Acinetobacter: Environmental and Biotechnological Applications

Abstract

Among microbial communities involved in different ecosystems such as soil, freshwater, wastewater and solid wastes, several strains belonging to the genus of Acinetobacter have been attracting growing interest from medical, environmental and a biotechnological point of view. Bacteria of this genus are known to be involved in biodegradation, leaching and removal of several organic and inorganic man-made hazardous wastes. It is also well known that some of Acinetobacter strains produce important bioproducts. This review summarizes the usefulness and environmental applications of Acinetobacter strains.

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African Journal of Biotechnology Vol. 2 (4), pp. 71-74, April 2003
Available online at www.academicjournals.org/AJB
ISSN 1684-5315 © 2003 Academic Journals
Acinetobacter: environmental and biotechnological
applications
Desouky Abdel-El-Haleem
Environmental Biotechnology Department, Genetic Engineering and Biotechnology Research Institute, Mubarak City for
Scientific Research and Technology Applications, New Burg-Elarab City, Alexandria, Egypt. Phone: (00203) 459-3421,
fax: (00203) 459-3423, e-mail: abdelhaleemm@yahoo.de
Accepted 28 March 2003
Among microbial communities involved in different ecosystems such as soil, freshwater, wastewater and solid
wastes, several strains belonging to the genus of Acinetobacter have been attracting growing interest from
medical, environmental and a biotechnological point of view. Bacteria of this genus are known to be involved in
biodegradation, leaching and removal of several organic and inorganic man-made hazardous wastes. It is also
well known that some of Acinetobacter strains produce important bioproducts. This review summarizes the
usefulness and environmental applications of Acinetobacter strains.
Key words: Acinetobacter, biodegradation, xenobiotic, oil, heavy metals, bioproducts, lipases, polysaccharides.
INTRODUCTION
Acinetobacter spp. are widespread in nature, and can be
obtained from water, soil, living organisms and even from
human skins. They are oxidase-negative, non-motile,
strictly aerobic and appear as gram-negative coccobacilli
in pairs under the microscope. They can use various
carbon sources for growth, and can be cultured on
relatively simple media, including nutrient agar or
trypticase soya agar. Also, most members of
Acinetobacter show good growth on MacConkey agar
with the exception of some A. lwoffii strains (Bergogne-
Bérézin and Towner, 1996).
Species of Acinetobacter have been attracting
increasing attention in both environmental and
biotechnological applications. Some strains of this genus
are known to be involved in biodegradation of a number
of different pollutants such as biphenyl and chlorinated
biphenyl, amino acids (analine), phenol, benzoate, crude
oil, acetonitrile, and in the removal of phosphate or heavy
metals. Acinetobacter strains are also well represented
among fermentable bacteria for the production of a
number of extra-and-intracellular economic products such
as lipases, proteases, cyanophycine, bioemulsifiers and
several kinds of biopolymers.
The focus of this review, therefore, concerns the use of
several Acinetobacter strains as a biocatalyst to
remediate various environmental contaminants and other
biotechnological applications.
BIOREMEDIATION OF CONTAMINANTS
Several investigations have shown that many toxic
compounds can enter the environment, disperse and
persist to a great extent (van der Meer et al., 1992). The
application of conventional physical or chemical
processes to cleanup industrial sites and water supplies
are extremely expensive and time consuming. In
contrast, bioremediation is relatively inexpensive and can
achieve the conversion of hazardous substances, using
microorganisms, into forms that are less or non-toxic
(Adams and Ribbons, 1988). Increase of bioremediation
capacity is being fostered by the knowledge of specificity
of microorganisms in biodegradation of certain chemicals.
In the last two decades, the field of microbiology has
increasingly focused on the use of microorganisms for
environmental clean up. Acinetobacter is one of the
bacteria can degrade and remove a wide range of
organic and inorganic compounds.
Biodegradation of xenobiotics and halogens
Xenobiotics pollutants such as benzene, toluene, phenol
and styrene, as well as halogenated organic compounds
such as pentachlorophenol and polychlorinated biphenyls
are often present in waste streams in fairly low
concentrations. They may also be present in larger
quantities as spills, or in the soil and water at abandoned
industrial sites. These compounds are generally highly
toxic and exceedingly difficult to dispose of.
