Chakravarty and Kundu AMB Expr (2016) 6:101
DOI 10.1186/s13568-016-0274-0
Improved production ofDaptomycin inan airlift bioreactor bymorphologically modied andimmobilized cells ofStreptomyces roseosporus
Ipsita Chakravarty and Subir Kundu*http://orcid.org/0000-0002-3516-5505
Web End =
http://orcid.org/0000-0002-3516-5505
Web End = Introduction
The health care sector is apprehensive and intimidated about the issue of drug resistance (Fluit et al. 2001). Researchers around the world are engaged with metabolic engineering and genetic improvement of several life-saving drugs that can combat this issue. There is also an urge to improve their production to reduce the cost of its industrial applicability. Therefore, availing it to a larger section of the society. Daptomycin is such a novel cyclic lipopeptide antibiotic
produced by Streptomyces roseosporus, which works effectively against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci due to its unique mechanism of action. This antibiotic has got the FDA approval only in 2003 for the treatment of patients with complicated skin and skin structure infections, right-sided endocarditis and bacteremia (Eisenstein et al. 2010; Chakravarty et al. 2015). The filamentous nature of S. roseosporus affects the rheology of the fermentation broth. This hampers its production to a great extent. Though, researchers have attempted to increase the yield through genetic engineering and metabolic flux analysis (Huang etal.
*Correspondence: [email protected]; [email protected] School of Biochemical Engineering, Indian Institute of Technology, Banaras Hindu University, Varanasi 221005, India
The Author(s) 2016. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/
Web End =http://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
Chakravarty and Kundu AMB Expr (2016) 6:101
Page 2 of 8
2012).There have been very few attempts towards the process strategies for improved production of Daptomycin. The physiological state and morphological differentiation of filamentous microorganisms can be related to changes in growth conditions during submerged cultivations (Kundu et al. 2000). Morphological features have been correlated with the production of secondary metabolites at several instances (OCleirigh etal. 2005; Papagianni and Mattey 2006). Improved antimicrobials production was observed through pellet formation (Choi etal. 2000). Also, whole- cell immobilization is a useful processing strategy to instantiate the potential of bioprocesses. Previous reports have contributed to explore how the phenomenon of immobilization affects growth and metabolite production (Kundu etal. 1992; Mahapatra et al. 2002). Higher cell concentration can be maintained by immobilization methods which enhance the productivity of the secondary metabolite as it is a non-growth associated product. Prolonged reusability is also an advantage of whole-cell immobilization (Srivastava and Kundu 1998; Mishra etal. 2005). The objective of the present work was to evaluate the production of Daptomycin through improved broth rheology and controlled hydrodynamics. Therefore, we have attempted some alternative strategies to improve antibiotic production and have tried to study the pattern of oxygen mass transfer using an air-lift bioreactor with different modes of cell growth.
Materials andmethods
Strain
Streptomyces roseosporus NBRC 12910 (Chiba, Japan) was used for these studies.
Medium andculture conditions
Streptomyces roseosporus was cultivated and maintained at 30C and 200rpm in culture broth medium containing (g/l): malt extract 3, glucose 10, yeast 3 and peptone 5 at pH 6.5. Fermentation was carried out in medium containing (g/l) Dextrin 30, Glucose 10, Soyabean our 20, Fe (NH4)2 SO4 0.6, KH2PO4 0.2, and pH 7 and incubated at 30C for 6days. Cofactors were added to the sterile culture medium after aseptic ltration. Eight cofactors were added which included nicotinic acid(4mg/l), riboavin(0.5 mg/l), heme(9 mg/l), thiamine(0.4 mg/l), biotin(0.1mg/l), cyanocobalamin(0.04mg/l), tetrahydrofolic acid(6 mg/l) and pyridoxal 5-phosphate(0.4 mg/l) (Yu etal. 2011). As for the airlift bioreactor, the pH was automatically controlled at 7.0 with 8M NaOH solution and the aeration speed of 1 v/v/m for 6days. N-decanoic acid was fed at 48h after inoculation (0.2g/l). All experiments were done in triplicate.
