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To test the effect of several inclusion levels of Citrobacter braakii phytase (CBP), on phytate P release, 420 50-wk-old-Bovans White hens were randomly allocated to 7 treatments with 5 replicates of 12 hens each. The experimental period lasted 12 weeks, first 8 for adaptation and last 4 for data collection. Feed and water were provided ad libitum. Treatments were: (1) a 0.12% basal corn-soybean meal diet deficient only in non-phytate P. Treatments 2 and 3 were added with constant increases of 0.11% inorganic P, to get a linear hen response to P addition. Treatments 4 to 7 were the addition of 300; 600; 1,200; and 1,800 phytase units (FYT)/kg to the basal diet. Variables analyzed were hen productive performance (HPP) and tibia resistance to fracture (TRF), and mineral content. Data were analyzed as a Complete Randomized Design (CRD). The results from treatments 1 to 3 were analyzed by a regression model to test for a significant linear response (P < 0.05). Then for every level of CBP added (treatments 4 to 7), the linear regression equation was solved to find out the equivalent value of released P. Based on hen health and welfare, the response variables that yielded realistic P equivalence values for the CBP levels used in the present trial were the tibia data. Following the significant (P < 0.001) linear response, the equations; TRF, kg (Y = 28.16X + 17.42 R2 = 0.84); Tibia Ca, % (Y = 11.6X + 14.2 R2 = 0.80); Tibia P, % (Y = 11.6X + 6.1 R2 = 0.81); and T ash, % (Y = 33.3X + 38.1 R2 = 0.80). Under the experimental conditions of this trial, the HPP variables were not a sensitive parameter to measure P release; whereas, tibia parameters showed the following average P release values per level of CBP inclusion in the corn- soybean meal diet; 300 FYT/kg = 0.099%, 600 FYT/kg = 0.141%, 1,200 FYT/kg = 0.182%, and 1,800 FYT/kg = 0.198%.

The plasmid-mediated colistin resistance gene mcr-1 has been found worldwide, but the diversity of organisms harbouring this gene is unknown. In this study, 12 colistin-resistant Citrobacter spp. isolates were obtained from diseased or dead chickens in China, and PCR analysis indicated that five were positive for mcr-1. One Citrobacter braakii strain (SCC4) with a multidrug-resistant phenotype was chosen for further analysis. SCC4 was resistant or intermediate-resistant to ten of the tested antibiotics, and the colistin minimum inhibitory concentration (MIC) was >4 µg/mL. A conjugation assay demonstrated successful transfer of colistin resistance to Escherichia coli strain J53 at a frequency of 10–7 cells per recipient cell. Whole-genome sequencing revealed that SCC4 contained 13 antibiotic resistance genes in its genome, and the mcr-1 gene resided on a 44-kb self-transmissible IncP-type plasmid of a recently discovered IncP-1 clade. In addition, the mcr-1 gene was part of an insertion element (ISApl1–mcr-1–orf–ISApl1) that was excised from the plasmid as a circular intermediate form. This is the first report of mcr-1-posiitve C. braakii of animal origin and these findings highlight the fact that the mcr-1 gene can be found in normal enteric flora as part of broad-host-range plasmids.

Two alchornoic acid derivatives, deepoxyalchornoic acid ( 1 ) and bisalchornoicester ( 2 ), together with six known compounds, alchornoic acid ( 3 ), gallic acid, 4- O -methylgallic acid, shikimic acid, kaempferol-3- O -galactoside and 2,3-dihydroxypropyl octacosanoate were isolated from a dichloromethane/methanol-soluble extract of the ripe fruits of the Cameroonian medicinal plant Alchornea cordifolia . The structures of the new fatty acid derivatives were elucidated by interpretation of their HRESIMS, 1D and 2D NMR spectroscopic data and by comparison with those reported in the literature. Some of the isolated compounds demonstrated inhibition activity against the microbial strains Escherichia coli, Klebsiella pneumoniae and Citrobacter braakii .

