited market availability because of its complex production
InadditiontoGSK,there are twoothergeneric manufacturers present in
the market since July 2003: the American company, Geneva, with its
generic co-amoxiclav, and an Israeli company, Teva. An Indian company,
Ranbaxy, has also received a marketing authorization for co-amoxiclav
tablets, but their product is not yet available in the U.S. market.
Ljubljana, January 6, 2003 – Lek D.D, a Slovene pharmaceutical
company, has entered the world's largest pharmaceuticals market with
itsleadingproduct, co-amoxiclav.Byenteringthe USmarket,Lek has,to
date, achieved its most far-reaching business goal. On its first workday,
January2,2003, Lek's affiliated company,LekPharmaceuticals,Inc.,sold
$27 million worth of co-amoxiclav. Lek D.D. began its co-amoxiclav
projectin1989 withitsownR&D efforts,developingitsowntechnology
for the production of CA and its own finished forms of co-amoxiclav,
which are in compliance with international regulatory requirements.
Lek D.D. is producing the active ingredient, CA, in its plant in Lendava
and the finished product in Prevalje. The consolidated sales of co-
amoxiclav in the US market exceeded $100 million in 2003 making the
US Lek's biggest market.
P.S. Saudagar et al. / Biotechnology Advances 26 (2008) 335–351
Thus commercial products such as Augmentin™ and Timentin™,
combinations of CA together with amoxicillin or ticarcillin, respec-
tively, have made CA a product valued in excess of a billion dollars/
annum and created a powerful incentive to understand the biochem-
istry and genetics which underpins the production of this compound.
Presently, Glaxo SmithKline, USA; Lek D.D, a Sandoz company,
Lendava; and CIPAN, a Portuguese company are manufacturing CA.
2. Clavulanic acid – an introduction
CA is a major β-lactam antibiotic and was discovered indepen-
dently by Brown et al. (1976) and Napier et al. (1981) as a product of
the organism Streptomyces clavuligerus. S. clavuligerus had been
isolated in 1971 in a screening programme for producers of β-lactam
compounds with improved resistance to β-lactamases (Nagarajan
et al.,1971). Ironically, the selection of S. clavuligerus in this screenwas
due toits abilitytoproduce cephamycin C, and CAwas onlydiscovered
when the strains from this first screen were subsequently examined
for the production of β-lactamase inhibitors. In addition, a number of
other species that produce clavam metabolites that are structurally
related to CA (they carry the fused bicyclic β-lactam/oxazolidine ring
system) have been described. However, CA with its 3R, 5R stereo-
chemistry is the only one among the clavam metabolites in showing
the β-lactamase inhibitory activity. All the others have the 3S, 5S
stereochemistry and show no β-lactamase inhibition, although some
have antibacterial or antifungal properties.
Several of the CA producers also produce cephamycins, while others
produce only clavams. The ability to produce the clavam compounds
appear to be more restricted in occurrence than the ability to produce
conventional β-lactam antibiotics since no example of the producer
species from outside the genus Streptomyces have been reported.
Production of CA is even more restricted, with S. clavuligerus reported
to date. Box (1978) reported that CA and its salts may be produced by
cultivating a strain of S. jumonjinensis. S. lipmanii, isolated in the same
initial screen as S. clavuligerus, produces several of the intermediates
CA appears to be active against a wide spectrum of Gram-positive
and Gram-negative bacteria; however, relative to other broad-
spectrum antibiotics such as thienamycin the activity is relatively
low. As a result, it has not been possible to use CA as a solely
administered antibacterial product. It is rather co-formulated with
other broad-spectrum antibiotics, which are susceptible to lactamase
(Brown, 1986). For example, in vitro studies indicate that minimum
inhibitory concentration (MIC) of ampicillin to exceed 500 μg/ml
when measured against a lactamase producing strain of S. aureus. On
supplementation with 5 μg/ml of CA, the MIC for combined addition
was reduced to 0.1 μg/ml.
Following its discovery, the chemical structure of CAwas identified
by Howarth et al. (1976) (Fig. 1). This compound was found to be an
analogof the basic penicillin structure. In CA molecule, oxygen atom is
substituted for sulphur, a characteristic of penicillin. As such, CA
derivatives all contain an oxazolidine ring structure. The combined
effective inhibition of β-lactamase and antibacterial activity of CA
make it very important, both clinically and economically.
3. Biosynthesis of clavulanic acid
Little has been reported on the biosynthetic pathway to produce
CA. However initial studies (Elson and Oliver, 1978) suggest its
synthesis S. clavuligerus not to follow classical tripeptide theory. The
absence of 6-amino group or an amino adipoyl side chain in CA,
coupled with glutamic acid incorporation into oxazolidine carbon
backbone, indicates that the traditional Arstein tripeptide is not
formed. Apparently another assembly mechanism is in place. This is
surprising as S. clavuligerus also produces penicillin N, cephamycin C,
and other β-lactam compounds (Elson and Oliver, 1978).
Based on the frequent occurrence of the CA or other clavams in
species that also produce conventional β-lactam antibiotics, and given
the structural similarities between the two groups of compounds,
there was some initial speculation that these two classes of
metabolites might arise from a shared biosynthetic pathway. This
hypothesis was eventually disproved, in part by the demonstration
that purified enzymes known to be responsible for penicillin/
cephamycin biosynthesis showed substrate specificity that was
incompatible with the precursors of CA. At the same time feeding
studies implicated that the amino acids such as ornithine or arginine
together with a 3C glycolytic pathway intermediate such as glycerate
or pyruvate as the precursors of CA. Recent studies now point to
arginine and pyruvate as direct precursors of CA (Valentine et al.,
1993; Pitlik and Townsend, 1997; Thirkettle et al., 1997). Aside from
feeding studies, progress on elucidating the biosynthetic pathway to
CA was hindered for many years by the lack of recognizable pathway
intermediate analogous to δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine in
penicillin/cephamycin biosynthesis, which would support in vitro
biosynthesis of CA. Early intermediates in the biosynthesis of CA were
first discovered in a study where metabolites containing guanidino
groups were purified form culture filtrate of S. clavuligerus mutant
blocked in CA production (Elson et al., 1993b). The metabolites were
recognized by their ability to participate in the Sakaguchi colour
reaction specific for compounds bearing guanidino groups. Based on
the structures of the compounds so isolated, labeled forms of three
putative intermediates were synthesized and shown to be incorpo-
rated directly into CA thereby confirming their involvement in
biosynthetic pathway (Elson et al.,1993a). Fig. 2 shows the schematic
representation of the biosynthetic pathway of CA (Jenson and
Paradkar, 1999). The earliest of these intermediates, N2-(2-carbox-
yethyl)-arginine, is an acyclic compound. The structure of this
intermediate suggest that the condensation of the pyruvate and
arginine precursors must occur by a mechanism very different from
the amide bond formation catalyzed by a non-ribosomal peptide
synthetase, such as that involved in the early steps of penicillin
biosynthesis. This is consistent with the genetic studies, which have
found no evidence for the existence of the gene encoding a peptide
synthetase within the CA gene cluster.
The second of the guanidino intermediates, deoxyguanidino
proclavaminic acid, is a monocyclic β-lactam compounds which
arise from carboxyethyl arginine via closure of the β-lactam ring.
The enzyme responsible for the formation of the β-lactam ring, called
β-lactam synthetase (BLS) has been described (Bachmann et al.,1998),
and shows some similarity to asparagine synthetase in both primary
amino acid sequence and in the reaction catalyzed. As such the
reaction is very different from that involved in the penicillin/
cephamycin production, since the ring closure requires the formation
of the amide bond. Greater parallels may be with the formation of the
β-lactam ring as it occurs in the carbapenem pathway. Like CA,
understanding of the carbapenem biosynthetic pathway is at early
stages of development when compared to penicillin/cephamycin
Deoxyguanidino proclavaminic acid is then hydrolyzed in the first
of the three reactions catalyzed by the enzyme clavaminate
synthetase (CAS) (Baldwin et al., 1993). CAS is an unusual iron
Fig.1. Structure of clavulanic acid (1) and representative structures of penicillins (2) and
P.S. Saudagar et al. / Biotechnology Advances 26 (2008) 335–351
containing, 2-ketoglutarate dependent molecular deoxygenase, remi-
niscent of the ring expansion and hydroxylation enzymes involved in
cephalosporin biosynthesis. The product, guanidinoproclavaminate
(the third of the guanidine intermediates) contains a hydroxyl group
in which the oxygen atom is derived from the molecular oxygen,
analogous to the hydroxylation of deacetoxycephalosporin C to
deacetylcephalosporin C. Despite this similarity in the reactions
catalyzed, the two enzymes show very little sequence similarity.
Guanidino proclavaminic acid is then converted to proclavaminic
acid by the action of an amidinohydrolase, which removes the
guanidino group from the arginine-derived end of the molecule. The
enzyme involved has been named proclavaminic acid amidinohydro-
lase (PAH). The existence of this enzyme was first deduced by
sequence analysis of the corresponding gene (Elson et al., 1993a;
Aidoo et al., 1994). In the monomeric form, the enzyme has a
molecular weight of 33.3 kDa as deduced from the gene sequence, but
the active enzyme may be multimeric since it was isolated as a 270-
kDa protein, which would correspond to an octameric structure.
When a gene encoding an arginase or amidinohydrolase type enzyme
was detected in CA gene cluster, ornithine was first believed to be the
intermediate precursor of CA rather than arginine. Therefore PAH was
investigated as an arginase capable of converting arginine to
ornithine. However arginase activity is very low in the cell extracts
of S. clavuligerus, unless the cells are given arginine as a nitrogen
source (Romero et al., 1986), and correspondingly PAH has no
detectable arginase activity. The interpretation of these observations
became clear once it was understood that arginine and not ornithine
was intermediate precursor of CA. The actual function for PAH in
catalyzing the conversion of guanidine proclavaminic acid to
proclavaminic acid (Fig. 2) was demonstrated using PAH purified
from S. clavuligerus (Elson et al., 1993a).