Morphological variations ofS. roseosporus
The growth conditions were altered for pelletization the cells. The morphological variation of S. roseosporus was carried out by changing the inoculum size, the nitrogen source and the aeration rate in the fermentation medium. Morphological changes during the fermentation were observed. Suspended pellets were imaged and pellet diameter was calculated, assuming that the pellets were perfectly spherical. Pellet size distribution was calculated by averaging 2050 pellets. Both visual observations and image processing tools were taken into consideration.
Immobilization ofS. roseosporus onrefractory brick akes andsilk sachets
Refractory bricks were mechanically crushed and sieved uniformly to particle size of 5mm. These were pretreated by boiling in water for 20min at 80C and washed with distilled water. They were placed in methanol for 3 h. Then, washed again with distilled water. For the immobilization of S. roseosporus on the support matrices, 2.0g of pretreated carrier were placed in a 250ml Erlenmeyer ask containing seed medium. After sterilization (121C and 15lbs/in2 pressure for 20min), the asks were inoculated with 0.5ml of homogenized mycelia (0.15 dry cell weight of mycelia g/l) under sterile conditions. An equal weight of silk sachets (4cm2cm) was sterilized, and a concentrated seed culture was put into each silk sachet aseptically. S. roseosporus is found to grow on carrier after 5days of incubation. The growth medium was then removed and the immobilized matrices were thoroughly washed with distilled water under sterile conditions. Experiments were carried out for Daptomycin production by transferring the immobilized matrices into the production medium under sterile conditions. They were loaded into an indigenously developed, presterilized airlift bioreactor containing fermentation media as depicted in Fig.1 and the dimensions are described in Table1.
Cell growth andproduct estimation
10mL broth sample was centrifuged and the supernatant was discarded. Pellets were washed thrice with distilled water. The centrifuged biomass was transferred to a pre-weighed aluminium cup kept at 85C for 24h until a constant weigh was achieved. The dry cell weight was determined by subtracting the weight of aluminium cup from the previous weight. At specied intervals, Daptomycin was analyzed using disk diusion method using M. luteus as the assay organism and conrmed by HPLC.
Cell leakage estimation
The free cells and cells leaked from the support matrix were collected by centrifugation at 10,000rpm for 20min
Chakravarty and Kundu AMB Expr (2016) 6:101
Page 3 of 8
Table 1 Table showing the dimensions ofthe airlift bioreactor
Parameters Volumetric capacity ofreactor
Column diameter
Column height
Ungassed liquid height
Draft tube height
Draft tube diameter
Height ofdraft tube abovethe fermenter base
Single sparger hole diameter
Dimensions 2.0 l 60 mm 650 mm 150 mm 350 mm 40 mm 80 mm 1 mm
and dried at 90 C. The initial weight of each support matrix was determined by drying specied quantity to a constant weight. The support matrices with cells were carefully washed with distilled water and dried again. The dierence between the weights of the support matrices before and after cell adsorption is considered to be the weight of adsorbed cells (Dsouza etal. 1986).
Evaluation ofthe rheology andhydrodynamics offermentation broth
The consequences of the stress conditions subjected in case of dierent morphological forms were assessed by the rheological properties which were measured using the Brookeld viscometer at dierent time intervals. The volumetric oxygen transfer coefficient was estimated by the dynamic gassing-out technique. Air ow rate was maintained at 1vvm (Ruchti etal. 1981).
Reusability ofimmobilized andpelletized cells
The cell pellets and the immobilized cells were collected at the 50 micron nylon mesh screen at the sampling port and replenished with fresh fermentation medium for further batches.
Microscopic analysis ofthe pelletized andthe immobilized cells
Scanning electron microscopy (SEM) was carried out for immobilized cells on refractory bricks to determine the morphological alterations and distribution of cells in the matrices during immobilization. The specimens were chemically xed and examined in Zeiss scanning electron microscope at 20kV. All images were digitally recorded. Pelletized cells and silk sachets containing S. roseosporus were observed by electron microscopy.