A number of studies have focused on the
biodegradation of phenol by various microorganisms.
Among phenol degraders are several strains of
Acinetobacter, which can use it as a sole energy and
carbon source (Briganti et al., 1997; Chibata and Tosa,
1981). Recently, out of twelve bacterial phenol-degraders

72 Afr. J. Biotechnol.
isolated from different Egyptian ecosystems, four are
closely related to Acinetobacter (Abd-El-Haleem et al.,
2002a). One of these has been used in two different
environmental application studies (Abd-El-Haleem et al.,
2002c; Beshy et al., 2002). Other xenobiotic compounds
such as toluene (Zilli et al., 2001), 4-hydroxybenzoate
(Allende et al., 2000), 2-chloro-N-isopropylacetanilide
(Martin et al., 1999), 4-hydroxymandelic and 4-hydroxy-3-
methoxymandelic acids (Rusansky et al., 1987), benzoic
and p-hydroxybenzoic (Delneri et al., 1995), 4-
chlorobenzoate (Adriaens and Focht, 1991) and 3-
chlorobenzoic acid (Zaitsev and Baskunov, 1985) can be
metabolized to their corresponding benzoates by various
Acinetobacter strains.
It has also been reported that certain Acinetobacter
strains can utilize biphenyls including chlorinated
biphenyls (Adriaens and Focht, 1991; Furukawa and
Chakrabarty, 1982). Singer et al. (1985) observed that
out of 36 pure isomers of polychlorinated biphenyls
examined, 33 were metabolized by Acinetobacter sp.
strain P6. Furthermore, some Acinetobacter spp. isolated
from mixed cultures has proven to be proficient at
complete mineralization of monohalogenated biphenyls
(Shields et al., 1985). Strains of Acinetobacter have also
been employed for degradation of lignin (Buchan et al.,
2001; Crawford,. 1975; Mak et al., 1990) and amino acids
(Kahng et al., 2002; Kim et al., 2001).
Degradation of oil
Several constituents of oily sludge are carcinogenic and
potent immunotoxicants (Mishra et al., 2001). Oily sludge
is a complex mixture of alkanes, aromatic compounds,
NSO (nitrogen-, sulfur-, and oxygen-containing
compounds), and asphaltene fractions (Bossert and
Bartha, 1984). Acinetobacter strains are considered one
of the most efficient oil degraders (Rusansky et al.,
1987).
Removal of phosphate and heavy metals
Biological phosphate removal from wastewater is an
efficient cost-effective alternative to chemical phosphorus
precipitation. This biological process is obtained by
recycling the sludge through anaerobic and aerobic
zones. It is dependent on the enrichment of activated
sludge with polyphosphate accumulating strictly aerobic
Acinetobacter sp. which could absorb phosphate up to
100 mg phosphorus per gram of dry biomass during
aerobic conditions and release it anaerobically (van
Groenestijn et al., 1989; Timmerman, 1984). It was
confirmed that Acinetobacter are primarily responsible for
biological phosphate removal (Auling et al., 1991;
Wagner et al., 1994). Acinetobacter strains also play an
important role in the removal of heavy metals (Boswell et
al., 2001; Francisco et al., 2002).
BIO-PRODUCTS FROM ACINETOBACTER
Bioemulsifiers
Bioemulsifiers, which contain both hydrophobic and
hydrophilic groups, are used widely in the food,
agrochemical, cosmetic, and pharmaceutical industries.
Several microorganisms including Acinetobacter strains
can synthesize a wide variety of bioemulsifiers
(Rosenberg and Ron, 1998). Among the so-called
“bioemulsans,” the most studied are those produced by
A. calcoaceticus RAG-1 (Rosenberg and Ron, 1998), A.
calcoaceticus BD4 (Kaplan et al., 1987), and A.
radioresistens KA53 (Navon-Venezia et al., 1995). The
low-molecular-mass bioemulsifiers are generally
glycolipids, such as trehalose lipids, sophorolipids, and
rhamnolipids, or lipopeptides, such as surfactin,
gramicidin S, and polymyxin. The high-molecular-mass
bioemulsifiers are amphipathic polysaccharides, proteins,
lipopolysaccharides, lipoproteins, or complex mixtures of
these biopolymers (Toren et al., 2001).