Proclavaminic acid was one of the first intermediates in the CA
biosynthetic pathway to be identified following the isolation from
mycelial extracts of S. clavuligerus together with CA (Elson et al.,1987).
the discovery of CAS. Proclavaminic acid is converted into clavaminic
acid in a two-step reaction involving the transient intermediate,
dihydroclavaminic acid (Baldwin et al., 1991; Salowe et al., 1991). Both
and 2-ketoglutarate as a cosubstrate, generating succinate as a side
product. It wasonly realized later that CAS is also responsible fora third
activity, the hydroxylation of deoxyguanidinoproclavaminic acid, which
occurs earlier in the pathway. Full characterization of CAS did not
become possible until chemical synthesis of proclavaminate was
available to provide reliable assay for enzyme (Baggaley et al., 1990).
The purification of CAS was first achieved by Salowe et al. (1990) and
revealed a surprising fact that there are two forms of the enzyme that
differonlyslightly inkinetic properties and molecularweight. Although
this remarkable enzyme catalyzes three distinct steps in the pathway,
there was no indication that the two forms of the enzyme differed in
two separate forms of this enzyme include a gene dosage effect to
provide additional amounts of a rate-limiting enzyme, or gene
Fig. 2. Scheme for the biosynthesis of clavulanic acid derived from pyruvate and arginine.
P.S. Saudagar et al. / Biotechnology Advances 26 (2008) 335–351
duplicationwithoneformof CASarisingasa partof theCAbiosynthetic
pathway, while a second form is associated with production of non-CA
Clavaminic acid has the fused bicyclic β-lactam/oxazolidine ring
system but differs from CA in stereochemistry of the ring system. In
this property it resembles all of the clavam metabolites accumulated
them its ineffectiveness as a β-lactamase inhibitor. The similar nuclear
structure and stereochemistry of clavaminic acid and other clavam
metabolites led to investigation of the possibility that all clavam
metabolite share a common biosynthetic pathway. Jane et al. (1993)
the stage of proclavaminic acid because labeled proclavaminate is
incorporated directly into clavams as well as into CA. Evidence that
other clavams has been provided by Egan et al. (1997) based in part on
the studies in S. antibioticus, where a single isoenzyme of CAS, called
CAS3 has been isolated (Jane et al., 1995). This strain of S. antibioticus
produces hydroxyethyl clavam and valclavam, but not CA. Hence, the
presence of a CAS activity implies that clavaminic acid is an
intermediate in the biosynthesis in one or both of the clavam
metabolites as well as its known involvement in CA biosynthesis.
The conversion of clavaminic acid into CA requires the inversion of
stereochemistry of the ring system as well as conversion of the side
chain substituent from an amino to hydroxyl group, but as with the
earliest step in the pathway, the reaction involved are unclear. The
existence of an aldehyde intermediate with the same stereochemistry
dehydrogenase called CA dehydrogenase (CAD), which reduces the
clavaldehyde to CA, has been documented (Nicholson et al.,1994).
4. Fermentative production of clavulanic acid
Specific nutritional requirements of microorganisms used in
industrial fermentation processes are as complex and varied as the
microorganisms in question. Not onlyare the types of microorganisms
diverse (bacteria, molds and yeast, normally), but also the species and
strains become very specific as to their requirements. Microorganisms
obtain energy for support of biosynthesis and growth from their
environment in a variety of ways. Besides a source of energy,
organisms require a source of materials for biosynthesis of cellular
matter and products in cell operation, maintenance and reproduction.
These materials must supply all the elements necessary to accomplish
this. Some microorganisms utilize elements in the form of simple
compounds; others requiremore complexcompounds, usually related
to the form in which they ultimately will be incorporated in the
cellular material. The four predominant types of polymeric cell
compounds are the lipids (fats), the polysaccharides (starch, cellulose,
etc.), the information-encoded polydeoxyribonucleic acid and poly-
ribonucleic acids (DNA and RNA), and proteins.
The biological significance of the main chemical elements in
microorganisms is given in Table 1.
CA isa β-lactamaseinhibitor whichis administered in combination
with penicillin group antibiotic to overcome certain types of antibiotic
resistance. Specifically, it is used to overcome resistance in bacteria
that secrete β-lactamase, which otherwise inactivate most penicillins.
CA has negligble intrinsic antimicrobial activity, despite sharing the
β-lactam ring that is characteristic of β-lactams. However, the
similarity in chemical structure allows the molecule to act as a
competitive inhibitor of β-lactamases secreted by certain bacteria to
confer resistance to β-lactam antibiotics. This inhibition restores the
antimicrobial activity of β-lactam antibiotics against β-lactamase-
secreting resistant bacteria.
Classical commercial fermentation processes for the production of
secondary metabolic products can be subdivided into three phases
(Omstead et al.,1985) and CA is no exception:
• The first stage is one of inoculum development and occurs
sequentially in a series of mixed reaction vessels.
• The second phase usually takes place in as single reaction and is the
stage in which antibiotics are microbiologically synthesized.
• The third phase is product recovery.
4.1. Development of inoculum
The physiological and genetic characteristic of strain plays an
important role in the improvement of productivity. Neves et al. (2001)
demonstrated the production profile of CA by S. clavuligerus to be
strongly dependent on inoculum activity.
The role of inoculum quality in successful execution of the
fermentation processes has long been recognized. Hockenhull (1963)
noted that unless the early fermentation growth phase is optimal, the
rate of product formation will most likely remain well below its
potential in spite of all efforts to alert it. The following criteria are
currently recognized as necessary to obtain satisfactory inoculum
(Stanbury and Whitaker,1984; Atkinson and Mavituna,1992):
• The inoculum must be in an active and healthy status to minimize
the duration of the lag phase in the subsequent fermentation.
• It must provide an inoculum of optimum size (3–10% of the medium
• The inoculum must be in a suitable morphological form.
• It must be free of contamination.
• The inoculated biomass must retain its product forming capabilities.
S. clavuligerus, as the majority of industrially important Strepto-
mycetes and fungi, is capable of asexual sporulation. It is therefore a
common practice to use a spore suspension as seed during the first
stage of inoculum development program (Hockenhull, 1980). How-
ever several vegetative culture steps are normally required in industry
to produce a large enough volume of preculture medium to inoculate
the production vessel. The major problem in using the vegetative
inoculum is the difficulty in obtaining a uniform standard preculture
due to morphological differentiation associated with the growth of
this type of microorganism.
Physiological functions of principal elements
Constituent of cellular water and organic cell materials
Constituent of organic cell materials
Constituent of proteins, nucleic acids and coenzymes
Constituent of cellular water and organic materials, as
O, electron acceptor in respiration of aerobes
Principal extracellular cation
Important divalent cellular cation, inorganic cofactor
for many enzymatic reactions, incl. those involving
ATP; actions in binding enzymes to substrates and
present in chlorophylls
Constituent of phospholipids, coenzymes and nucleic
Constituent of cysteine, cystine, methionine and
proteins as well as some coenzymes as CoA and
Principal intracellular and Extracellular anion
Principal intracellular cation, cofactor for some
Important cellular cation, cofactor for enzymes as
Inorganic cofactor cation, cofactor for enzymes as
Constituent of cytochromes and other heme or non-
heme proteins, cofactor for a number of enzymes
Constituent of vitamin B, and its coenzyme derivatives
Inorganic constituents of special enzymes
P.S. Saudagar et al. / Biotechnology Advances 26 (2008) 335–351
In antibiotic industry, inoculation of the production tanksis typically
based on the constant age procedures. Low reproducibility production
systems are usually obtained as a result of variable biomass concentra-
tion and metabolic activity of the preculture at the time of inoculation.
This experimental variability can be reduced, and by monitoring the
activity of the preculture tank, ideally in every fermentation cycle, the
process performance can be enhanced (Buckland, 1984; Guthke and
In order to improve the performance of the fermentation system, it
is not sufficient to control the environment to which the microorgan-
ism is exposed. It is also necessary to monitor the actual culture
metabolic activity (e.g. shifts from different primary substrates or
from growth to product formation). Off-gas analysis is one such
technique that provides valuable data on the culture state. It is robust
and reliable enough to work on-line in an industrial environment. It is
also unaffected by the presence of solid substrates and to some extent
to the changes in the physiochemical properties of the cultivation
medium such as viscosity or the pH (Meyer et al., 1985; Mou and
Cooney, 1983; Royce and Thornhill, 1992).
Neves et al. (2001) studied the effect of preculture variability on CA
fermentation. Two sets of fermentations (A and B) were investigated at
were inoculated using late exponential growth phase mycelia. Type B
fermentations were inoculated using mycelia harvested at stationary
phase. Productivities throughout type A fermentations were consis-
tently higher than type B, reaching a maximum at about 70 h and then
decayingtothesamefinal productivitiesat 140h of typeB runs.Several
scheduling alternatives, based on the combinations of the two inocula
types and different fermentation lengths, were compared in terms of
overall process economics. An increase of ca. 22% on the overall process
profit is predicted using late exponential growth phase inocula and a
fermentation duration of 96 h. A new operating strategy was thus
proposed for inoculum production based on the control of preculture
activity using off-gas analysis. This method ensures higher productivity
and a better batch-to-batch reproducibility of CA fermentation than
traditional methods based on constant age inocula.
antibiotic synthesis in S. clavuligerus. The authors documented that
antibiotic biosynthetic pathways were activated shortly after spore
germination, but the inoculum density affected the timing and the
kinetics of activation. Rapid activation was favoured by high
inoculum density or by growth in the medium conditioned by
previous incubation of S. clavuligerus spores or mycelium. A heat
resistant conditioning factor able to accelerate the acquisition of the
antibiotic biosynthetic capacity when added to the low-density
cultures was released in suspensions of spores inwater. Conditioning
factor was also obtained in suspensions of spores from different
Streptomyces species orof Bacillus cells, indicating that the signal was
not produced specifically by S. clavuligerus. Fractionation of the
conditioning factor showed that its effect was not due to the single
molecule. The fractions contained amino acids in amounts that
roughly correlated with their respective conditioning power.
Furthermore, the conditioning effect was reproduced by supple-
menting defined medium with amino acids and peptides in
concentrations that mimicked those found in the conditioning factor.
4.2. Strain improvement
Strain improvement is an essential partof process development for
microbial fermentation products.