Results
Preliminary screening ofsupport matrices
The signicance of whole cell immobilization was evaluated by screening ve dierent conventional and non-conventional support matrices viz., ultra porous refractory brick akes, silk sachets, polyurethane foam, loofah sponge and ceramic foam as shown in Fig. 2. The reusability, retention capacity, immobilization time, mechanical stability and economic viability prompted the use of refractory bricks and silk sachets. Since, airlift bioreactor was used, buoyancy and repeated utility of the matrix was taken into regard.
Chakravarty and Kundu AMB Expr (2016) 6:101
Page 4 of 8
Eect ofnitrogen sources, inoculum size andaeration rate onpellet formation
The morphological variation of S. roseosporus was carried out by changing the inoculum size, the nitrogen source and reducing aeration rate in the fermentation medium as shown in Table 2. Several visual dierences were observed in terms of the branching of hyphae and hyphal arrangement (Fig.3).
Correlation ofcell morphology andDaptomycin production
The production prole of immobilized cells revealed that the antibiotic production started at 48h of fermentation with immobilized cells on refractory bricks and reached maximum level (700 mg/l) by 132 h. On further incubation, no improvement in antibiotic production was observed. In case of free cell fermentation, the maximum antibiotic production (750mg/l) was observed by 132h as shown in Fig. 4. The Daptomycin production prole (Fig.4) with immobilized cells on silk sachets indicate a progress in cell mass and antibiotic titre up to 132h. The maximum antibiotic yield with immobilized cells on silk sachets reached 620 mg/l. Small and uy cell pellets were utilized for Daptomycin production. The growth and Daptomycin production proles with pellets indicate
a progress in cell mass and antibiotic production up to 132h. The maximum antibiotic yield with pellets reached 810mg/l.
Reusability ofcells pelletized andimmobilized cells
Repeated batch fermentation process was carried out for Daptomycin by S. roseosporus cells immobilized on refractory brick and silk sachets as shown in Fig.5. These modes were found to be superior due to low cell leakage and stable for repeated use. Signicant process engineering advantages are evident from immobilized cells in repeated batch operations. Reusability of immobilized cells was achieved by aseptic removal of fermentation medium and replacement with afresh medium for S. rose-osporus production. Daptomycin production was studied up to ten reuse cycles. The fermentation was continued for several batches until the carrier material disinte-grated. There was an increase in Daptomycin production up to eighth cycle for refractory brick immobilized cells and later a gradual decrease in antibiotic production was noticed. Similarly, for the silk sachets, the production could be repeated for six cycles.
Microscopic view ofdierent cell morphology
Streptomyces roseosporus cells as free cells and immobilized cells were studied by electron microscopy. The SEM photograph showed that cells were randomly distributed in the pores of the carrier matrix as seen in Fig.6. The photographs show the inner pores of carriers used, which were densely populated by the immobilized cells. The porous network was covered with the S. roseosporus cells which were adsorbed on to the matrix surface. The electron microscopic view of the immobilized cells revealed their dense and compressed structure. The mycelial network was compact and condensed to a smaller area. The silk sachets packaged the mycelium well enough but did not compress them much. The elongated, free mycelium were distributed over the space while the pelletized cells congregated under stress conditions.
Table 2 Table showing the optimization ofvarious conditions forpellet formation
Air ow rate=0.70vvm Nitrogen source Inoculum size
4% 5%
Soyabean meal Entangled mycelia laments
Dispersed mycelia laments
Peptone Small and smooth pellets Small irregular mycelia clumps
Yeast extract Big and uy pellets Entangled mycelial clumps
Chakravarty and Kundu AMB Expr (2016) 6:101
Page 5 of 8
Rheological characterization ofthe broth
Figure 7 shows that morphological changes and rheo-logical properties of S. roseosporus were interdependent phenomena. The three phase broth viscosity increased over the time for both free and pelletized cells. The specic cell growth rate was more for the free forms of cells than immobilized cells. The free cells accumulated as dense aggregates and resulted in viscous broth during the course of fermentation. The viscosity increased three times almost the initial value. This majorly aected the oxygen mass transfer. The pellets were comparatively stable but the dense growth of cells over the time led to 1.5 times increased viscosity and reduced mass transfer. The slow and steady growth of immobilized cells helped them overcome the viscosity issue and hence improve the hydrodynamics of the system.