A. calcoaceticus RAG-1 is an industrially important
strain which has been extensively characterized with
respect to its growth on hydrocarbons and its production
of a high molecular mass bioemulsifier, emulsan
(Rosenberg and Ron, 1998). The RAG-1 emulsan is a
non-covalently linked complex of a
lipoheteropolysaccharide and protein. The
polysaccharide, called apoemulsan, has a molecular
weight of about 990 kD. A. calcoaceticus BD4, initially
isolated by Taylor and Juni (1961), produces a large
polysaccharide capsule. When released into the medium,
the capsular polysaccharide forms a complex with
proteins which then becomes an effective emulsifier. The
BD4 emulsan apparently derives its amphipathic
properties from the association of an anionic hydrophilic
polysaccharide with proteins (Bryan et al., 1986). A.
radioresistens strain KA53 (Navon-Venezia et al., 1995)
produce a bioemulsifier designated alasan. It has a
molecular weight of 100 to 200 kD, and an emulsifying
activity which increases with preheating at 60 to 90°C
(Toren et al., 2001, 2002).
Polysaccharides, polyesters and lipases
Several strains of Acinetobacter produces extracellular
polysaccharides with sizes up to several million Daltons.
These polysaccharides can consist of D-galactose, D-2-
acetamido-2-deoxy-D-glucose, 3-(L-2-hydroxypropionam-
ido)-3,6-dideoxy-D-galactose (Haseley et al., 1997),
rhamnose, 3-deoxy-3-(D-3-hydroxybutyramido)-D-quino-
vose, S-(+)-2-(4'-Isobutylphenyl)propionic acid or
lipopolysaccharide (Haseley et al., 1997; Kunii et al.,
2001; Yamamoto et al., 1990). Furthermore, some
Acinetobacter strains are able to grow on ethanol and
synthesize exopolysaccharides called ethapolan (Johri et
al., 2002; Pirog et al., 2002; Pyroh et al., 2002).

Several Acinetobacter strains are also known to
accumulate wax esters, polyhydroxyalkalonic acids and
cyanophycin (Krehenbrink et al., 2002; Pirog et al., 2002;
Spiekermann et al., 1999; Vinogradov et al., 2002).
Various types of these biopolymer are widely used in the
manufacture of fine chemicals such as cosmetics,
candles, printing inks, lubricants, and coating.
Furthermore, a vast number of Acinetobacter lipases
have a wide range of potential applications in the
hydrolysis, esterification, and transesterification of
triglycerides, and in the chiral selective synthesis of
esters (Chen et al., 1999; Li, et al. 2000, 2001).
ACINETOBACTER AS A BIOREPORTER
The use of bioluminescent bioreporter as an inexpensive
and real-time method for detecting and monitoring
contaminants in the environment is one of the most
promising nano-technologies. Bioreporters refer to intact,
living microbial cells that have been genetically
engineered to produce a measurable signal in response
to a specific chemical or physical agent. The genetic
construct consists of an inducible promoter gene fused to
a reporter gene such as luciferase and green fluorescent
protein (Applegate et al., 1998; Hay et al., 2000)
Recently, we (Abd-El-Haleem et al., 2002b) were able
to construct a bioluminescent reporter strain,
Acinetobacter sp. DF4 for the detection of phenol by
inserting the phenol 3-hydroxyacyl-CoA-dehydrogenase
(3-hcd) promoter upstream of the bioluminescence genes
luxCDABE. When it was introduced into the chromosome
of Acinetobacter sp. DF4, the resulting strain, produced a
sensitive bioluminescence response to phenol at
concentrations ranging from 5.0 to 100 ppm. With such
specificity, Acinetobacter strains are prime candidates for
whole-cell bioreporter monitoring of phenol.
CONCLUSIONS AND PERSPECTIVE
There are numerous applications of Acinetobacter strains
in hazardous waste treatment or as producers of
economically important bio-products. Potential
improvements are expected from genetic engineering of
Acinetobacter strains from natural environments with
increased robustness for environmental and industrial
applications.
REFERENCES
Abd El-Haleem D, Moawad H, Zaki E, Zaki S (2002a). Molecular
characterization of phenol-degrading bacteria isolated from diffrent
Egyptian ecosystems. Microb. Ecol. 43:217-224.