• It enables cost reduction by developing strain with increased
productivity (measured as titer or total yield).
• Confers ability to use inexpensive raw materials, or
• Imparts more desirable special characteristics such as improved
• Ability to produce under certain conditions of temperature or
The industrial production of CA is carried out by large-scale
fermentation of S. clavuligerus. Strains capable of supporting a high
level of CA productivity have been derived from the wild-type S.
clavuligerus through a conventional strain development program ex-
tending over many years. Biochemical analysis of these strains has
revealed that they still have the genetic capability to produce both
cephamycin C and antipodal clavams. However, these are undesirable
products in an industrial CA-producing strain. Therefore, it was of
interest to determine if elimination of the pathway for the biosynthe-
tically unrelated metabolite cephamycin C and the pathway for the
biosynthetically related antipodal clavams would have beneficial ef-
fects on CA productivity in these high-titer industrial strains.
Li and Townsend (2006) and Townsend and Li (2007) reported on
rational strain improvement for enhanced CA production by genetic
engineering of the glycolytic pathway in Streptomyces clavuligerus.
Two genes (gap1 and gap2) whose protein products are distinct
glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) were inacti-
vated in S. clavuligerus by targeted gene disruption. A doubled
production of CAwas consistently obtained when gap1 was disrupted,
and reversed by complementation. Addition of arginine to the
cultured mutant further improved CA production giving a greater
than 2-fold increase over wild-type, suggesting that arginine became
limiting for biosynthesis.
In S. clavuligerus, gene constructs of the biosynthetic genes ces1,
and ces2 resulted in recombinant strains with 60% and 100% higher CA
production, respectively, compared to the wild-type strain (Perez-
Redondo et al., 1999). Townsend et al. (2001) provided a method for
increasing the production of CA by gene insertion with orf2 from the
CA biosynthetic pathway in S. clavuligerus; and by manipulation of
fermentation conditions, especially the concentration of D-glyceralde-
hyde-3-phosphate, a substrate of N-(2-carboxyethyl) arginine
synthase, the protein encoded by orf2.
Disruption of negative regulatory gene(s) or increased expression
of positive regulatory gene(s) also can result in the elevated
production of secondary metabolites. Paradkar et al. (1998) observed
a 2 to 3-fold increase in CA production by introducing additional
copies of positive regulatory genes in the wild-type (Paradkar et al.,
1998; Perez-Llarena et al., 1997; Perez-Redondo et al., 1998). Barton
et al. (2000, 2004) reported on the process for improving the
manufacture of 5R clavams e.g. CA. They either disrupted or deleted
open reading frames from DNA essential for 5S clavam biosynthesis
such that the production of 5S clavams by S. clavuligerus is reduced
and CA production is at least maintained. They also isolated
polynucleotide comprising open reading frames encoding one or
more enzymes involved in CA biosynthesis and introduced the said
polynucleotide in a suitable microorganism for improved production
of CA. Anders et al. (2005) reported on isolation of DNA comprising
one or more genes specific for 5S clavam biosynthesis, vectors
comprising such DNA and streptomyces hosts capable of improved
CA production. Cephamycin C production was blocked in wild-type
cultures of the CA-producing organism S. clavuligerus by targeted
disruption of the gene (lat) encoding lysine ε-aminotransferase.
Specific production of CA increased in the lat mutants derived from
the wild-type strain by 2.0 to 2.5-fold. Similar beneficial effects on CA
production were noted when gene disruption was used to block the
production of the non-CA clavams produced by S. clavuligerus.
Therefore, mutations in lat and in cvm1, a gene involved in clavam
production, were introduced into a high-titer industrial strain of S.
clavuligerus to create a double mutant with defects in production of
bothcephamycin C and clavams. Production of both cephamycin C and
non-CA clavams was eliminated in the double mutant, and CA titers
increased about 10% relative to those of the parental strain. This
represented the first report of the successful use of genetic
P.S. Saudagar et al. / Biotechnology Advances 26 (2008) 335–351
engineering to eliminate undesirable metabolic pathways in an
industrial strain used for the production of an antibiotic important
in human medicine.
Another approach is to inactivate pathways that compete for key
precursors, intermediates, cofactors and energy supply. The inactiva-
tion of the clavam pathway, which shares the common intermediate
clavaminic acid with the CA pathway, has been shown to give an
elevated yield of CA in S. clavuligerus (Paradkar et al., 1998).
S. clavuligerus can produce at least 21 secondary metabolites
including the commercially important CA. The changes in the
nutrients and their concentrations have different effects on the
accumulation of different metabolites, which are controlled by the
4.3. Effect of carbon source on clavulanic acid production
The preferred carbon source for CA production by S. clavuligerus is
a lipid (Butterworth, 1984). Carbohydrates are the simplest energy
sources for growth and secondary metabolite production. However,
rapid catabolism of glucose and other carbohydrates has been shown
to decrease the rate of antibiotic biosynthesis. Carbon catabolites have
been reported to inhibit penicillin production in P. chrysogenum
(Revilla et al., 1984) and cephalosporin synthesis in S. clavuligerus
(Aharonowitz and Demain, 1978) Addition of a low solubility carbon
source such as oil is a method of avoiding carbon catabolite regulation.
The addition of oil to a growth medium is also preferred on an energy
basis, as typical oil contains approximately 2.4 times the energy of
glucose on a weight-to-weight basis (Stowell,1985). Oils are preferred
in terms of volume when compared to carbohydrates; for example, it
would take 1.24l of soybean oil toadd 10 kcal of energy toa fermenter,
where as it would take 5 l of glucose or sucrose to add same amountof
energy assuming that they are being added as 50% w/w.
Disadvantages of using oils in media include problems that arise
due to residual oil levels higher than that of the carbohydrates (which
in batch process often equates to zero) (Stowell, 1985), and a higher
oxygen requirement for oil metabolism when compared to carbohy-
drates. Residual oil levels may lead to the problems associated with
the increased medium viscosity and warrant additional downstream
processing. Because of the inability of the S. clavuligerus to utilize
simple carbohydrates such as glucose and the reported increase in the
secondary metabolite biosynthesis in other organisms, the preferred
carbon source for CA production is a lipid.
Large et al. (1998) studied the effect of oil containing C16and C18
unsaturated and saturated fatty acids in conjugation with modified
starch as the carbon sources in the process medium. These authors
report CA production in relation to the viscosity of the medium. Their
results suggest production of approximately 80 mg/l CA in a medium
containing an unspecified lipid.
Lee and Ho (1996) reported that palm and palm-kernel oils and
their olein and stearin fractions to be suitable as the main carbon
sources for growth and production of CA by S. clavuligerus. However
oleic and lauric acid were not utilized for growth. A spontaneous
mutant that was selected for higher cephamycin C production also
produced more CA with these oils in the medium.
Maranesi et al. (2005) studied the utilization of vegetable oil in
production of CA by S. clavuligerus ATCC 27064. The medium
containing soybean oil and starch as carbon and energy source gave
the best production results. This medium, with the starch replaced by
glycerol, and with various soybean oil concentrations (16, 23 and 30 g/
l) was utilized to further investigate CA production. Medium contain-
ing 23 g/l soybean oil led to the highest CA productivity (722 mg/l in
120 h) and that containing 30 g/l gave the highest CA titer (753 mg/l in
130 h). Also, substitution of corn and sunflower edible oils furnished
similarly good results in terms of CA titer and productivity. The
authors concluded that easily available vegetable oil to be a very
promising substrate for CA production, since it is converted slowly to
glycerol and fatty acids, which are the main carbon and energy source
for the microorganism.
Ortiz et al. (2007) investigated the influence of the type of soybean
derivatives as nitrogen sources, as well as the simultaneous influence
of theconcentrationsofnitrogenandcarbonsourcesin theproduction
of CA, by S. clavuligerus. Firstly, two runs in shake flasks were
performed utilizing soybean flour and soybean protein isolated in the
culture medium with concentration of 1.6 g/l total nitrogen. The CA
production in the culture medium with soybean flour was much
higher, about double the production obtained with soybean protein
isolated. Additional experiments to study the quantitative influences
of the concentrations of soybean flour as nitrogen source and soybean
oil as carbon source, on CA production were performed. Maximum CA
production of 906 mg/l was obtained with soybean flour 40 g/l and
soybean oil 16 g/l.
Romero et al. (1984) reported an inhibition of biosynthesis of CA
when glycerol, glutamic acid, ammonium and phosphate in the
medium were over 165 mM, 20 mM, 20 mM,10 mM, respectively. On
the other hand in the absence of glycerol no CA was formed, but
cephamycin C, another metabolite of S. clavuligerus was produced.
Glycerol is essential for the biosynthesis of CA.
Elson and Oliver (1978) have clearly demonstrated that glycerol
fed in the fermentation medium was incorporated particularly into
the β-lactam ring of CA using labeled
indicates that the carbon skeleton of the β-lactam ring of CA was
derived from glycerol without any intermediate rearrangement of
the three carbon atoms. Chen et al. (2003) also demonstrated that in
fed-batch culture glycerol feeding rather than ornithine feeding to
be rate limiting for the CA synthesis. Chen et al. (2002) investigated
the effect of feeding glycerol on CA production by S. clavuligerus.
They reported that glycerol at 10–20 g/l increased CA production by
S. clavuligerus in shake flask cultures. The biosynthesis of CA con-
tinued up to 132 h by feeding glycerol and production increased to
270 mg/l as compared to 115 mg/l without feeding. In fermenter
batch culture, degradation of CA began after 72 h. With glycerol
feeding in fed-batch culture, CA productionwas not only increased to
about 280 mg/l but also remained stable up to 130 h.