Variation ofvolumetric oxygen transfer coefficient
The variation of volumetric oxygen transfer coefficients by immobilized cells on dierent adsorbent along with free cells. It was observed that, with free cells, the volu-metric oxygen transfer coefficients could be maintained at 20h1 at 144h as depicted in Fig.8. This eectively resulted in reduced Daptomycin. However, with the use of immobilized modes, volumetric oxygen transfer improved as three phase uid viscosity of the broth was much better controlled.
Volumetric oxygen mass transfer coefficients observed at 132h were 85 and 80h1 for refractory bricks and silk respectively whereas pellet was low. Broth viscosity was controlled in case of immobilized cells.
Chakravarty and Kundu AMB Expr (2016) 6:101
Page 6 of 8
Discussion
The lamentous nature of actinomycetes leads to its shear-sensitivity. Therefore, airlift bioreactor was chosen. Daptomycin production with free cells, pelletized cells and immobilized cells on various support matrices depicted the correlation of morphological variations with antibiotic production. The production of Daptomycin by immobilized cells was compared with that of two morphological forms of the cells i.e. free and pelletized cells. Two types of Pellet growth occurred in media using pep-tone as nitrogen source and yeast extract at 4% inoculum
size. Smaller pellets formed by peptone supplemented media with 4 % inoculum were more advantageous for antibiotic production due to their high surface to volume ratio (Nielsen et al. 1995). Faster consumption of pep-tone as a nitrogen source and low inoculum size led to comparatively slower growth of cells and nitrogen starvation. The cells started utilizing cellular nitrogen leading to stress conditions. S. roseosporous is a strict aerobe. It is necessary to provide sufficient dissolved oxygen during the fermentation. The dissolved oxygen tension in bulk uid was increased by lowering the air ow rate to
Chakravarty and Kundu AMB Expr (2016) 6:101
Page 7 of 8
2. The cyclization and elongation of the peptide chain where an adenylation domain selects the amino acid monomer to be incorporated and activates the carboxylate with ATP to make the aminoacyl-AMP. Next, the A domain installs an aminoacyl group on the thiolate of the adjacent T domain. The condensation (C) domain catalyzes the peptide bond forming reaction, which elicits chain elongation.
The dense network of the free cells led to three phase uid viscosity which hampered the oxygen mass transfer into the cells. For the immobilized system, the three phase broth viscosity was much better controlled and the hydrodynamics improved over the time. Volumetric oxygen mass transfer coefficients observed at 132h were 85h1 and 80h1 for refractory bricks and silk respectively whereas that for pellet and free cells was low. The present study conrmed that Daptomycin production with whole cell immobilization strategy in an airlift bio-reactor showed improved results. Therefore, whole-cell immobilization can be considered as a useful strategy for enhanced production of this life-saving drug. Further, eorts can be taken to improve the mass transfer characteristics of the free cell systems.
Abbreviations
NBRC: National Biological resource Centre, Chiba, Japan; VVM: volume of air ow per unit of volume of media per minute (volume per volume per minute); SEM: scanning electron microscopy.
Authors contributions
SK designed the research and developed the experimental setup; IC performed the research and wrote the paper. Both authors read and approved the nal manuscript.
Acknowledgements
The authors would like to thank the Central Instrumentation Facility Centre, IIT (BHU), Varanasi.
Competing interests
The authors declare that they have no competing interests.
Ethics approval
This article does not contain any studies concerned with experiment on human or animals.
Funding
Government (Federal) Funding.