Abd El-Haleem D, Ripp S, Scott C, Sayler G (2002b). A luxCDABE-
Based bioluminescent bioreporter for the detection of phenol. J. Ind.
Microbiol. Biotechnol. 29:233-237.
Abd El-Haleem D., Beshey U, Abdelhamid A, Moawad H, Zaki S
(2002c). Effects of mixed nitrogen sources on biodegradation of
Abd-El-Haleem 73
phenol by immobilized Acinetobacter sp. strain W-17. African J.
Biotechnol. 2:8-12.
Adams D, Ribbons, D (1988). The metabolism of aromatic ring fission
products by Bacillus stearothermophilus IC3. J. Gen. Microbiol.
134:3179-3185.
Adriaens P, Focht D (1991). Cometabolism of 3,4-dichlorobenzoate by
Acinetobacter sp. strain 4-CB1. Appl. Environ. Microbiol. 5: 173-179.
Allende L, Gibello A, Fortun A, Mengs G, Ferrer E, Martin M. (2000). 4-
Hydroxybenzoate uptake in an isolated soil Acinetobacter sp. Curr
Microbiol. 40:34-39.
Applegate BM, Kehrmeyer SR, Sayler GS (1998). A chromosomally
based tod-luxCDABE whole-cell reporter for benzene, toluene,
ethylbenzene, and xylene (BTEX) sensing. Appl. Environ. Microbiol.
64:2730-2735.
Auling G, Pilz F, Busse HJ, Karrasch S, Streichan M, Schon G (1991).
Analysis of the polyphosphate-accumulating microflora in
phosphorus-eliminating, anaerobic-aerobic activated sludge systems
by using diaminopropane as a biomarker for rapid estimation of
Acinetobacter spp. Appl Environ Microbiol 57:3585-3592.
Bergogne-Bérézin E, Towner K (1996). Acinetobacter spp. as
nosocomial pathogens: microbiological, clinical, and epidemiological
features. Clin. Microbiol. Rev. 9:148-165.
Beshey U, Abd El-Haleem D, Moawad H, Zaki S (2002). Phenol
biodegradation by free and immobilized Acinetobacter. Biotechnol.
Lett. 24:1295-1297.
Bossert I, Bartha R (1984). The fate of petroleum in the soil
ecosystems,. In R. M. Atlas (ed.), Petroleum microbiology. Macmillan,
New York, N.Y. pp. 435-473.
Boswell CD, Dick RE, Eccles H, Macaskie LE (2001) Phosphate uptake
and release by Acinetobacter johnsonii in continuous culture and
coupling of phosphate release to heavy metal accumulation. J. Ind.
Microbiol. Biotechnol. 26:333-340.
Briganti F, Pessione E, Giunta C, Scozzafava A (1997). Purification,
biochemical properties and substrate specificity of a catechol 1,2-
dioxygenase from a phenol degrading Acinetobacter radioresistens.
FEBS Lett. 416:61-4.
Bryan BA, Linhardt RJ, Daniels L (1986). Variation in composition and
yield of exopolysaccharides produced by Klebsiella sp. strain K32
and Acinetobacter calcoaceticus BD4. Appl. Environ. Microbiol.
51:1304-1310.
Buchan A, Neidle EL, Moran MA (2001) Diversity of the ring-cleaving
dioxygenase gene pcaH in a salt marsh bacterial community. Appl.
Environ. Microbiol. 67:5801-5809.
Buchan L (1981). The location and nature of accumulated phosphorus
in seven sludges from activated sludge plants which exhibited
enhanced phosphorus removal. Water Sci. Technol. 17:127-138.
Chen JY, Wen CM, Chen TL (1999). Effect of oxygen transfer on lipase
production by Acinetobacter radioresistens. Biotechnol. Bioeng.
62:311-316.
Chibata I, Tosa T (1981). Use of immobilized cells. Annu. Rev. Biophys.
Bioeng. 10:197-216.
Crawford L (1975). Mutualistic degradation of the lignin model
compound veratrylglycerol-beta-(o-methoxyphenyl) ether by bacteria.
Can. J. Microbiol. 21:1654-1665.
Delneri D, Degrassi G, Rizzo R, Bruschi CV (1995). Degradation of
trans-ferulic and p-coumaric acid by Acinetobacter calcoaceticus
DSM 586. Biochim. Biophys. Acta. 1244:363-371.
Francisco R, Alpoim MC, Morais PV (2002). Diversity of chromium-
resistant and -reducing bacteria in a chromium-contaminated
activated sludge. J. Appl. Microbiol. 92:837-43.
Furukawa K, Chakrabarty AM (1982). Involvement of plasmids in total
degradation of chlorinated biphenyls. Appl. Environ. Microbiol.
44:619-626.
Haseley SR, Holst O, Brade H (1997). Structural studies of the O-
antigenic polysaccharide of the lipopolysaccharide from
Acinetobacter. Eur. J. Biochem. 247:815-819.
Hay AG, Applegate BM, Bright NG, Sayler GS (2000). A bioluminescent
whole-cell reporter for detection of 2,4-dichlorophenoxyacetic acid
and 2,4-dichlorophenol in soil. Appl. Environ. Microbiol. 66:4589-
4594.
Johri AK, Blank W, Kaplan DL (2002). Bioengineered emulsans from
Acinetobacter calcoaceticus RAG-1 transposon mutants. Appl.

74 Afr. J. Biotechnol.
Microbiol. Biotechnol. 59:217-223.
Kahng HY, Cho K, Song SY, Kim SJ, Leem SH, Kim SI. (2002).
Enhanced detection and characterization of protocatechuate 3,4-
dioxygenase in Acinetobacter lwoffii K24 by proteomics using a
column separation. Biochem. Biophys. Res. Commun. 295:903-909.
Kaplan N, Zosim Z, Rosenberg E (1987). Reconstitution of emulsifying
activity of Acinetobacter calcoaceticus BD4 emulsan by using pure
polysaccharide and protein. Appl. Environ. Microbiol. 53:440-446
Kim SI, Yoo YC, Kahng HY (2001). Complete nucleotide sequence and
overexpression of cat1 gene cluster, and roles of the putative
transcriptional activator CatR1 in Acinetobacter lwoffii K24 capable of
aniline degradation. Biochem. Biophys. Res. 288:645-649.
Krehenbrink M, Oppermann-Sanio B, Steinbuchel A (2002). Evaluation
of non-cyanobacterial genome sequences for occurrence of genes
encoding proteins homologous to cyanophycin synthetase and
cloning of an active cyanophycin synthetase from Acinetobacter sp.
strain DSM 587. Arch. Microbiol. 177:371-380.
Kunii K, Nakamura S, Sato C, Fukuoka S (2001). A new extraction
method for Acinetobacter species ODB-L2 rough form
lipopolysaccharide from culture broth. Microbios 105:153-161.
Li YC, Wu JY, Chen TL (2001). Production of Acinetobacter
radioresistens lipase with repeated batch culture in presence of
nonwoven fabric. Biotechnol. Bioeng. 76:214-218.
Li SC, Wu JY, Chen CY, Chen TL (2000). Semicontinuous production of
lipase by Acinetobacter radioresistens in presence of nonwoven
fabric. Appl. Biochem. Biotechnol. 87:73-80.
Mak NK, Mok YK, Chui VW , Wong MH. (1990). Removal of lead from
aqueous solution by Acinetobacter calcoaceticus. Biomed. Environ.
Sci. 3:202-210.
Martin M, Mengs G, Allende JL, Fernandez J, Alonso R, Ferrer E
(1999). Characterization of two novel propachlor degradation
pathways in two species of soil bacteria. Appl. Environ. Microbiol.
65:802-806.
Mishra S, Jyot J, Kuhad RC, Lal B (2001). Evaluation of inoculum
addition to stimulate in situ bioremediation of oily-sludge-
contaminated soil. Appl. Environ. Microbiol. 67:1675-1681.
Navon-Venezia S, Zosim Z, Gottlieb A, Legmann R, Carmeli S, Ron EZ,
Rosenberg E (1995). Alasan, a new bioemulsifier from Acinetobacter
radioresistens Appl. Environ. Microbiol. 61:3240-3247.
Pirog P, Kovalenko MA, Kuz'minskaia luV (2002). Exopolysaccharide
production and peculiarities of C6-metabolism in Acinetobacter sp.
grown on carbohydrate substrates. Mikrobiologiia 71:215-221.
Pyroh TP, Hrinberh TO, Malashenko luR (2002). Strategy of obtaining
microbial exopolysaccharides possessing stable preset properties.
Mikrobiol. 64: 81-94.
Rosenberg E, Ron Z (1998). Surface active polymers from the genus
Acinetobacter, In D. L. Kaplan (ed.), Biopolymers from renewable
resources. Springer, New York, N.Y. pp. 281-289.
Rusansky S, Avigad R, Michaeli S, Gutnick DL (1987). Involvement of a
plasmid in growth on and dispersion of crude oil by Acinetobacter
calcoaceticus RA57. Appl. Environ. Microbiol. 53:1918-1923.
Shields MS, Hooper SW, Sayler GS (1985). Plasmid-mediated
mineralization of 4-chlorobiphenyl. J. Bacteriol. 163:882-889.
Singer ME, Tyler SM, Finnerty WR (1985). Growth of Acinetobacter sp.
strain HO1-N on n-hexadecanol: physiological and ultrastructural
characteristics. J. Bacteriol. 162:162-169.
Spiekermann P, Rehm BH, Kalscheuer R, Baumeister D, Steinbuchel A
(1999). A sensitive, viable-colony staining method using Nile red for
direct screening of bacteria that accumulate polyhydroxyalkanoic
acids and other lipid storage compounds. Arch. Microbiol. 171:73-80.
Taylor H, Juni E (1961). Pathways for biosynthesis of a bacterial
capsular polysaccharide. I. Characterization of the organism and
polysaccharide. J. Bacteriol. 81:688–693.
Timmerman MW (1984). Biological phosphorus removal in wastewater
treatment. Microbiol. Sci. 1:149-152.
Toren A, Navon-Venezia S, Ron EZ, Rosenberg E (2001). Emulsifying
Activities of Purified Alasan Proteins from Acinetobacter
radioresistens KA53. Appl. Envir. Microbiol. 67:1102-1106.
Toren A, Orr E, Paitan Y, Ron EZ, Rosenberg E (2002). The active
component of the bioemulsifier alasan from Acinetobacter
radioresistens KA53 is an OmpA-like protein. J. Bacteriol.184:165-
170.
van der Meer JR., de Vos WM, Harayama S, Zehnder AJ (1992).
Molecular mechanisms of genetic adaptation to xenobiotic
compounds. Microbiol. Rev. 56:677-694.
van Groenestijn JW, Bentvelsen MM, Deinema MH, Zehnder AJ (1989).
Polyphosphate-degrading enzymes in Acinetobacter spp. and
activated sludge. Appl. Environ. Microbiol. 55:219-223.
Vinogradov EV, Duus JO, Brade H, Holst O (2002). The structure of the
carbohydrate backbone of the lipopolysaccharide from Acinetobacter
baumanii strain ATCC 19606. Eur. J. Biochem. 269:422-430.
Vinogradov EV, Petersen BO, Thomas-Oates JE, Duus J, Brade H,
Holst O (1998). Characterization of a novel branched tetrasaccharide
of 3-deoxy-D-manno-oct-2-ulopyranosonic acid. The structure of the
carbohydrate backbone of the lipopolysaccharide from Acinetobacter
baumannii strain nctc 10303 (ATCC 17904). J. Biol. Chem.
273:28122-28131.
Wagner M., Erhart R, Manz W, Amann R, Lemmer H, Wedi D, Schleifer
KH (1994). Development of an rRNA-targeted oligonucleotide probe
specific for the genus Acinetobacter and its application for in situ
monitoring in activated sludge. Appl. Environ. Microbiol. 60:792-800.
Yamamoto K, Ueno Y, Otsubo K, Kawakami K, Komatsu K (1990).
Production of S-(+)-ibuprofen from a nitrile compound by
Acinetobacter sp. strain AK226. Appl. Environ. Microbiol. 56:3125-
3129.
Zaitsev GM, Baskunov BP (1985). Utilization of 3-chlorobenzoic acid by
Acinetobacter calcoaceticus . Mikrobiologiia. 54:203-208.
Zilli M, Palazzi E, Sene L, Converti A, Borghi MD (2001). Toluene and
styrene removal from air in biofilters. Process Biochem.10:423-429.