Saudagarand Singhal (2007a) studied the fermentative production
of CA by S. clavuligerus MTCC 1142. They investigated the effect of
media components (i.e., carbon source, nitrogen source and inoculum
concentration) and environmental factors such as pH for CA produc-
tion. They optimized the concentration of soybean flour, soybean oil,
dextrin, yeast extract and K2HPO4 were optimized using L25
orthogonal array method. Attempts to increase the CA synthesis by
manipulating the anaplerotic flux on C3 and C5 precursors by
supplementing the medium with arginine, ornithine, proline, valine,
leucine, isoleucine, pyruvic acid and α-ketoglutarate were reported to
be successful. Supplementing the optimized medium with 0.1 M
arginine and 0.1 M leucine increased the yield of CA further to 1.1 mg/
ml and 1.384 mg/ml respectively compared to 0.14 mg/ml before
13C to the precursor. This
4.4. Effect of nitrogen source on production of clavulanic acid
The nutrient medium used for the cultivation of S. clavuligerus may
contain 0.1 to 10% (w/v) of complex nitrogen source such as yeast
extract, corn steep liquor, vegetable protein, seed protein or hydro-
lysate of such proteins. The protein from soybean has been shown to
be themostimportantnutrient forCA production(Butterworth,1984).
Gouveia et al. (1999) studied the effect of nitrogen source in the
production medium on the CA production. Batch cultures using two
types of synthetic culture medium and two types of complex culture
medium containing soybean derivatives were employed. To allow
comparison of the various media, all of them were formulated with
4.0 g total nitrogen/l. It was observed that the production of CA using
synthetic medium reached values slightly greater than those usually
P.S. Saudagar et al. / Biotechnology Advances 26 (2008) 335–351
found in the literature. However, in trials with complex media, it was
found that when Samprosoy 90NB (protein extract of soybean) was
utilized, production of CA went up to 920 mg/l, twice as high as when
soy meal was used, and notably higher than values reported in the
literature (300±500 mg/l) for complex medium. The authors
concluded that increasing the concentration of total nitrogen results
in a larger production of CA regardless of the culture medium
substrate (synthetic or complex).
Gouveia et al. (2001) studied the optimization of medium
composition for CA production by S. clavuligerus. The authors reported
that among the four different commercially available nitrogen sources
containing soybean derivatives (corn steep liquor containing 4% w/w
total nitrogen, protein extract of soybean containing 15% w/w total
nitrogen, yeast extract containing 12.8% total nitrogen, and bacter-
iological peptone containing10% w/w total nitrogen), a protein extract
of soybean gave the highest yields for CA.
Mayerand Deckwer (1996) studied the CA production in a medium
containing glycerol, soy meal or soy meal extract. It was observed that
the growth and CA production took place simultaneously, and in
stationary phase the CA production was stagnant or reduced.
Although the growth was three fold faster in a media containing soy
meal extract, CA was only produced in the stationary phase.
Wang et al. (2005) studied the optimization of medium compo-
nents for the production of CA by S. clavuligerus using both factional
factorial design and response surface methodology. The factional
factorial design experiment results shown that soy meal powder,
FeSO4·7H2O, and ornithine were the most important medium
components (confidence levels above 85%). Response surface meth-
odology for optimization was further investigated and the optimal
concentrations for soy meal powder, FeSO4·7H2O and ornithine were
found to be 38.102 g/l, 0.395 g/l, 1.177 g/l respectively to give yield of
672 mg/l. The yield of CA was increased by 50% by culturing strain S.
clavuligerus in the nutritionally optimized fermentation medium.
Visser-Luirink et al. (2006) reported on fermentation of CA using
ammonium salts. They found an increase in production of CA when
the ammonium concentrationwas maintained equal to or higher than
50 mg/l. On the other hand, the ammonium concentration must be
low enough for reducing the repression of secondary metabolism and
avoiding toxicity of ammonium ions. The residual ammonium
concentration in a fermentation broth was controlled by adding an
ammonium source continuously or intermittently. Furthermore,
ammonium concentration can be reduced, for instance, by manipula-
tion of the temperature or pH, which leads to an increased growth rate
and an increase in the rate of ammonium consumption. Valentine et
al. (1997) also reported an improved production of CAwhen the levels
of ammonium and urea were kept low in the fermentation medium so
as to avoid repression of enzymes e.g. urease.
Table 2 summarizes the effect of different carbon and nitrogen
sources on CA production as reported in the literature.
4.5. Effect of phosphate
Phosphate is a crucial growth-limiting nutrient and regulates the
synthesis of antibiotics belonging to different biosynthetic groups.
Industrial production of antibiotics is carried out at growth-limiting
concentrations of inorganic phosphate. Phosphate at 0.3–300 mM
generally supports extensive cell growth, but at 10 mM and above
suppresses the biosynthesis of many antibiotics (Martin, 1976).
Production of clavulanic acid by different strains of Streptomyces clavuligerus grown on different carbon and nitrogen sources
StrainCarbon source Nitrogen sourceClavulanate concentration
(mg/g of cell)
Proline, glutamic acid
Proline, glutamic acid
Soy meal extract, meat peptone
Soy meal, meat peptone
Proline, glutamic acid
Romero et al. (1984)
Romero et al. (1984)
ATCC 27064 Glycerol
Mayer and Deckwer
Lee and Ho (1996)
DSM 738Glycerol 300
NRRL 3585 Sucrose
NRRL 3585NH4Cl 35130.47 Ives and Bushell
Gouveia et al. (1999)NRRL 3585 GlycerolSoy meal, Bacteriological peptone
Samprosoy 90NB, Bacteriological
Soy meal extract, peptone, ornithine,
Samprosoy 90NB, Malt extract,
Samprosoy 90NB, Malt extract,
Soybean flour, ornithine
Samprosoy 90NB, Yeast extract
ATCC 27064 Glycerol311
Chen et al. (2003)
ATCC 27064 Glycerol404
8.8 Baptista-Neto et al.
Rosa et al. (2005)ATCC 27064 Glycerol 614
Wang et al. (2005)
Baptista-Neto et al.
Maranesi et al. (2005)ATCC 27064Glycerol,
Soybean flour 753
ATCC 27064 Soybean flour906
Ortiz et al. (2007)
ATCC 27064Samprosoy 90NB, Malt extract,
Teodoro et al. (2006)
Values estimated from original works.
P.S. Saudagar et al. / Biotechnology Advances 26 (2008) 335–351
Phosphate addition not only interferes with antibiotic synthesis, but
also after several hours causes a reversal of non-growing, antibiotic
producing cells back to growing, non-producing state (Liu et al.,1975).
Phosphate plays an important role in CA synthesis. The phosphate
content of the medium controls production of cephamycin C and CA.
Aharonowitz and Demain (1978) reported CA production to be
reduced by 80% at phosphate concentration of ≥100 mM in the
fermentation medium, whereas cephamycin production was reduced
by 50% at phosphate concentration of ≥50 mM in the fermentation
medium. Romero et al. (1984) have described similar inhibitoryeffects
of phosphate concentration on production of CA by S. clavuligerus.
Rodriguez et al. (2000) reported on the process for the fermentative
production of CA and/or its salts by controlling the concentration of
soluble phosphate. They kept an initial soluble phosphate concentra-
tion between 150 and 600 mg/l, and after the initial stage maintained
it between 20 and 150 mg/l by adding soluble phosphate or removing
soluble phosphate from the medium. Similar results were reported by
Kranjc and Racman (1997) and Gupta (1998).
Lubbe et al. (1985) demonstrated the biosynthesis of expandase to
be repressed by inorganic phosphates, while the biosynthesis of
cyclase and epimerase was not. Lebrihi et al. (1987) described the
phosphate repression of cephamycin and CA production by S.
clavuligerus. They concluded that phosphate represses the biosynth-
esis of cephamycin synthetase and CA synthetase. In the presence of
2 mM phosphate, the specific activities of expandase, ‘cephamycin
synthetase’ and ‘CA synthetase’ were higher than in presence of
75 mM phosphate. The specific activity of ‘cephamycin synthetase’
was maximal with an initial concentration of 10 mM, whereas the
specific activity of expandase was maximal with 1 mM phosphate. A
correlation between cephamycin synthetase specific activity and
expandase specific activity was established at a phosphate concentra-
tion higher than 10 mM. The authors concluded that expandase was
an important enzyme in the mechanism by which phosphate
concentration affects biosynthesis of CA and cephamycins.
4.6. Effect of precursors
Townsend and Ho (1985) investigated the effect of amino acids on
CA production. They concluded that ornithine and arginine, amino
acids in the urea cycle, were better utilized than the other amino acids
for CA production. In addition, the oxazolidine ring of CA was derived
directly from the urea-cycle amino acids, i.e. ornithine first and then
arginine. Detailed isotopic-labeling experiments have placed strict
constrainson themechanismof the couplingreactionsthatlink the C3
and C5 building blocks drawn from primary metabolism to initiate CA
biosynthesis. Until presently, the primary metabolic precursors of C3
and C5 intermediates are well known to be derived from glycerol and
The primary metabolic precursors of CA are known to be arginine/
ornithine and a C3 intermediate. Feeding of glycerol during fermenta-
tion could maintain cell metabolism, and thereby effectively enhance
the CA production. Elson and Oliver(1978) indicated that the β-lactam
ring, i.e. the C3 unit, originated from the TCA cycle via gluconeogen-
esis. Glycerol when included in the medium was very well
incorporated in CA. The long-range
experiments suggested that the β-lactam ring might be derived via
the incorporation of intact glycerol.
The biosynthesis of CA begins with the condensation of arginine
and a three-carbon glycolytic intermediate (C3 precursor). It is
followed by a series of reactions to form proclavaminic acid, then
converted to clavaminic acid and finally to CA (Townsend and Ho,
1985; Valentine et al., 1993 and Stirling and Elson, 1979). The C3
precursor appears to be the rate-limiting factor for CA synthesis with
excess arginine failing to increase CA production (Ives and Bushell,
1997). Fang et al. (1996) observed an increase in β-lactams produced
by S. clavuligerus when concentrations of lysine above 10 mM were
13C–13C spin–spin coupling
added to cultures. Although the β-Lactam concentration increased
with increasing lysine concentration, the biomass was unaffected by
Ives and Bushell (1997) fed various amino acids to chemostat
cultures of S. clavuligerus and concluded that addition of amino acids
in the aspartate family (which are derived from pyruvate) relieve the
drain on C3 precursors. Lysine is a member of the aspartate family of
amino acids. These factors add further evidence to the rate-limiting
role of the C3 precursor, and to the C3 precursor being derived from
pyruvate. When supplied externally, the aspartate family relieves the
drain on pyruvate allowing growth and CA production to occur
simultaneously. Lysine present in the culture medium prevents
biosynthesis of lysine by the microorganism, and thus relieves the
drain of pyruvate in lysine production.
Saudagar and Singhal (2007b) reported on the nutritional
requirements and environmental conditions for submerged culture
of S. clavuligerus for CA production using orthogonal matrix method
(Taguchi L16 design) and also fed-batch fermentation for CA
production by feeding glycerol, arginine and threonoine to the
fermentation medium intermittently. CA production was increased
by 18% with the span of feeding glycerol and reached a maximum at
1.30 mg/ml with 120 h glycerol feeding as compared to 1.10 mg/ml in
the control. The production also increased with the span of feeding
amino acids and reached a maximum of 1.31 and 1.86 mg/ml with
feeding arginine and threonine, respectively in 120 h.
Both ornithine and arginine have been considered to be precursors
of CA. Chen et al. (2003) reported that there was no increase in CA
biosynthesis after arginine feeding (initially or intermittently), and
hence it may not be regarded as a rate-limiting substrate. In contrast
with arginine, the addition of ornithine, initially or intermittently,
revealed a remarkable enhancing effect. Due to the unusual presence
of the urea cycle in prokaryote, any added ornithine would provide an
abundant supply of arginine, the C5 precursor to CA (Fig. 3). Not only
did it reduce the anaplerotic carbon flux of C3 to the synthesis of C5-
amino acid, it also removed ammonia from cells resulting in toxic
intracellular concentration (Mendz and Hazell, 1996).
Furthermore, Romero et al. (1986) reported ornithine to strongly
inhibit cephamycin biosynthesis, another β-lactam antibiotic, pro-
duced by the same strain. Therefore, the feeding of ornithine could
allow more C3 precursor for the CA biosynthesis. Chen et al. (2003)
suggested ornithine rather arginine to be a better stimulator for CA
biosynthesis, although arginine is a later metabolic intermediate
relative to ornithine in the biosynthesis of CA by S. clavuligerus.
4.7. Effect of the operation mode of the bioreactor
Several types of bioreactor operation are being used in the
production of secondary metabolite such as batch mode, fed-batch
and continuous cultivation. The fed-batch method is more frequently
used due to its higher productivity and ease of operation. Fed-batch
strategies control the growth rate, prolonging the stationary phase, and
overcoming substrate inhibition and metabolite repression. Continuous
Fig. 3. Effect of glycerol and ornithine on the biosynthesis of clavulanic acid (Valentine
et al., 1993).
P.S. Saudagar et al. / Biotechnology Advances 26 (2008) 335–351
processing time, since dead time due to charge and discharge ope-
rations, bioreactor preparation and sterilization is avoided. However
productivity is often lowered by genetic variations liable to occur when
operation continues over a long time. Also, the probability of con-
tamination is much higher than in batch or fed-batch systems.
Baptista-Neto et al. (2005a) compared CA productivity in batch, fed-
batch and continuous fermentors by cultivating S. clavuligerus in a
complex medium. The operating conditions in all cases were a tem-
perature of 28 °C, aeration at 0.5 vvm, and agitation at 800 rpm. The CA
much higher than 194 mg/l in the batch culture and 293 mg/l in the
continuous culture. The highest productivityof 10.6 mg/l-h was obtained
in the continuous cultivation, as compared to 8.8 mg/l-h in the fed-batch
process and 3.5 mg/l-h in the batch process, suggesting that continuous
culture of S. clavuligerus is a promising strategy for CA production.
Baptista-Neto et al. (2005b) compared CA production in continuous
culture with and without cell recycling and in batch process by S.
clavuligerus Continuous cultivations with high cell concentration using
cell recycling were performed utilizing a hollow fiber ultrafiltration
moduleto separate cells fromthefiltratebroth.Thecontinuous cultures
without cell recycling and the batch cultivations were performed
conventionally. The highest productivity of 22.2 mg/l-h was attained in
the continuous cultivation with cell recycling, while highest CA
concentration of 470 mg/l was obtained in the batch process.
Kirk et al. (2000) studied the metabolic flux of S. clavuligerus to
purpose the authors conducted continuous cultivations with limited
sources of carbon, nitrogen and phosphorus and obtained specific pro-
ductivities of 0, 0.32 and 3.65 mg of CA per g of cells per hour,
respectively. De Almeida and Regalo (2000) reported the improved
process for CA production. They carried out fermentation with
continuous or semicontinuous feeding of one or more organic nitrogen
complex sources, preferably soybean meal, so as to control the protein
concentration in the filtered broth within certain limits during the time
course of the fermentation.
Teodoro et al. (2006); Mayer and Deckwer (1996) and Chen et al.
(2002) studied the influence of feeding conditions, feeding procedures
and the effect of glycerol feeding respectivelyon CA production in fed-
batch cultivation with medium containing glycerol. It was observed
that the production of CA increased with a decrease in the flow rate
and an increase of the glycerol concentration in the feed medium.
Chen et al. (2003) reported the enhancement of CA production by S.
clavuligerus with ornithine feeding in shake cultivations. Fed-batch
cultivations were started at 60 h after inoculation by feeding a solution
of glycerol and ornithine with a flow rate of 17.0 ml/h over a period of
100 h. A CA concentration of 311 mg/l was obtained, a 2.7-fold increase
as compared to in batch cultivation (115 mg/l).
Saudagar et al. (2008) reported on the immobilization of S.
clavuligerus on loofah sponge for the production of CA. Immobilization
of S. clavuligerus onto loofah sponge discs was studied with respect to
the optimization of the inoculum size (number of discs) and its
reusability for CA production. Best yield of 1.125 mg/ml CAwas reached
and 120 h duration in the first cycle. ImmobilizationofS. clavuligerus on
to loofah sponge discs permit repeated use under the specified
fermentation conditions for CA production. Although a decrease in the
final production level of CAwas observed with every reuse, the time for
initiation of CA productionwas decreased as the cells are already in the
production stage after the first cycle.
5. Scale-up studies for clavulanic acid production
Scale-up is very important during the process development.
Expanding a lab scale unit to a commercial one is a challenge due to
difficulty in assessing the factors affecting the scale-up process during
the cultivation. As a result, many large-scale processes give a lower
yield than that predicted on the basis of laboratory studies. The
traditional method for the scaling-up a fermentation system is usually
based on empirical criteria such as constant power input per unit
volume, a constant mass transfer coefficient, constant mixing time
and a constant impeller tip velocity (Shuler and Kargi, 1992). In the
stirred fermentors, oxygen transfer to the microbial strains is affected
by the aeration rate, agitation rate and the broth characteristics
(viscosity, surface tensionetc.). The influenceof dissolved oxygen(DO)
level and shear condition on biomass and product yields in specific
processes involving S. clavuligerus have been reported in the literature
(Rosa et al., 2005; Rollins et al., 1988).
Rollins et al. (1988) studied the effect of DO level on cephamycin C
production by S. clavuligerus in a 10 L bioreactor. The authors con-
cluded that controlling the DO at 50% and 100% saturation level
increased the rate of specific cephamycin C production, two and
threefold, respectively, compared to the experiments without DO
control.Rosaet al.(2005)studiedtheeffectof DOandshear, expressed
as impeller tip velocity, on CA production. Cultivations were per-
formed in a 4 L fermentor equipped with 6 flat blade turbine impellers
(0.076mmin diameter)anda stainless steelringgassparger,at speeds
of 600, 800 and 1000 rpm and a fixed air flow rate (0.5 vvm). DO level
had no remarkable effect on CA production, whereas most significant
improvement in CA production was related to high stirrer speeds i.e.
possibly fluid shear.
On the contrary, Tarbuck et al. (1985) studied the effect of stirrer
speed on CA production in both batch and fed-batch fermentations in
a 10 L fermentor equipped with Rushton turbine impellers. When the
stirrer speed was increased from 350 to 500 rpm in batch cultivations,
CA production decreased from 36 mg/l to 18 mg/l. An air flow rate of
0.25-vvm and stirrer speed of 350 rpm could give maximum CA
concentration of only 36 mg/l.
Roubos et al. (2001) proposed a quantitative approach for
Observations during 10-batch cultivation with S. clavuligerus on a
defined media indicated the organism to be very sensitive to fluid shear
when grown in batch cultures with increasingstirrer speeds. The stirrer
speed was increased to keep the DO level above 50% air saturation. A
quantitative approach based on the calculation of elemental balances
and a simple mathematical model was proposed to characterize the
biomass lysis. A linear relation between biomass yield and observed
specific growth rate was determined. Results showed cell lysis to occur
at high degradation rate, e.g. μmax=0.16/h and kd=0.07/h, when the gas
power input increased above 1.1, 1.7, or 2.0 kW/m2, respectively
depending on the medium composition. The overall biomass yield on
the substrate was dramatically reduced in all experiments (N30%).
Large et al. (1998) studied the effect of agitation rate on lipid
utilization and CA production in 5 l batch cultivation of S. clavuligerus.
Increasing the tip speed from 1.88–2.83 ms−1improved biomass
production, while 3.77 ms−1was detrimental to growth. Lipase
activity decreased with increasing tip speeds. An optimum tip speed
was found for CA production. Tip speed did not significantly alter the
rate of lipid utilization or the total lipid remaining at the end of the
process. Jiang et al. (2004) optimized CA fermentation and reported
that CA reached more than 3000 μg/ml at temperature, 28 °C; stirring
rate, 400 r/min; airflow, 0.5vvm; dissolved oxygen concentration
maintained at higher than 40%; fermentation period, 4 days.
Morphological characteristics of mycelial-submerged cultures
have been established as one of the key bioprocess parameters. The
morphological type and the related physiology strongly depend on
environmental conditions in the bioreactor, and in turn affect the
rheological properties of the broth and thereby bioreactor perfor-
mance. Accordingly, the productivity and energy consumption of the
process are functions of the morphology.
Pinto et al. (2004) studied the effects of varying inoculum age and
production scale on the morphology and viability of S. clavuligerus by
P.S. Saudagar et al. / Biotechnology Advances 26 (2008) 335–351
analyzing visible and fluorescent light images acquired throughout
pilot-plant and pre-industrial scale fermentations. Changes in produc-
tion scale revealed that in 5 l fermentor, the maximum hyphal area
obtained was double the value obtained in 0.5 l fermentor. This was
fermentor caused by higher tip speeds. The morphological quantifica-
tion based on elongation and branching rates allowed fermentations to
be pattern classified into distinct physiological time zones namely
elongation, branching, fragmentation,etc.The generalpatternobserved
for fermentations inoculated with late exponential phase inocula was
except that both the elongation and branching periods started earlier in
the former case. Using the available staining technique and image
acquisition system, the viability seemed to be generally high and con-
stant throughout the time course of all the studied fermentations.
6. Stability of clavulanic acid
As with other β-lactam compounds, CA in its crude form is
chemically unstable. Several authors investigated the stability of CA in
buffered aqueous solutions at various pH (Haginaka et al.,1981; Mayer
and Deckwer,1996; Bersanetti et al., 2005). These authors observed CA
degradation to follow pseudo- first-order kinetics, and that this
degradation was catalyzed by the buffering salts used to maintain
Haginakaet al. (1981),whoinvestigatedCA stabilityat 35°Candan
ionic strength (μ) of 0.5 at different pH, observed the degradation rate
constant to be highly dependent on the pH. CA had maximal stability
at pH 6.39. The authors also studied the catalytic effects of buffer
species at constant pH and ionic strength, and found the degradation
rate constant (Kd) to increase with increasing buffer concentration.
The degradation rate constant (Kd) is highly dependent on the pH, i.e.,
a logarithmic plot of Kdvs pH is parabolic with a minimum at pH 6.4
and a steep slope toward acidic or basic conditions. CA is about 10
times less stable than penicillin G at neutral pH, five times less stable
in the alkaline region, and has similar stability in the acidic region.
Furthermore, a linear relation between Kd and the phosphate
concentration was found at pH between 5 and 8.
Temperature has a majoreffect on CA degradation. Bersanetti et al.
(2005) reported that the stability of the antibiotic at 20 °C and ionic
strength (µ) 0.5 is highest at a pH around 6.0; and that degradation
occurred more rapidly inbasic solutions than inacidic solutions. The
authors showed the relation between the hydrolysis rate constant of
CA in aqueous solution and pH in the range from 2 to 10. They found
ionic strength to have no influence on this rate in the range studied.
Mayer and Deckwer (1996) studied the simultaneous production
and decomposition of CA during S. clavuligerus cultivation on
complex medium containing either soy meal extract or soy meal
particles. The in vitro and in vivo CA stability was investigated. The
authors concluded that the inactivation rate constants for the invivo
were considerably higher (2 to 10-fold) than those in vitro. While
acid-catalyzed hydrolysis seemed to be responsible for most of the
measured instability of CA in vitro, as indicated by the pH
dependency of the degradation rate constant, one or several
additional mechanisms were active in CA degradation during the
stationary phase of cultures in the soy meal extract medium.
Bersanetti et al. (2005) studied the kinetics of CA degradation,
established best conditions of temperature and pH to be used in CA
recovery processes. CA degradation rate from various sources was
6.2 and 7.0 which are in the range where the authors observed the
lowest degradation rates (Haginaka et al., 1981; Mayer and Deckwer
1996). The rate constants were determined on the assumption that the
process followed first-order kinetics. CA in aqueous solution, or that
present in culture medium was used. The hydrolysis rate constant as a
function of temperature followed Arrhenius equation at both the pH.
and that the Arrhenius equation could be applied to establish a
relationship between the degradation rate constant and temperature,
atbothpH.CAinfermentationmedium is muchmoreunstablethanthe
standard solution and from a commercially available medicine. Also, it
was observed that CA was more stable at pH 6.2 than at pH 7.0,
irrespective of the CA source.
Roubous et al. (2002) studied CA degradation in S. clavuligerus fed-
batch cultivations. Three different types of experiments were used to
elucidate CA degradation under fed-batch cultivation conditions as
• First, the influenceof individual medium compounds was examined.
• Second, degradation was monitored during the exponential growth
phase in batch cultivations.
• Third, CA degradation was studied in the supernatant of samples
taken during a fed-batch.
• In addition, data from six fed-batch cultivations were studied to
derive information on CA degradation during the production phase.
These cultivations were based on a mineral medium, containing
glycerol, glutamate, ammonium, and phosphate as the main nutrients.
Ammonium concentration had a large influence on the degradation
rate constant. In addition, either changes in the substrate availability
or high concentrations of ammonium or glycerol were mainly res-
ponsible for increasing the degradation rate constant. Finally, a linear
and a fuzzy logic model were made to predict CA degradation rates in
Lynch and Yang (2004) examined the role of CA on biomass
accumulation and production of CA in batch cultures of the
organism. The authors suggested that the rate of degradation was
equivalent to the rate of production of CA following a period of
initial additive degradation. The results indicated that CA is both
produced and degraded in cultures of S. clavuligerus and that the
products of degradation are used by the organism, resulting in
further production of the antibiotic.
7. Downstream processing of clavulanic acid
Development of new and efficient separation processes is based on
more effectively exploiting differences in the actual physicochemical
properties of the product such as surface charge/titration curve, surface
hydrophobicity, molecular weight, biospecificity towards certain ligands
(e.g. metal ions, dyes), PI and stability, compared to those of the
contaminant components in the crude broth (Asenjo, 1993). The main
physicochemical factors involved in the development of separation
processes are shown in Table 3.
CA or its salts may be extracted directly from the culture medium
in various ways, but normally the cells of the S. clavuligerus are first
removed from the culture medium by filtration or centrifugation
before such extraction procedures are commenced. Whole broth
extraction may also be employed.
7.1. Solvent extraction
Solvent extraction from cold clarified culture medium adjusted to
acid pH, and methods utilizing the anionic nature of CA at neutral pH
such as the use of anion exchange resins, have been found to be
particularly useful for isolation of CA. A further useful method is to
form an ester of CA, purify the ester and regenerate the acid or its salt
be divided into a primary isolation process followed by a further puri-
fication process. Suitable primary isolation processes include solvent
extraction of the free CA. In the solvent extraction process the CA is
extracted into an organic solvent from cold clarified culture medium,
which may be the whole broth adjusted to an acid pH value.
P.S. Saudagar et al. / Biotechnology Advances 26 (2008) 335–351
In one solvent extraction process for CA, the clarified medium was
chilledandthepHlowered intotheregionof pH1–2 bytheadditionof
acid while mixing with a substantially water immiscible organic
solvent. Suitable acids used to lower the pH include hydrochloric,
sulphuric,nitric,or phosphoric acids.Suitable organic solvents include
n-butanol, ethyl acetate, n-butyl acetate and methyl isobutyl ketone,
and other similar solvents. Methyl isobutyl ketone is a particularly
suitable solvent in the extraction of the acidified culture filtrate. After
separation of the phases, CA is found in the organic phase. CA may be
back extracted from the organic solvent into an aqueous solution or
suspension of an alkali metal or alkaline earth metal base, such as
sodium hydrogen carbonate, potassium hydrogenphosphate buffer or
calcium carbonate, or water, while maintaining the pH at approxi-
mately neutrality (pH 7). Simon (1997) reported extraction of CA from
an organic solvent phase into an aqueous medium phase, using a
mixing region in which the phases are mixed rapidly under high
turbulence and shear stress. The aqueous extract, after separation of
the phases, may be concentrated under reduced pressure. Freeze-
drying may also be employed to provide a solid crude preparation of
the salt of CA. Such solid preparations are stable when stored as a dry
solid at −20oC. This process may be modified in known ways by for
example, additional purification steps applied to the organic solvent
phase to remove high molecular weight impurities from the impure
A further secondary purificationprocess for CA is that described by
Cook et al. (1981) and Cook and Wilkins (1995) in which a solution of
impure CA in an organic solvent is contacted with t-butylamine to
form the t-butylamine salt of CA, which is then isolated, thereby
separating the CA from impurities remaining in the organic solvent.
The salt is then converted back to CA or into a derivative of CA such as
an alkali metal salt or an ester. Zhang and McKnight (2003) also
reported a similar process for the preparation of a pharmaceutically
acceptable metal salt of CA. They extracted CA from aqueous
fermentation broth into an organic solvent, converted it into an
intermediate tertiary butylamine clavulanate at a pH below 6.0, and
finally converted the amine salt to potassium clavulanate. Other
known secondary purification processes for CA involve the use of
other organic amines such as diethylamine, tri- (lower alkyl) amines,
dimethylaniline and N,N'-diisopropylethylenediamine to form salts
and/or other derivatives thereof with the CA. These purification
processes have the inherent disadvantage that they can introduce
traces of the amine, or leave residual traces of salts of CA with the
amine, in the final product.
Such back extraction processes present a problem when CA is
prepared, as CA is particularly water-sensitive. In conventional back
extraction processes CA can remain in contact with water for a long
time, typically around an hour or more as the solution concentration
of CA builds up under the relatively gentle mixing and separating
conditions generally used. This can lead to extensive hydrolytic
Cardoso (1998) and Summeret al. (1999) reported a process for the
isolation of a pharmaceutically acceptable alkali metal salt of CA from
the fermentation broth. The steps included filtration of the fermented
broth, extraction of the CA in a water immiscible or partly water
immiscible solvent at pH 1.2–2.0, and precipitation of the alkali metal
7.2. Membrane technology
Alves et al. (2002) studied isolation of antibiotics from industrial
fermentation broths using membrane technology. This technology
involves one, two or three membrane operations in sequence de-
pending on the cases. The first operation, directed towards the solid–
liquid separation of fermented broths is, in general, a microfiltration
(MF) (Tanaka et al., 1993; Bowen and Hall, 1995; Adikane et al., 1999)
or an ultrafiltration (UF) (Morao et al., 2001). In both cases, a con-
centrate containing all the biomass of the broths and a permeate
containing the antibiotic, salts and water are obtained. The quality of
permeate thus obtained, determines the subsequent operation(s). In
some applications, second ultrafiltration acting as a purification step
for the first permeates, is needed. Diafiltration (DF) is usually needed
to improve the performance of UF.
The permeates are generally very dilute solutions of the antibiotic,
and need a concentration step prior to solvent extraction. Nanofiltra-
tion (NF) is a suitable operation for this concentration task. Alves et al.
(2002) established the procedure for the isolation of CA from
fermentation broths using membrane technology through:
• The study of the performance of UF for the step of solid–liquid
separation of CA from fermentation broths as function of the MWCO
of the membranes, of the material and of the geometry of the
• The study of the effect of an intermediate purification step of the
first permeates on the final extraction of CA.
The need of an intermediate purification step of the permeates
of the first operation is established based on the results obtained
with the solvent extraction of the concentrated (nanofiltered)
antibiotic. If the solvent extraction of the NF concentrates coming
from a single UF operation occurs without difficulties, the quality of
permeate of the first ultrafiltration is good enough. On the contrary,
if the extraction occurs with an extended interphase, a posterior
purification is needed. This purification can be achieved with
further more tight ultrafiltration(s), and is followed by a concen-
tration operation using nanofiltration prior to the solvent extrac-
Extraction was carried out using two tubular ceramic membranes
with molecular weight cut-offs (MWCO) of 15 and 150 kDa and two
flat sheet organic membranes with MWCO of 20 and 5 kDa in order to
establish the sequence of operations needed to achieve a good phase
separation when the permeates are subjected to solvent extraction.
Ultrafiltration fluxes are higher for the 150 kDa membrane, but the
quality of its permeate is not good enough for acceptable phase
separation: Hence, for solid–liquid separation a further purification of
its permeates using the 20 kDa membrane is required. The purification
of permeates of the membrane of 20 kDa using a membrane of 5 kDa
was also investigated. This last operation did not improve the process.
Ultrafiltration experiments using either the membranes of 15 kDa or
of 20 kDa directly for the solid–liquid separation showed that a good
performance tobe obtained for both membranes. High fluxes and high
Physiochemical basis for development of a separation process
Physiochemical basisSeparation process
Aqueous two-phase partitioning
Reverse micelle extraction
Hydrophobic interaction chromatography
Reversed phase chromatography
Aqueous two-phase partitioning
Size Gel filtration
Isoelectric point Chromatofocusing
Supercritical fluid extraction
Ref: Asenjo and Chaudhari (1996).
P.S. Saudagar et al. / Biotechnology Advances 26 (2008) 335–351
yields of antibiotic as well as a good phase separation were obtained,
when their permeates were subjected to solvent extraction.
7.3. Ion exchange chromatography
The CA purification process includes a series of steps: at the end of
fermentation, the broth is first clarified by filtration or centrifugation
and the S. clavuligerus mycelium is discarded (Butterworth, 1984).
Primary extraction includes processes such as adsorption, ion
exchange chromatography or liquid–liquid extraction. These two-
phase separation methods have, until today, been the main choice for
low molecular weight compounds presented in diluted solutions, as is
thecaseof antibioticsproduced byfermentation,andthis isbecauseof
their low costs and relatively high yields (Mayer and Deckwer, 1996).
In the case of CA, the reported yields for ion exchange, adsorption and
liquid–liquid operations are generally low. In fact, it undergoes rapid
degradation under normal processing conditions (Mayer and
Deckwer, 1996). Thus, to design effective and selective adsorption
separation, the effect of operating conditions such as temperature, pH,
and adsorbate concentration on the adsorption kinetics and equili-
brium must be known.
Barboza et al. (2003) studied the effect of temperature on the
kinetics of adsorption and desorption of CA by ionic exchange. The
work on thermodynamics (ΔH° and ΔS°) of adsorption of CA involved
equilibrium and kinetic studies, with batch shake experiments using
Amberlite IRA 400 ion exchanger resin carried out at four different
temperatures. A model of the CA adsorption process, that took mass
transfer limitation into account, was proposed.
The kinetics of adsorption and desorption of CA by ionic exchange
was well represented by the model proposed by Barboza et al. (2003),
which allows for important transport parameters such as the
resistance of the external film and, principally, the effective diffusivity
to be determined, in addition to the equally important intrinsic
constants of adsorption and desorption, since the kinetics is the
limiting stage. The literature on the identification of intrinsic kinetics
and the influence of temperature thereon is scant. Temperature
showed a more significant effect on the equilibrium constant (KD) and
on the intrinsic constants k1and k3. This effect, though perhaps not
highlysignificant per se, is quite characteristic and especially influence
the desorption stage, which is facilitated at higher temperatures. The
methodology presented by Barboza et al. (2003) to estimate the
adsorption enthalpy proved to be appropriate, showing a ΔH° value of
29.15 kJ mol−1. These values portray an exothermic process in the
formation of the CA complex on the sites of the resin.
Besides the thermodynamic characterization of sorption systems,
as described above, their dynamic properties are equally important to
help the selection and optimization of a purification process. They
determine the characteristic time ranges within which the sorption
step can be carried out to ensure a good performance of the
equipment, give support to the process scale-up and, in the specific
case of CA, they give support to the evaluation of the performance of
each system with regard to losses due to the product degradation
occurring during the sorption step.
Mayer et al. (1996) studied diffusivity of CA in different porous
systems (Amberlite IRA400, activated carbon and two ion–pair
adsorption systems based on Amberlite XAD4 and activated carbon
as matrixes and water-soluble quaternary ammonium salts as ion
pairing substances) using batch adsorption experiments. The results
described in their work enabled comparison between the different
sorption systems tested for the purification of CA from fermentation
supernatants. Of the different matrices used in the study, activated
charcoal showed the poorest characteristics, basically due to its
microporous structure, which hampered the intraparticle diffusion of
CA and consequently slowed down the adsorption process. The
presence of benzyl tributyl ammonium chloride (BTBA) ameliorated
the recovery in the elution step (Mayer et al., 1996), probably by
decreasing the proportion of the direct adsorption mechanism of CA
on activated charcoal when compared to the adsorption based on ion–
pair interactions although, does not promote the diffusion properties
to this material. These adsorption systems allow far more reversible
interactions than those between CA and activated charcoal.
With respect to their desorption properties, the use of Amberlite
XAD4-ABDA (alkyl benzyl dimethyl ammonium chloride) as the
sorption system allowed recovery higher than 99%, whereas the use
of Amberlite IRA-400 is inevitably linked to low recoveries due to an
in situ accelerated decomposition of CA (Mayer et al., 1996). In
comparison to activated charcoal, the matrixes based on polymeric
macroreticular materials such as XAD4 and IRA400 exhibited good
structural properties, which enabled rapid adsorption. The system
XAD4-ABDA based on ion–pair adsorption showed some advantages
when compared to IRA-400, which can be summarized as
• The diffusivities are less influenced by the temperature as that from
the ion exchanger and
• The presence of competitive medium components is less deleterious
to the kinetic course of the adsorption, which means that it is more
selective to CA than IRA400 and is less affected by higher ionic
Mosbach et al. (2003) reported a novel process for the removal of
impurities from CA using a selective a molecularly imprinted polymer
as an adsorbent.
8. Pharmacological actions of clavulanic acid
The effectiveness of a β-lactamase inhibitor/β-lactam combination
against Gram-negative pathogens depends on many interplaying
factors, one of which is the penetration of the inhibitor across the
outer membrane. Farmer et al. (1999) measured the relative penetra-
tions of CA, sulbactam, tazobactam and BRL 42715 into two strains of
Escherichia coli producing TEM-1 β-lactamase, two strains of Klebsiella
pneumoniae producing either TEM-1 or K-1, and two strains of Enter-
obacter cloacae each producing a Class C β-lactamase. It was shown
that CA penetrated the outer membranes of all these strains more
readily than the other β-lactamase inhibitors. For the strains of E. coli
and K. pneumoniae, CA penetrated approximately 6–19 times more
effectively than tazobactam, 2–9 times more effectively than sulbac-
tam, and 4–25 times more effectively than BRL 42715. The superior
penetration of CAobserved in this study contributed to the efficacy of
CA/β-lactam combinations in combating β-lactam resistant bacterial
Hoban et al. (2003) compared the in vitro activity of amoxycillin–
CA with four comparator oral antimicrobial agents; ampicillin,
azithromycin, cefuroxime and trimethoprim–sulphamethoxazole
against 4536 recent clinical isolates covering 29 species isolated in
the US and Canada between 1997 and 1999. Based upon minimum
inhibitory concentrations (MICs), amoxycillin–CA was the most active
agent against many Gram-positive species and phenotypes including
methicillin susceptible Staphylococcus aureus (MSSA) Staphylococcus
epidermidis, Enterococcus faecalis, Streptococcus pyogenes, Streptococ-
cus pneumoniae including penicillin intermediate and macrolide
resistant strains and was as active as ampicillin against Streptococcus
agalactiae, penicillin resistant S. pneumoniae and viridans streptococci.
Against Enterobacteriaceae amoxycillin–CA generally displayed weak
activity with only Proteus mirabilis and Proteus vulgaris displaying
levels of susceptibility above the 90th percentile. Amoxycillin–CA had
significant activity against many species of Gram-negative non-
Enterobacteriaceae including Haemophilus influenzae, Haemophilus
parainfluenzae and M. catarrhalis but negligible activity against Bur-
kholderia cepacia, Pseudomonas aeruginosa and Stenotrophomonas
maltophilia. Amoxycillin–CA continues to retain excellent activity
against the majority of targeted pathogens despite 20 years of clinical
P.S. Saudagar et al. / Biotechnology Advances 26 (2008) 335–351
Akalin (1996) reported on clinical implications of aminopenicil-
lins with β-lactamase inhibitors. Aminopenicillin/β-lactamase inhi-
bitor combinations (ampicillin/sulbactam and amoxicillin/CA) are
well established in the therapy of a wide range of infections in both
hospital and primary-care settings as a result of their very broad-
spectrum activity and good tolerance. These agents are particularly
suited to the prophylaxis and treatment of polymicrobial infections.