Received: 12 September 2016 Accepted: 17 October 2016
Table 3 Comparison of Daptomycin production by free andimmobilized cells ofS. roseosporus
Carrier material Daptomycin conc. (mg/l) (after rst batch)
Daptomycin conc. (mg/l) (after nal batch)
Reusability upto (cycles)
Pellets 810 1430 2
Free cells 750 750 Nil
Refractory brick 700 4895 8
Silk sachets 620 3623 6
0.70 vvm which led to high oxygen tension on the surface and in the centre of the pellets. Previous literature(s) support that improving DO tension in submerged cultivations is favorable for the pellet formation, as described in (Du et al. 2003). The consolidated data in Table 3 shows that in a single batch, pelletized cells and free cells depicted considerable production ability.
Pellet growth oered advantages over the free cells and the regulation of hyphal extension and pellet size are of great importance. But the reusability and controlled uid viscosity of immobilized cells had an edge over them. This is benecial for antibiotic fermentation which is non growth associated (Ramakrishna and Prakasham 1999). The increased production of Daptomycin in immobilized system over free cells is accounted due to changes in permeability of cell walls well as reusability of the matrices (Morikawa etal. 1979). For pelletized cells, the reusability was limited up to two cycles as the cells degenerated, showed dense aggregates and hence increased broth viscosity. 1430mg/l Daptomycin was produced using pellets for 2 batches. Immobilized cells on refractory bricks and silk sachets led to 4895mg/l and 3623mg/l Daptomycin production respectively. The cell leakage, increased viscosity and cell degeneration over the time led to lesser reusability of silk sachets than refractory bricks. The high oxygen demand of the Daptomycin production processes may be attributed to its demand in the biosynthetic pathway. The biosynthetic pathway of Daptomycin shows that there are two crucial oxygen consuming steps in the pathway (Tally and De Bruin 2000; Robbel and Marahiel 2010):
1. The initiation step of Daptomycin formation requires oxygen where lipidation by DptE and DptF takes place. Decanoic acid is activated by the putative adenylating enzyme DptE under ATP consumption. The fatty acid is then transferred onto acyl carrier protein DptF. The C domain of DptA is predicted to catalyze the condensation reaction between fatty acid and N-terminal tryptophan.
References
Chakravarty I, Kundu K, Kundu S (2015) Daptomycin: discovery, development and perspectives. In: Mndez-Vilas A (ed) The battle against microbial pathogens: basic science, technological advances and educational programs, vol 2. Formatex Publications, Badajoz, Spain, pp 895903
Chakravarty and Kundu AMB Expr (2016) 6:101
Page 8 of 8
Choi DB, Park EY, Okabe M (2000) Dependence of apparent viscosity on mycelial morphology of Streptomyces fradiae culture in various nitrogen sources. Biotechnol Progr 16:525532
Dsouza SF, Melo JS, Deshpande A, Nadkarni GB (1986) Immobilization of yeast cells by adhesion to glass surface using polyethylenimine. Biotechnol Lett 8:643648
Du LX, Jia SJ, Lu FP (2003) Morphological changes of Rhizopus chinesis 12 in submerged culture and its relationship with antibiotic production. Process Biochem 38:16431646
Eisenstein BI, Oleson FB, Baltz RH (2010) Daptomycin: from the mountain to the clinic, with essential help from Francis Tally, MD. Clin Infect Dis 50:1015
Fluit AC, Wielders CLC, Verhoef J, Schmitz FJ (2001) Epidemiology and susceptibility of 3051 Staphylococcus aureus isolates from 25 university hospitals participating in the European SENTRY study. J Clin Microbiol 39:37273732
Huang D, Wen J, Wang G, Yu G, Jia X, Chen Y (2012) In silico aided metabolic engineering of Streptomyces roseosporus for daptomycin yield improvement. Appl Microbiol Biotechnol 94:637649
Kundu S, Mahapatra AC, Srivastava P, Kundu K (1992) Studies on cephalosporin-C production using immobilised cells of Cephalosporium acremonium in a packed bed reactor. Process Biochem 27:347350
Kundu S, Gupta S, Bihari V, Agrawal SC (2000) Studies on free and immobilized cells of C. acremonium on the production of cephalosporins. Indian J. Microbiol 40:141143