The aim of this work was to identify the main species of Enterobacteriaceae present in two different varieties of raw ewe’s milk cheese, to study their evolution during manufacturing and ripening, and to assess their potential to produce gas from lactose. Enterobacteriaceae activity may influence cheese ripening; it can even be responsible for some specific characteristics of artisanal cheeses. Nevertheles some of these microorganisms are potential pathogens. Enterobacteriaceae growth and disappearance were different between the soft and the semi–hard cheese variety, as well as the proportions of the species detected. Citrobacter freundii, Citrobacter braakii and Klebsiella oxytoca were the predominant species in milk and curd, whereas Hafnia alvei was predominant throughout ripening. The highest gas production was generated by Enterobacter nimipresuralis , C. braakii and Enterobacter cloacae strains, followed by Enterobacter aerogenes, H. alvei, K. oxytoca and C. freundii strains. According to the species found, their levels during the initial ripening stages and gas production ability, C. braakii, C. freundii, K. oxytoca and H. alvei can be considered as responsible for early blowing in soft and semi–hard ewe cheeses. The identification of the Enterobacteriaceae species most likely responsible for early blowing can lead to a better understanding of raw milk cheese ripening, and therefore, to the improvement of its quality.

Sodium lauryl ether sulfate (SLES) is an anionic surfactant used in the formulation of several detergent products. The high concentration of organic materials and SLES present in the industrial wastewater poses a real problem. In the previous study, Dhouib et al. (Dhouib et al., 2003) showed that Citrobacter braakii has a large capacity to degrade anionic surfactants in the wastewater treatment of a cosmetics industry. The scale-down has made possible to isolate, from the aerobic membrane bioreactor (MBR) inoculated with Citrobacter braakii since after one year, four strains A4, A10, A13 and A14 which were identified as Pseudomonas aeruginosa , Pseudomonas stutzeri , Bacillus safensis and Staphylococcus arlettae , respectively. These isolates are able to degrade 100% of SLES at a concentration of 1000 mg l −1 in the BSM medium. Physical-chemical parameters have been followed during 104 days in the influent, bioreactor and effluent. Biomass concentration increased slightly in the reactor and reached 13 g VSS l −1 . The COD and surfactants average removals were about 92% and 99.6%, respectively.

The main objective of this study is to demonstrate the potential of microbial induced calcite precipitation (MICP) as rapid and effective process in the synthesis of fluorescent cadmium sulfide nanoparticles. A urease-producing bacterial isolate AX5, identified as Citrobacter braakii, from calcite rich shells of abalone that could tolerate cadmium at the concentration of 250 mg/l was used. In order to investigate the role of MICP in enhancing the synthesis of CdS nanoparticles, the biomimetic synthesis was carried out by C. braakii in modified nutrient broth without amending any additional organic or toxic surfactants back into the environment. The large amount of monodispersed CdS nanoparticles was harvested and found to bind with biocalcite. XRD analysis confirmed the presence of CdS nanoparticles bound with biocalcite minerals in the precipitation. HRTEM and SEM micrographs showed spherical CdS nanoparticles of particle size in the range of 50–100 nm. The other characteristics of harvested CdS nanoparticles were studied with UV–vis, PL spectra, FTIR, and fluorescence microscopy. The findings of present study have important insights into the application of MICP as a promising mechanism for the biosynthesis of CdS nanoparticles. Keywords: Biocalcite, Nanoparticles, Citrobacter braakii, Urease, CdS

Presence of extended spectrum beta-lactamases (ESBL) in bacteria is a growing health concern of global significance. The local, regional, national, and international epidemiological studies for extended spectrum beta-lactamases-producing Enterobacteriaceae and their encoding genes in foods are still incomplete. The objective of this study was to determine the occurrence of extended spectrum beta-lactamases-producing Enterobacteriaceae and the characteristics of their encoding genes from a total of 250 samples of various foods of animal-origin (100 raw chicken meat, 100 raw cow milk, and 50 raw cow milk cheese) sold in Turkey. Overall, 55 isolates were positive as extended spectrum beta-lactamases-producing Enterobacteriaceae. The most prevalent extended spectrum beta-lactamases-producing strain were identified as Escherichia coli (80%), followed by Enterobacter cloacae (9.1%), Citrobacter braakii (5.5%), Klebsiella pneumoniae (3.6%), and Citrobacter werkmanii (1.8%) by Vitek® MS. The simultaneous production of extended spectrum beta-lactamases and AmpC was detected in five isolates (9.1%) in E. coli (80%) and E. cloacae (20%). The frequency rates of blaTEM, blaCTX-M, and blaSHV were 96.4%, 53.7%, and 34.5%, respectively. The co-existence of bla -genes was observed in 82% of extended spectrum beta-lactamases producers with a distribution of blaTEM & blaCTX-M (52.7%), blaTEM & blaSHV (20%), blaTEM & blaCTX-M & blaSHV (12.7%), and blaSHV & blaCTX-M (1.8%). The most prevalent variant of blaCTX-M clusters was defined as blaCTX-M-1 (97.2%), followed by blaCTX-M-8 (2.8%). In summary, the analysed foods were found to be posing a health risk for Turkish consumers due to contamination by Enterobacteriaceae with a diversity of extended spectrum beta-lactamases encoding genes.

Phytases are important commercial enzymes that catalyze the dephosphorylation of myo-inositol hexakisphosphate (phytate) to its lower inositol phosphate (IP) esters, IP6 to IP1. Digestion of phytate by Citrobacter braakii 6-phytase deviates significantly from monophasic Michaelis-Menten kinetics. Analysis of phytate digestion using isothermal titration calorimetry (ITC) using the single injection method produced a thermogram with two peaks consistent with two periods of high enzyme activity. Continuous-flow electrospray ionization time-of-flight mass spectroscopy (ESI-ToF-MS) provided real-time analysis of phytase catalysis. It was able to show that the first two cleavage steps were rapid and concurrent but the third cleavage step from IP4 to IP3 was slow. The third (IP4 to IP3), fourth (IP3 to IP2) and fifth (IP2 to IP1) cleavages were effectively sequential due to the preferred association of the more phosphorylated species with the phytase catalytic site. This created a bottleneck during the cleavage of IP4 to IP3 until the point at which IP4 was exhausted and was followed by the rapid cleavage of IP3 to IP2, which was observed as the second peak in the ITC thermogram. This work illustrates the importance of an orthogonal approach when studying non-specific or complex enzyme catalyzed reactions.

Among the Citrobacter genus, the most commonly isolated bacteria from human specimens are Citrobacter freundii and Citrobacter koseri, and previous cases of infection due to Citrobacter braakii have been rarely reported. We present a case of bacteremia due to C. braakii in a 38-year-old woman with cervical cancer. She was admitted to our hospital with complaints of a fever, chills, and nausea. Blood culture results showed gram-negative bacilli identified as C. braakii via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, although biochemical testing findings were suggestive of C. freundii. Since a rare pathogen was detected in the present case and the results of additional biochemical studies were suggestive of both C. braakii and Citrobacter farmeri, genetic analysis was conducted. Finally, the gram-negative bacilli were confirmed as C. braakii, a member of the C. freundii complex since 1993, by 16S ribosomal RNA gene sequencing analysis. The gastrointestinal tract was considered the portal of entry, because the patient had a rectal fistula and other cultures such as urine and vaginal discharge incubated species other than C. braakii. The patient recovered after receiving treatment with ciprofloxacin for 14 days. The epidemiology and clinical characteristics of C. braakii infection are still unknown because of the limitations in accurate identification by using currently available commercial biochemical testing and previously, only 6 cases of C. braakii infection have been reported. Physicians should focus on this species, because it causes community-acquired infections, although further studies are needed to clarify the clinical characteristics of C. braakii infections.

Phosphorus (P) in most diets for sows is poorly digested because pigs do not produce salivary or gastric phytase to hydrolyze P from phytate (inositol hexaphosphate), thus leading to a relatively high amount of P in the manure. An experiment was carried out to study the efficacy of a novel bacterial 6-phytase produced by a strain of Aspergillus oryzae expressing two synthetic genes, both mimicking a phytase gene from a Citrobacter braakii strain. The apparent total tract digestibility coefficient (CATTD) of P, calcium (Ca), crude protein (CP), and gross energy, concentrations of plasma variables, and sow performance were used as response variables. In total, 45 crossbred gestating sows (Large white×Landrace) were individually housed with their litter. Three diets were prepared: (1) positive control (PC; 5.2 g available P/kg), regular sow diet containing inorganic P; (2) negative control (NC; 2.0 g available P/kg) without inorganic P; and (3) NC + 500 FTU/kg diet (NC + PHY). Each diet was fed to 15 sows for 21 days (from day 108 of gestation to day 15 of lactation). To the diets, 3.0 g titanium dioxide/kg was added as a marker to determine CATTD of nutrients. Blood samples were collected from the anterior vena cava at day 1 and 15 of lactation to determine plasma minerals (P, Ca, magnesium, copper, iron, and zinc) and plasma urea, glucose, total protein, albumin, and globulin. At day 15 of lactation, CATTD of P did not differ between PC and NC. Phytase supplementation to NC increased (P

A 14-d experiment was conducted to study the effects of 2 dietary variables on efficacy of a 6-phytase from Citrobacter braakii on broiler growth performance and tibia mineralization. Diets were formulated with or without nutrient matrix values for phytase as negative or positive control (NC or PC, respectively) and with 2 Ca:total P (tP; 2:1 or 2.5:1). The diets were supplemented with 0, 1,000, or 2,000 phytase units (FYT)/kg, thus producing a 2 × 2 × 3 factorial arrangement. Birds and feed were weighed on d 7 and 21, and tibia bones were collected from all the birds on d 21. The main effects of nutrient matrix, Ca:tP, and phytase supplementation were significant (P0.05) for all the growth performance responses (except for G:F for which there was no effect of matrix). The Ca:tP × phytase and matrix × phytase interactions were significant (P0.05) for weight gain. In the PC diets, phytase increased weight gain (P0.05) relative to the control only in diets with 2,000 FYT/kg, whereas in NC diets weight gain increased (P0.01) only from 0 to 1,000 FYT/kg levels. Broilers consuming diets with 2.5:1 Ca:tP had lower (P0.05) tibia ash, whereas phytase increased (P0.01) tibia ash, Ca, P, and Zn but decreased (P0.01) tibia K. Phytase supplementation of diets with 2:1 Ca:tP increased (P0.05) tibia P in birds receiving 1,000 FYT/kg relative to the control with no further increase at 2,000 FYT/kg, whereas each level of phytase supplementation increased (P0.05) tibia P in the diets with 2.5:1 Ca:tP. It was concluded that the best response to lower phytase supplementation (1,000 FYT/kg) was in NC diets with narrow Ca:tP, whereas the best response to higher level of phytase supplementation (2,000 FYT/kg) was achieved in diets in PC diets with wide Ca:tP.

Glycerol dehydrogenase (GlyDH) catalyzes the regioselective oxidation of glycerol to yield 1,3-dihydroxyacetone (DHA); an important building block in chemical industry. Three recombinant GlyDHs from Geobacillus stearothermophilus, from Citrobacter braakii and from Cellulomonas sp. were stabilized by covalent immobilization. The highest activity recoveries (40–50%) of the insoluble preparations were obtained by immobilizing these enzymes in presence of polyethylene glycol (PEG). Noteworthy, these immobilized preparations were more stable and less inhibited by DHA than their soluble counterparts. In particular, GlyDH from G. stearothermophilus immobilized on agarose activated with both amine and glyoxyl groups and crosslinked with dextran aldehyde was 3.7-fold less inhibited by DHA than its soluble form and retained 100% of its initial activity after 18 h of incubation at 65 °C and pH 7. This is one of the few examples where the same immobilization protocol has minimized enzyme product inhibition and maximized thermal stability., This work has been supported by the Spanish Ministry of Economy and Innovation (project CTQ2009-07568). We would like to thank IKERBASQUE, Basque foundation for Science for the funding to PhD Fernando López Gallego. We also thank to Comunidad de Madrid gouverment for funding the PhD fellowship for J. Rocha-Martín.

Objective The objective of the present study was to analyze the presence of qnr genes among different clinical isolates and, subsequently, antibacterial activity of drugs was tested against all of the clinical isolates. Next, we examined the effect of different concentrations of ethylenediaminetetraacetic acid (EDTA) and half of minimum inhibitory concentration (MIC) of drugs on conjugation. Methods Analysis of the presence of qnr genes ( qnrA , qnrB and qnrS ) was done using previously reported primers by polymerase chain reaction. The antibacterial activity of all drugs was determined by MIC and antimicrobial susceptibility (AST) according to the Clinical and Laboratory Standards Institute guidelines (CLSI, 2009). Conjugation was carried out according to the broth mating method. Results The qnrA gene was detected in Acinetobacter baumannii and Citrobacter braakii , whereas qnrB gene was found in Escherichia coli , A. baumannii , C. braakii , Pseudomonas aeruginosa and Klebsiella pneumoniae . However, qnrS was not detected in any of the clinical isolates. The results revealed that Potentox has potential antimicrobial activity against all of the clinical isolates compared to other drugs. Conjugal transfer of qnrB gene from donor to recipient was inhibited at 10 mM EDTA. Similarly, when various drugs at half of MIC were evaluated on conjugation, interestingly, only Potentox could inhibit conjugal transfer of qnrB gene, whereas rest of the drugs failed to inhibit conjugation. Conclusion Potentox has enhanced in vitro antibacterial activity compared to other drugs against quinolone resistant clinical isolates. 10 mM EDTA effectively prevents the conjugal transfer of qnrB gene from donor to recipient. When the same concentration of EDTA was used as a solvent for Potentox, it has again inhibited the conjugal transfer of qnrB gene from donor to recipient. Therefore, inhibition of conjugation can be a novel antimicrobial approach to combat spreading of antibiotic resistance which can be achieved only with Potentox the novel antibiotic adjuvant entity (US patent - US8178501B2).

Cytochrome P450cin (CYP176A1) is a bacterial P450 isolated from Citrobacter braakii that catalyses the hydroxylation of 1,8-cineole to (1R)-6β-hydroxycineole. P450cin uses two redox partners in vitro for catalysis: cindoxin, its physiological FMN-containing redox partner, and Escherichia coli flavodoxin reductase. Here we report the construction of a tricistronic plasmid that expresses P450cin, cindoxin and E. coli flavodoxin reductase and a bicistronic plasmid that encodes only P450cin and cindoxin. E. coli transformed with the bicistronic vector effectively catalysed the oxidation of 1,8-cineole, with the endogenous E. coli flavodoxin reductase presumably acting as the terminal electron transfer protein. This in vivo system was capable of producing enantiomerically pure (1R)-6β-hydroxycineole in yields of ∼1 g/L culture, thus providing a simple, one-step synthesis of this compound. In addition, the metabolism of (1R)- and (1S)-camphor, structural homologues of 1,8-cineole was also evaluated in order to investigate the ability of this in vivo system to produce compounds for mechanistic studies. Significant quantities of five of the six possible secondary alcohols arising from methylene oxidation of both (1R)- and (1S)-camphor were isolated and structurally characterised. The similarity of the (1R)- and (1S)-camphor product profiles highlight the importance of the inherent reactivity of the substrate in determining the regiochemistry of oxidation in the absence of any specific enzyme–substrate binding interactions.

Histidine, lysine, ornithine and tyrosine decarboxylase activities were tested in 79 strains of Enterobacteriaceae (41 of Hafnia alvei, 17 of Serratia liquefaciens, 5 of Enterobacter cloacae, 4 of Citrobacter braakii, 2 of Proteus vulgaris, 2 of Proteus mirabilis, 2 of Providencia stuartii, 2 of Klebsiella terrigena, 1 of Rahnella aquatilis, 1 of Salmonella arizonae, 1 of Citrobacter youngae and 1 of Escherichia coli) isolated from Botillo, a Spanish traditional sausage. In general, the strains were positive for all four activities, with the exception of two strains of H. alvei and the E. coli strain, which did not display histidine decarboxylase activity. The strains of P. mirabilis and P. stuartii did not exhibit any of the four activities tested. Accumulation of putrescine and cadaverine was studied throughout growth of the 75 strains that displayed ornithine and lysine decarboxylase activities. Biogenic amines were produced particularly in the exponential phase, with maximum accumulation occurring after between 12 to 72 h, depending on the biogenic amine and microbial species considered. Maximum accumulation of putrescine varied greatly between species and within the same species, and ranged from 18 mg/l in the R. aquatilis strain to 7325 mg/l in a H. alvei strain. Maximum accumulation of cadaverine varied less than that of putrescine, and ranged from 30 mg/l in the R. aquatilis strain to 1935 mg/l in a S. liquefaciens strain.

The first steps in the biodegradation of 1,8-cineole involve the introduction of an alcohol and its subsequent oxidation to a ketone. In Citrobacter braakii, cytochrome P450(cin) has previously been demonstrated to perform the first oxidation to produce (1R)-6beta-hydroxycineole. In this study, we have cloned cinD from C. braakii and expressed the gene product, which displays significant homology to a number of short-chain alcohol dehydrogenases. It was demonstrated that the gene product of cinD exhibits (1R)-6beta-hydroxycineole dehydrogenase activity, the second step in the degradation of 1,8-cineole. All four isomers of 6-hydroxycineole were examined but only (1R)-6beta-hydroxycineole was converted to (1R)-6-ketocineole. The (1R)-6beta-hydroxycineole dehydrogenase exhibited a strict requirement for NAD(H), with no reaction observed in the presence of NADP(H). The enzyme also catalyses the reverse reaction, reducing (1R)-6-ketocineole to (1R)-6beta-hydroxycineole. During this study the N-terminal His-tag used to assist protein purification was found to interfere with NAD(H) binding and lower enzyme activity. This could be recovered by the addition of Ni(2+) ions or proteolytic removal of the His-tag.

Cytochrome P450cin (CYP176A1) is a bacterial P450 isolated from Citrobacter braakii that catalyzes the hydroxylation of cineole to (S)-6β-hydroxycineole. This initiates the biodegradation of cineole, enabling C. braakii to live on cineole as its sole source of carbon and energy. P450cin lacks the almost universally conserved threonine residue believed to be involved in dioxygen activation and instead contains an asparagine at this position (Asn-242). To investigate the role of Asn-242 in P450cin catalysis, it was converted to alanine, and the resultant mutant was characterized. The characteristic CO-bound spectrum and spectrally determined KD for substrate binding were unchanged in the mutant. The x-ray crystal structures of the substrate-free and -bound N242A mutant were determined and show that the only significant change is in a reorientation of the substrate such that (R)-6α-hydroxycineole should be a major product. Molecular dynamics simulations of both wild type and mutant are consistent with the change in regio- and stereoselectivity predicted from the crystal structure. The mutation has only a modest effect on enzyme activity and on the diversion of the NADPH-reducing equivalent toward unproductive peroxide formation. Product profile analysis shows that (R)-6α-hydroxycineole is the main product, which is consistent with the crystal structure. These results demonstrate that Asn-242 is not a functional replacement for the conserved threonine in other P450s but, rather, is critical in controlling regioselective substrate oxidation.

The treatment of an anionic surfactant rich wastewater by a powerful Citrobacter braakii strain was investigated at large scale. This bacterium was inoculated into a 4.5 m3 continuous stirred tank reactor (CSTR) without a membrane. At volumetric loading rates ranged between 0.44 and 0.96 g COD l−1 d−1, a weak C. braakii population density was observed in the reactor which results in slow anionic surfactants degradation kinetics. The augmented bacterium was diluted and replaced by protozoa and other forms of bacteria after a prolonged experience run. The CSTR was then coupled to a cross-flow ultrafiltration membrane. This allowed complete biomass recycling and partially retention of the surfactants in the reactor. C. braakii became again dominant in the medium. At volumetric loading rates of 0.6–1.5 g l−1 d−1, the anionic surfactant concentration in the permeate varied from 0 to 40 mg l−1 with a mean value of 18 mg l−1. After 2 months of running, mixed liquor volatile suspended solids (MLVSS) in the reactor increased from 0.71 to 5.92 g l−1 and the permeate-flux slightly decreased from 28.75 to 23.75 l h−1 m−2. The reclaimed water could be reused either for the washing of the material and soil, and to irrigate the parkland of the factory.

An O-specific heterohexasaccharide fragment of Citrobacter braakii O7a, 3b, 1c, α- d -Manp-(1→3)-α- d -Manp-(1→2)-[α- d -Glcp-(1→3)]-α- d -Manp-(1→2)-α- d -Manp-(1→2)-α- d -Manp was synthesized as its methyl glycoside. Acetylation of allyl 4,6-O-benzylidene-α- d -mannopyranoside, followed by debenzylidenization and benzoylation gave allyl 2,3-di-O-acetyl-4,6-di-O-benzoyl-α- d -mannopyranoside (3), and subsequent deacetylation of 3 with CH3COCl–MeOH gave the monosaccharide acceptor 4. Condensation of isopropyl 2,3,4,6-tetra-O-benzyl-1-thio-β- d -glucopyranoside (6) with 4 selectively afforded the α-(1→3)-linked disaccharide 7. Condensation of 7 with the (1→3)-linked disaccharide donor 9, followed by deallylation and trichloroacetimidation, afforded the tetrasaccharide donor 12. Coupling of 12 with disaccharide acceptor 13, followed by debenzylation and deacylation, furnished the target heterohexasaccharide 16.

Anionic surfactants, as core components of detergent and cosmetic products formulations, contribute significantly to the pollution profile of sewage and wastewaters of all kinds. In Tunisia more than 100 000 m 3 per year of surfactant containing wastewater are released. Treatment by conventional processes such as activated sludge is problematic due to the low kinetics of degradation by unsuitable consortia and to foam production. Bioremediation involves the isolation of suitable microorganisms, adequate process development and in situ biodegradation. The objective of this work was to develop a high performance process for the treatment of anionic surfactant containing wastewater. Screening of bacteria degrading the anionic surfactant sodium lauryl ether sulfate (SLES) was carried out by a conventional enrichment-culture technique. A Citrobacter braakii strain degrading many anionic surfactants was isolated and tested in bioaugmentation for industrial wastewater treatment. It was used for the continuous degradation of highly concentrated SLES synthetic medium and cosmetic industry wastewater. By testing several dilution rates, it was concluded that C. braakii is able to degrade up to 0.065 g l � 1 h � 1 . The hydraulic retention time (HRT) was nearly 20 h. This process was improved by coupling the bioreactor to a mineral microfiltration membrane. This allowed an increase in the SLES elimination rate from 0.065 to 0.15 g l � 1 h � 1 and to reduce the HRT from 20 to 5 h. Moreover, this integrated system generates microorganism and surfactant free wastewaters able to be rejected without public menace or reused in situ in the industrial process. # 2002 Elsevier Science Ltd. All rights reserved.