Clinical studies have demonstrated their efficacy in the treatment of
diabetic foot infections, intra-abdominal infections, aspiration-
related lung infections, brain abscesses and pelvic inflammatory
disease, and in the prophylaxis of infections following abdominal,
pelvic and head and neck surgery. Recent studies have also revealed
aminopenicillin/β-lactamase inhibitors to provide therapy or pro-
phylaxis, which is more cost-effective than with comparative anti-
8.1. Clavulanic acid in lower respiratory tract infections
Legnani (1997) studied the role of oral antibiotics in treatment of
community-acquired lower respiratory tract infections. Amoxicillin/CA
has been one of the first choice treatments for community-acquired
lower respiratory tract infection since its introduction nearly 25 years
ago. Since then, it has become the “gold standard” against which most
new oral antimicrobials are compared, but none of these newer agents
has demonstrated a superior efficacy. On the contrary, two recent
floxacin in the treatment of acute exacerbations of chronic bronchitis
have demonstrated a higher efficacy rate for amoxicillin/CA.
Uwaydah et al. (2006) reported a study in which a total of 123
clinical isolates of S. pneumoniae were collected from all over Lebanon
and tested for their susceptibility to penicillin: 30.1% were susceptible
(MIC≤0.06 μg/mL), 56.1% were intermediately susceptible (MIC 0.09–
1.0 μg/mL) and 13.8% were resistant (MICN1.0 μg/mL). The oxacillin
disk-screening test detected all penicillin-resistant isolates, but erro-
neously designated two penicillin-intermediate isolates as penicillin
susceptible. All isolates were consistently susceptible to levofloxacin,
but cross-resistance between penicillin and the three tested cepha-
losporins was frequently noted. The in vitro activity of amoxicillin/CA
paralleled that of penicillin; however, 92.7% of the isolates were
designated as susceptible based on the recommended interpretive
cut-off point (MIC≤2/1 μg/mL).
The increasing rate of treatment failure with penicillin and
other β-lactam antibiotics in pharyngotonsillitis caused by group A
β-hemolytic streptococci (GABHS) has prompted the search for
alternative antimicrobials. Both clindamycin and amoxicillin/CA
have excellent clinical activity in pharyngotonsillitis. Mahakit et al.
(2006) compared the clinical and bacteriologic efficacy and
tolerability of oral clindamycin with those of oral amoxicillin/CA
in the outpatient treatment of acute recurrent GABHS pharyngo-
tonsillitis. In this study, in patients with acute recurrent GABHS
pharyngotonsillitis, oral clindamycin 300 mg BID and oral amox-
icillin/CA 1 g BID achieved comparable rates of bacteriologic eradi-
cation at 12 days and 3 months and comparable clinical cure rates
at 3 months. Patients who received clindamycin had significantly
greater clinical cure rates at 12 days. Both regimens were well
Blondeau et al. (2001) measured the susceptibility of Canadian
isolates of threerespiratory tract pathogens (H.influenzae,M.catarrhalis
and S. pneumoniae) to several currently approved antimicrobial agents
by two different methods. The susceptibility of isolates to seven
fluoroquinolones was also determined. β-lactamase was produced by
M. catarrhalis isolates. For S. pneumoniae 83/374 (22.2%) isolates were
penicillin resistant and of these 2.1% (8/374) showed high level
resistance (MIC≥2 mg/l). Regardless of methodology, all fluoroquino-
lones were highly active against H. influenzae (MIC90≤0.031 mg/l) and
susceptibleto cefaclorand cefprozil. Azithromycin susceptibilityranged
from 82.6 to 99.2% depending on the method. M. catarrhalis isolates
were uniformly susceptible to all agents tested except amoxicillin.
Koeth et al. (2004) developed a new pharmacokinetically
enhanced, oral formulation of amoxicillin/CA to overcome resistance
in the major bacterial respiratory pathogen S. pneumoniae, while
maintaining excellent activity against H. influenzae and M. catarrhalis,
including β-lactamase producing strains. This study was conducted to
provide in vitro susceptibility data for amoxicillin/CA and 16
comparator agents against the key respiratory tract pathogens. The
extended release formulation of amoxicillin/CA has potential for
empirical use against many respiratory tract infections worldwide due
to its activity against species resistant to many agents currently in use.
8.2. Clavulanic acid in urinary tract infections
Adjei and Opoku (2004) studied the effect of CA on urinary tract
infections in African infants. Urinary tract infection (UTI) causes
significant illness in the first 2 years of life. The diagnosis in most
developing countries is often overlooked due to the tedious nature of
obtaining urine from young infants who would not void voluntarily.
Misdiagnosis very often led to avoidable ill health and long-term renal
damage. There is a need to diagnose UTI in febrile infants to alert
clinicians. A prospective study of febrile infants aged up to 12 months
on admission was undertaken in a 6-months period. Urine specimen
was obtained by supra pubic aspiration and investigated. Out of 150
urine samples screened for UTI, 45 (30%) had positive bacterial
growth. E. coli (32%) and Proteus sp. (22%) formed more than 50% of
the total isolates. The Gram-positive bacteria isolated was S. aureus
representing 11%. All isolates were susceptible to cefuroxime and
resistant to ampicillin. Susceptibility to amoxicillin/CA was 77.8% and
to nitrofurantoin was 67%.
8.3. Clavulanic acid in alveolar osteitis
Alveolar osteitis, also known as dry socket, results when a normal
clot fails to form in the socket of a recently extracted tooth. This
condition is usually very painful, and always considered as an
emergency. Oringer (2003) reported the combination of oral amox-
icillin plus CA and chlorhexidine rinse to reduce the incidence of
alveolar osteitis associated with the extraction of mandibular third
molars. The incidence of alveolar osteitis was 20.9% in the chlorhex-
idine-rinse-only group, 8.9% in the chlorhexidine/amoxicillin-plus-
clavulanic-acid group, and 23.7% in the saline-rinse-only group. The
incidence of alveolar osteitis was significantly less (P=.001, χ2test) in
the group that rinsed with chlorhexidine and received the antibiotic.
Chlorhexidine rinse alone did not significantly reduce the incidence of
dry socket as compared with the control group.
Delilbasi et al. (2002) reported on effects of 0.2% chlorhexidine
following mandibular third molar extractions and investigate adverse
reactions to chlorhexidine. The authors concluded that it would be
more beneficial to use chlorhexidine solution with a β-lactamase in-
hibitor-containing antibiotic to enhance its effectiveness for the pre-
vention of alveolar osteitis.
8.4. Clavulanic acid in endodontic abscesses
Baumgartner and Xia (2003) studied antibiotic susceptibility of
bacteria associated with endodontic abscesses. Antibiotics to treat
endodontic infections are routinely prescribed based on previously
established susceptibility tests. The increased resistance to the
currently recommended antibiotics has been a concern. Antibiotic
susceptibility tests on a panel of bacteria isolated from endodontic
P.S. Saudagar et al. / Biotechnology Advances 26 (2008) 335–351
infections was performed. The bacteria in this study were aseptically
aspirated with a needle from endodontic abscesses, cultivated, and
identified at the species level. Each of the 98 species of bacteria was
tested forantibiotic susceptibility to a panel of six antibiotics using the
E-test. The antibiotics were penicillin V, amoxicillin, amoxicillin+CA,
clindamycin, metronidazole, and clarithromycin. The percentages of
susceptibility for the 98 species were penicillin V: 83/98 (85%),
amoxicillin: 89/98 (91%), amoxicillin+CA: 98/98 (100%), clindamycin:
94/98 (96%), and metronidazole: 44/98 (45%). Metronidazole had the
greatest amount of bacterial resistance; however, if used in combina-
tion with penicillin V or amoxicillin, the susceptibility increased to
93% and 99%, respectively.
8.5. Clavulanic acid in pregnancy
Czeizel et al. (2001) studied the human teratogenic potential of
augmentin (amoxicillin+CA) treatment during pregnancy, and
concluded it unlikely to increase the risk of congenital abnormalities
in newborn infants when used in usual therapeutic dose. However,
the number of cases and controls was limited; therefore, further
multicenter–multinational studies are needed for the final risk
8.6. Clavulanic acid in diarrhea
No controlled trial has examined the clinical efficacy of antibiotics
in small bowel bacterial overgrowth. Attar et al. (1999) reported
norfloxacin and amoxicillin–CA to be effective in the treatment of
bacterial overgrowth-related diarrhea.
Stricker et al. (2006) studied on puerperal mastitis in adolescents.
Mastitis in non-lactating adolescents is rare and its cause is still
unclear. This retrospective study summarizes 22 such episodes, in 3 of
which S. aureus was isolated. Serum prolactin levels were normal.
Most patients were successfully treated with oral amoxicillin–CA.
Three patients with bilateral breast cysts had a recurrence.
Zechini et al. (2006) reported a case of subacute endocarditis in a
55-year-old patient affected by left atrial myxoma and with a severe
mitral regurgitation. Lactococcus lactis subsp. lactis was isolated from
blood cultures and infection was eliminated by treatment with
England (1999) reported on effect of CA-potentiated amoxycillin on
semen quality at two different doses (12.25 mg/kg and 25 mg/kg orally
twice daily for 28 days) and libido in dogs. No significant difference in
either parameter between control dogs and treated dogs were found.
Despite the marked effect of certain antibiotic agents upon spermato-
to twice the therapeutic dosage recommended by the manufacturer
without a deleterious effect upon semen quality.
9. Toxicity associated with clavulanic acid
Amoxicillin/CA is a widely used antibiotic. Hepatic dysfunction is a
rare adverse reaction associated with this combination antibiotic.
Nathani et al. (1998) report the case of a 40-year-old woman with a
somewhat unusual presentation of amoxicillin/clavulanate-related
cholestatic hepatotoxicity and multiple duodenal erosions whose
diagnosis was delayed until inadvertent rechallenge with the anti-
biotic combination. The diagnosis may be missed because the onset of
signs/symptoms may occur several weeks after the cessation of
therapy. The hepatic dysfunction may be severe and more prevalent in
elderly patients, is usually reversible, although chronic liver disease
and deaths have been reported. Immunological hypersensitivity is
considered to be the most likely mechanism resulting in liver injury.
Amoxicillin/clavulanate should be used with caution in patients with
underlying liver disease and in the elderly.
Hepatotoxity associated with amoxicillin/CA is usually a self-
limited disease with complete recovery. Chawla et al. (2000) reported
a rapidly progressing liver disease with ductopenia and portal fibrosis
in a 3-year-old boy treated with Augmentin.
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