Mahapatra AC, Kundu K, Nigam VK, Prasad MV, Kundu S (2002) Cephalosporin-
C production by free and immobilized cells of Cephalosporium acremonium in dierent modes of bioreactors. Indian J Microbiol 42:319322 Mishra P, Srivastava P, Kundu S (2005) A comparative evaluation of oxygen mass transfer and broth viscosity using Cephalosporin-C production as a case strategy. World J Microbiol Biotechnol 21:525530
Morikawa Y, Karube I, Suzuki S (1979) Penicillin G production by immobilized whole cells of Penicillium chrysogenum. Biotechnol Bioeng 21:261270
Nielsen J, Johansen CL, Jacobsen M, Krabben P, Villadsen J (1995) Pellet formation and fragmentation in submerged cultures of Penicillium chrysogenum and its relation to penicillin production. Biotechnol Progr 11:9398
OCleirigh C, Casey JT, Walsh PK, Oshea DG (2005) Morphological engineering of Streptomyces hygroscopicus: regulation of pellet morphology through manipulation of broth viscosity. Appl Microbiol Biotechnol 68:305310
Papagianni M, Mattey M (2006) Morphological development of Aspergillus niger in submerged citric acid fermentation as a function of the spore inoculum level. Application of neural network and cluster analysis for characterization of mycelial morphology. Microb Cell Fact 5:1
Ramakrishna SV, Prakasham RS (1999) Microbial fermentations with immobilized cells. Curr Sci 77:87
Robbel L, Marahiel MA (2010) Daptomycin, a bacterial lipopeptide synthesized by a nonribosomal machinery. J Biol Chem 285:2750127508
Ruchti G, Dunn IJ, Bourne JR (1981) Comparison of dynamic oxygen electrode methods for the measurement of KLa. Biotechnol Bioeng 23:277290
Srivastava P, Kundu S (1998) A comparative evaluation of Cephalosporin C production using various immobilization modes. J Gen Appl Microbiol 44:113117
Tally FP, De Bruin MF (2000) Development of Daptomycin for gram-positive infections. J Antimicrob Chemother 46:523526
Yu G, Jia X, Wen J, Wang G, Chen Y (2011) Enhancement of Daptomycin production in Streptomyces roseosporus LC-51 by manipulation of cofactors concentration in the fermentation culture. World J Microbiol Biotechnol 27:18591868
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer
AMB Express is a copyright of Springer, 2016.
Abstract
The increased threat of drug resistance has challenged the existence of several conventional and non-conventional antibiotics in the recent times. Daptomycin is a novel cyclic-lipopeptide antibiotic produced by Streptomyces roseosporus that has progressed as a significant anti-MRSA (methicillin-resistant Staphylococcus aureus) antibiotic. But, the economic practicality of this highly valued secondary metabolite is deterred by its poor production and tedious processing methodology. The present study aims at strategic improvement of Daptomycin production through morphological variations of S. roseosporus cells. Free cells, pelletized cells and immobilized cells on ultra porous refractory brick and silk sachets were investigated for the production of Daptomycin in a lab-scale 2.0 l air-lift bioreactor. The effect(s) of nitrogen source, inoculum size and oxygen stress were analyzed for pellet formation of S. roseosporus. Interestingly, free cells produced 750 mg/l of Daptomycin in a single batch. But, the three phase broth viscosity increased due to vigorous growth of free cells which hampered the oxygen transfer rate. The cell degeneration over the time deterred pellet reusability. 1430 mg/l Daptomycin was produced using pellets for 2 batches. On the contrary, mechanical stability, buoyancy and reusability of refractory bricks and silk sachets were beneficial. Daptomycin production was recorded for 6-8 batches. Immobilized cells on refractory bricks and silk sachets led to 4895 mg/l and 3623 mg/l Daptomycin production respectively. Cell immobilization improved the three phase broth rheology and hence, the hydrodynamics within the reactor. Therefore, whole-cell immobilization could contribute to the ameliorated production of this life-saving drug.
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer