SciELO - Scientific Electronic Library Online

vol.19 número6Enzyme activity and thermostability of a non-specific nuclease from Yersinia enterocolitica subsp. palearctica by site-directed mutagenesisAntioxidant activity and protective role on protein glycation of synthetic aminocoumarins índice de autoresíndice de materiabúsqueda de artículos
Home Pagelista alfabética de revistas  

Servicios Personalizados




Links relacionados

  • En proceso de indezaciónCitado por Google
  • No hay articulos similaresSimilares en SciELO
  • En proceso de indezaciónSimilares en Google


Electronic Journal of Biotechnology

versión On-line ISSN 0717-3458

Electron. J. Biotechnol. vol.19 no.6 Valparaíso nov. 2016 


A fast and simple assay to quantify bacterial leukotoxin activity


Tobias Oppermanna,1 ,Stefan Schwarzb,1, Nadine Busseb, Peter Czermaka,b,c,d*

a Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project group Bioresources, Winchesterstrasse 2,35394 Giessen, Germany
b Institute ofBioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen, Wiesenstrasse 14,35390 Giessen, Germany
c Department of Chemical Engineering, Kansas State University, Durland Hall 1005, Manhattan, KS 66506-5102, USA
d Faculty of Biology and Chemistry, University ofGiessen, Ludwigstrasse 23, 35390 Giessen, Germany


Background: Mannheimia haemolytica is the primary bacterial pathogen in causing bovine respiratory disease with tremendous annual losses in the cattle industry. The leukotoxin from M. haemolytica is the predominant virulence factor. Several leukotoxin activity assays are available but not standardized regarding sample preparation and cell line. Furthermore, these assays suffer from a high standard error, a prolonged time consumption and often complex sample pretreatments, which is important from the bioprocess engineering point of view.

Results: Within this study, an activity assay based on the continuous cell line BL3.1 combined with a commercial available adenosine triphosphate viability assay kit was established. The leukotoxin activity was found to be strongly dependent on the sample preparation. Furthermore, the interfering effect of lipopolysaccharides in the sample could be successfully suppressed by adding polymyxin B. We reached a maximum relative P95 value of 14%, which is more than seven times lower compared to current available assays as well as a time reduction up to 88%.

Conclusion: Ultimately, the established leukotoxin activity assay is simple, fast and has a high reproducibility. Critical parameters regarding the sample preparation were characterized and optimized making complex sample purification superfluous.

Keywords: ATP assay, Bacterial pathogen, Bovine respiratory disease, Cattle industry, Critical parameters, Lipopolysaccharides, Mannheimia haemolytica, Microbial biotechnology, Polymyxin B, Quantification, Virulence.


1. Introduction

Mannheimia haemolytica is the primary bacterial pathogen causing bovine respiratory disease (BRD) and its primary virulence factors are leukotoxin (LKT) and lipopolysaccharides (LPS).

The LKT of M. haemolytica belongs to the repeat-in-toxin (RTX) family. All serotypes of M. haemolytica produce a 102-105 kDa heat labile LKT during the logarithmic phase of growth. In contrast to other RTX toxins the LKT of M. haemolytica is specific for ruminant leukocytes and the cytotoxicity is limited to ruminant lymphocytes, macrophages, neutrophils and platelets due to the specific expression of (3.2 integrins as a binding partner for the LKT. Especially, the leukocyte function associated antigen 1 (LFA1) is involved in causing the leukotoxic effect. LFA1 is a heterodimer compound of a CD11a and a CD18 subunit. Binding of LKT to both subunits causes the highest cytotoxic effect. The effect of LKT is strongly dose-dependent. Low concentrations activate neutrophils and macrophages, induce the release of histamine by mast cells and inhibit the mitogen mediated lymphoid proliferation. The consequences are respiratory burst, degranulation and release of pro inflammatory cytokines (tumor necrosis factor-a (TNF-a), interleukin-1 (IL-1) and interleukin-8 (IL-8)). High concentration causes apoptosis of bovine leukocytes by extrinsic and intrinsic mechanisms and lead to pore formation, cell swelling and ultimately to necrosis.

Besides LKT, LPS is a major actor of the cytotoxic effect. LKT and LPS are the most prominent components in the supernatant of M. haemolytica and are able to complex increasing the cytotoxicity compared to native LKT. LPS can also bind to (2 integrins whereby the CD18 subunit does not seem essential. The spectrum of efficacy of LPS and LKT overlaps. Both can stimulate alveolar macrophages to produce reactive oxygen and nitrogen mediates. Furthermore, LPS can also induce the production of IL-1, IL-8, leukotriene 4 and TNF-a,resulting in inflammation and apoptotic cell death.

The majority of LKT activity assays are based on the measurement of cytotoxicity. LKT sensitive cells are incubated with various concentrations of the LKT followed by a cytotoxicity assay. In general isolates of peripheral blood monocular cells (PBMCs) from cattle and the bovine B-lymphoblastoid cell line (BL3) are utilized. Currently available cytotoxicity assays for determining the LKT activity are the Chromium-51 release assay, lactate dehydrogenase (LDH) releaseassay, neutral red uptake assay, 3-[4,5-dimethyl(thiazol-2-yl)-3,5-diphenyl] tetrazolium bromide (MTT) and the nitroblue tetrazolium (NBT) assay. Non-cytotoxicity assays are based on the morphological change of BL3 cells after incubation with LKT and the inhibition of the luminol dependent chemiluminescence of LKT incubated bovine neutrophils.

All LKT activity assays outlined above suffer from a high standard error and a high time consumption caused by a complex sample pretreatment. The overlapping cytotoxic effects and a molar ratio of LPS/LKT of ca. 60:1 in concentrated culture supernatants from M. haemolytica place a high demand on the sample preparation to measure just the single effect of LKT. However, assays enabling activity measurements of the pure LKT are beneficial for bioprocess applications. Furthermore, a time and temperature dependent decrease in LKT activity is mentioned in the literature but remained uncharacterized with respect to activity assays. Therefore, especially the sample preparation could influence the LKT activity leading to a reduced reproducibility and comparability.

This study provides a novel assay for a fast and reproducible determination of the LKT activity. The assay is based on a commercial available adenosine triphosphate (ATP) viability assay kit. Important parameters affecting the LKT activity such as time and temperature throughout the sample preparation were characterized and optimized. In order to repress the interfering effect of LPS the addition of polymyxin B (PB) as a LPS inhibitor was evaluated making further complex and time consuming sample purification superfluous.

2. Material and methods

2.1. Preparation of the LKT activity standard

The LKT was obtained by growth of Mannheimia haemolytica (ATCC® 43,270, American Type Culture Collection, USA) in RPMI-1640 medium (R6504, Sigma-Aldrich, Germany) in a 0.5 L stirred tank reactor (MiniBio 500, Applikon, Netherlands) under similar conditions as described previously. Harvest occurred at the end of the exponential growth phase. The supernatant was centrifuged (5000 xg, 10 min, 4°C), afterwards filtered through a 0.22 jjmbottle top filter (SCGVT05RE, Merck Millipore, Germany) and aliquoted and frozen at -85°C in cryo-vials (72.379, Sarstedt, Germany).

2.2. Preparation ofthe cell culture

The BL3.1 cells were grown in RPMI-1640 (RPMI 1640 FG 1385, Biochrom, Germany) supplemented with 10% (v/v) FBS (FBS Superior, Biochrom, Germany) at 37°C and 5% CO2 in T75-flasks (REF 83.3911.502, Sarstedt, Germany) with a working volume of 25 mL. The cells were passaged three days before the activity assay was performed. Cell counts and viability were determined in a hemocytometer using the trypan blue exclusion assay. Criteria for passaging was a cell viability of > 80%. The cell suspension was diluted to a viable cell density of 0.15-0.30 * 106 cells/mL to ensure a high growth rate as well as a high viability.

2.3. General procedure of the ATP assay

Within this study a viable cell density of ≥ 0.60 * 106 cells/mL and a viability of ≥ 95% was set to be optimal for the assay. The cell suspension was diluted in fresh medium to a viable cell density of 0.60 * 106 cells/mL. The LKT activity standard (LKTAS) vial was thawed in a water bath (23°C) until the ice was nearly gone. Afterwards the LKTAS was supplemented with 1% (v/v) of a 5 mg/mL concentrated solution of polymyxin B (Cat# 420,413, Calbiochem, USA) in PBS (Biochrom, Germany) and incubated for 15 min on ice. A twofold serial dilution of the cell suspension (0.60 * 106 cells/mL) served as a calibration and RPMI-1640 supplemented with 10% (v/v) FBS was used as a blank. As the positive control 50 µL of a 4% Triton X-100 (Sigma-Aldrich, Germany) solution dissolved in PBS was supplemented with 50 µL of the cell suspension. A twofold serial dilution with PBS was carried out for all samples in a white 96 well plate (Nunc 136102, Thermo Fisher Scientific, Germany) to a final volume of 50 µL/well. Each sample well was then supplemented with 50 µL of the cell suspension and the plate was incubated for 2 h at 37°C and 5% CO2. Afterwards, 100 µL of the working solution of the viability assay (CellTiter-Glo®, Promega, Germany) was added to each well. The plate was shaken for 3 min and remained for further 10 min in the 30°C prewarmed plate reader (Synergy HT, BioTek Instruments, USA), followed by the luminescence measurement.

The calibration line for cell count was forced through zero and a cell number of 3 * 104 cells/well was the upper value of the calibration. The data were linearized by plotting the logarithm of the dilution factor of the sample to the base of 2 on the abscissa according to [Equation 1 ].

One unit/mL of LKT activity is defined as the concentration of biological active leukotoxin which causes death of 50% of the target cells. As an alternative calculation for the LKT activity the EC50 of the dose response function in OriginPro 8.5 was tested (data not shown) and rejected because the relative P95 value was higher compared to the linearization method. The P95 value represents the interval of the sample data which covers the true value 95% of the time.

2.4. Optimization of the sample preparation

The assay was carried out as previously described. To determine the optimal sample preparation various incubations conditions after thawing (10 min at 37°C + 50 min on ice/1 h at 23°C/ 1 h on ice) were evaluated.

2.5. Optimization ofthe LKT incubation time

The assay was carried out as previously described. The LKT incubation time was varied in an independent test serial between 1, 2 and 3 h.

2.6. Neutralization ofLPS

Two experiments with minor changes from the general procedure of the ATP assay were carried out to proof a sufficient neutralization of LPS with PB. For the first experiment the effect of various PB concentrations were evaluated. The incubation time was set to was incubated for 70 min at 37°C for a complete LKT inactivation. Afterwards the standard was split and one half was supplemented with 1% (v/v) PB in PBS at a concentration of 5 mg/mL.

Table 1
Determined LKT units of the mean of triplicates and measurement deviation.

3. Results

3.1. Statistical evaluation ofthe assay

For the determination of the statistical values, nine samples with the same LKT concentration were analyzed in three independent test series. An averaged relative P95 value of 14% was calculated (Table 1).

3.2. Optimization ofthe sample preparation

The toxicity of the LKT standard shows a strong sensitivity to the sample incubation condition and time after thawing (Fig. 1). The storage for 1 h on ice did not show a significant reduction of biological LKT activity compared to an immediate use. Nevertheless, a temperature dependent reduction of the LKT activity is especially favored at T ≥ 37°C. The higher the temperature the faster the LKT inactivation.

Fig. 1. Temperature and time depended effect of the sample preparation on the LKT activity at a relative LKTAS concentration of 0.25. The LKTAS was either incubated after thawing for 10 min at 37°C, 1 h at 23°C, 1 h on ice prior the general ATP assay procedure or immediately used.

3.3. Optimization ofthe incubation time

Regarding the incubation time, no differences of the cytotoxicity could be observed between 1 and 3 h (Fig. 2). However, an artificial effect on the maximum death rate of the positive control (Triton X-100) depending on the incubation time could be seen (data not shown). At an incubation time of 1 h the death rate of the positive control was 96% instead of the anticipated 100%. An insufficient degradation of intracellular ATP after cell death could be assumed causing an interference in the viability assay and an inaccurate death rate of 96% is the result. Therefore, an incubation time of 2 h was set to be optimal.

Fig. 2. Incubation time dependent effect on the LKT activity. Throughout the general ATP assay procedure the LKT incubation time was set to 1 h, 2 h and 3 h.


3.4. Neutralization ofLPS

All samples supplemented with PB showed no difference to each other (Fig. 3). Consequently, a saturation with PB can be assumed at concentrations ranging from 0.025 mg/mL to 0.075 mg/mL. A PB concentration of 0.05 mg/mL was selected based on a safety factor to ensure neutralization under conditions with varying LPS concentrations.

Fig. 3. Effect ofdifferent PB concentrations on the LKT activity. Throughout the general ATP assay procedure the LKTAS was supplemented with either 0.025 mg/ml PB, 0.05 mg/mL PB, 0.75 mg/mL PB or without PB.

A further proof of a complete LPS neutralization through PB can be seen in Fig. 4. The incubation of the LKTAS at 37°C for 70 min led to an inactivation of the LKT and the remaining cytotoxicity of 22% can be attributed to the LPS. A further supplementation with 0.05 mg/mL PB led to a complete loss of cytotoxic activity. Therefore, a neutralization of LPS could be assumed.

Fig. 4. Effect of different temperature and PB sample pretreatments on the LKT activity at a relative LKTAS concentration of 0.5. Throughout the general ATP assay procedure the LKTAS was incubated for 70 min at 37°C and either supplemented without or with 0.05 mg/mL PB and compared to an immediately used LKTAS.

4. Discussion and conclusion

Current available LKT activity assays are based on continuous cell lines and isolated leukocytes. BL3.1 cells have the lowest variability compared to other continuous leukocyte cell lines. The usage of BL3.1 cells makes the extraction of fresh leukocytes superfluous, reducing the effort enormously and ensure a reproducible and high quality of the target cells.

Most important is a standardized sample preparation and assay procedure as demonstrated in this study. Especially, the time and temperature throughout the sample preparation had a strong influence on the LKT activity. A gentle thawing procedure in combination with a sample preparation on ice is mandatory. This leads to a higher accuracy and reproducibility compared to currently available activity assays. We reached a maximum relative P95 value of 14%, which is more than seven times lower compared to previous data. The time consumption for the ATP assay is ~30 min. Compared to the neutral red assay and MTT this corresponds to a reduction of 75% and respectively 88%. A further huge advantage of our established assay is the direct inactivation of LPS with PB, making further complex and time consuming sample purification superfluous.

In summary, the established LKT activity assay is simple, fast and sensitive overcoming all drawbacks of currently available activity assays. A complete automation of the ATP assay is possible making the assay well suited for process monitoring (e.g. downstream) for industrial LKT production. A transfer into a 384 well plate format is conceivable for a high-throughput screening system and could further reduce the time consumption and material costs.

Financial support

The researchers would like to thank the Hessen State Ministry of Higher Education, Research and the Arts for the financial support within the Hessen initiative for scientific and economic excellence (LOEWE).

Conflict of interest

The authors declare that they have no conflict of interest.



1. Mohamed RA, Abdelsalam EB. A review on Pneumonic pasteurellosis (Respiratory Mannheimiosis) with emphasis on pathogenesis, virulence mechanisms and predisposing factors. Bulg J Vet Med 2008;11:139-60.         [ Links ]

2. Singh K, Ritchey JW, Confer AW. Mannheimia haemolytica: Bacterial-host interactions in bovine pneumonia. Vet Pathol 2011;48:338-48.         [ Links ]

3. Highlander SK, Fedorova ND, Dusek DM, Panciera R, Alvarez LE, Rinehart C. Inactivation of Pasteurella Mannheimia) haemolytica leukotoxin causes partial attenuation of virulence in a calf challenge model. Infect Immun 2000;68:3916-22.         [ Links ]

4. Narayanan SK, Nagaraja TG, Chengappa MM, Stewart GC. Leukotoxins of gram-negative bacteria. Vet Microbiol 2002;84:337-56.         [ Links ]

5. Li J, Clinkenbeard KD. Lipopolysaccharide complexes with Pasteurella haemolytica leukotoxin. Infect Immun 1999;67:2920-7.         [ Links ]

6. Frey J, Kuhnert P. RTX toxins in Pasteurellaceae. Int J Med Microbiol 2002;292: 149-58.^221-00200.         [ Links ]

7. Dassanayake RP, Maheswaran SK, Srikumaran S. Monomeric expression of bovine p2-integrin subunits reveals their role in Mannheimia haemolytica leukotoxin-induced biological effects. Infect Immun 2007;75:5004-10.         [ Links ]

8. Leite F, Brown JF, Sylte MJ, Briggs RE, Czuprynski CJ. Recombinant bovine interleukin-1 (J amplifies the effects of partially purified Pasteurella haemolytica leukotoxin on bovine neutrophils in a J2-integrin-dependent manner. Infect Immun 2000;68:5581-6.         [ Links ] 

9. Lawrence PK, Nelson WR, Liu W, Knowles DP, Foreyt WJ, Srikumaran β2 integrin Mac-1 is a receptor for Mannheimia haemolytica leukotoxin on bovine and ovine leukocytes. Vet Immunol Immunopathol 2008;122:285-94.         [ Links ]

10. Thumbikat P, Dileepan T, Kannan MS, Maheswaran SK. Mechanisms underlying Mannheimia haemolytica leukotoxin-induced oncosis and apoptosis of bovine alveolar macrophages. Microb Pathog 2005;38:161-72.         [ Links ]

11. Thumbikat P, Briggs RE, Kannan MS, Maheswaran SK. Biological effects of two genetically defined leukotoxin mutants of Mannheimia haemolytica. Microb Pathog 2003;34:217-26.         [ Links ]

12. Atapattu DN, Czuprynski CJ. Mannheimia haemolytica leukotoxin induces apoptosis of bovine lymphoblastoid cells (BL-3) via a caspase-9-dependent mitochondrial pathway. Infect Immun 2005;73:5504-13.         [ Links ]

13. Wang JF, Kieba IR, Korostoff J, Guo TL, Yamaguchi N, Rozmiarek H, et al. Molecular and biochemical mechanisms of Pasteurella haemolytica leukotoxin-induced cell death. Microb Pathog 1998;25:317-31.         [ Links ]

14. Jeyaseelan S, Hsuan SL, Kannan MS, Walcheck B, Wang JF, Kehrli ME, et al. Lymphocyte function-associated antigen 1 is a receptor for Pasteurella haemolytica leukotoxin in bovine leukocytes. Infect Immun 2000;68:72-9.         [ Links ]

15. Lafleur RL, Malazdrewich C, Jeyaseelan S, Bleifield E, Abrahamsen MS, Maheswaran SK Lipopolysaccharide enhances cytolysis and inflammatory cytokine induction in bovine alveolar macrophages exposed to Pasteurella Mannheimia) haemolytica leukotoxin. Microb Pathog 2001;30:347-57.         [ Links ]

16. Dileepan T, Thumbikat P, Walcheck B, Kannan MS, Maheswaran SK. Recombinant expression of bovine LFA-1 and characterization of its role as a receptor for Mannheimia haemolytica leukotoxin. Microb Pathog 2005;38:249-57.         [ Links ]

17. Ackermann MR, Brogden KA. Response of the ruminant respiratory tract to Mannheimia (Pasteurella) haemolytica. Microbes Infect 2000;2:1079-88.         [ Links ]

18. Zecchinon L, Fett T, Desmecht D. How Mannheimia haemolytica defeats host defence through a kiss of death mechanism. Vet Res 2005;36:133-56.         [ Links ]

19. Li J, Clinkenbeard KD, Ritchey JW. Bovine CD18 identified as a species specific receptor for Pasteurella haemolytica leukotoxin. Vet Microbiol 1999;67:91-7. http: //         [ Links ]

20. Gopinath RS, Ambagala TC, Deshpande MS, Donis RO, Srikumaran S. Mannheimia (Pasteurella) haemolytica leukotoxin binding domain lies within amino acids 1 to 291 of bovine CD18. Infect Immun 2005;73:6179-82.         [ Links ]

21. Wright SD, Jong MT. Adhesion-promoting receptors on human macrophages recognize Escherichia coli by binding to lipopolysaccharide. J Exp Med 1986;164: 1876-88.         [ Links ]

22. Lafleur RL, Abrahamsen MS, Maheswaran SK The biphasic mRNA expression pattern of bovine interleukin-8 in Pasteurella haemolytica lipopolysaccharide-stimulated alveolar macrophages is primarily due to tumor necrosis factor alpha. Infect Immun 1998;66:4087-92.         [ Links ] 

23. Hsuan SL, Kannan MS, Jeyaseelan S, Prakash YS, Malazdrewich C, Abrahamsen MS, et al. Pasteurella haemolytica leukotoxin and endotoxin induced cytokine gene expression in bovine alveolar macrophages requires NF-kB activation and calcium elevation. Microb Pathog 1999;26:263-73.         [ Links ]

24. Yoo HS, Maheswaran SK, Lin G, Townsend EL, Ames TR. Induction of inflammatory cytokines in bovine alveolar macrophages following stimulation with Pasteurella haemolytica lipopolysaccharide. Infect Immun 1995;63:381-8.         [ Links ] 

25. Greer CN, Shewen Pe. Automated colorimetric assay for the detection of Pasteurella haemolytica leucotoxin. Vet Microbiol 1986;12:33-42.         [ Links ]

26. Chang YF, Young R, Post D, Struck DK. Identification and characterization of the Pasteurella haemolytica leukotoxin. Infect Immun 1987;55:2348-54.         [ Links ] 

27. Waurzyniak BJ. Enhancement of Pasteurella haemolytica A1 leukotoxin activity by bovine serum albumin. Michigan: Michigan State University East Lansing; 1991.         [ Links ] 

28. Craig FF, Dalgleish R, Sutherland AD, Parton R, Coote JG, Gibbs HA, et al. A colourimetric, microplate assay for the leucotoxin of Pasteurella haemolytica. Vet Microbiol 1990;22:309-17.         [ Links ]

29. Gentry MJ, Confer AW, Panciera RJ. Serum neutralization of cytotoxin from Pasteurella haemolytica, serotype 1 and resistance to experimental bovine pneumonic pasteurellosis. Vet Immunol Immunopathol 1985;9:239-50.         [ Links ]

30. Vega MV, Maheswaran SK, Leininger JR Ames TR. Adaptation of a colorimetric microtitration assay for quantifying Pasteurella haemolytica A1 leukotoxin and antileukotoxin. Am J Vet Res 1987;48:1559-64.         [ Links ]

31. Chang YF, Renshaw HW. Pasteurella haemolytica leukotoxin: Comparison of 51chromium-release, trypan blue dye exclusion, and luminol-dependent chemiluminescence-inhibition assays for sensitivity in detecting leukotoxin activity. Am J Vet Res 1986;47:134-8.         [ Links ]

32. Baluyut CS, Simonson RR Bemrick WJ, Maheswaran SK. Interaction of Pasteurella haemolytica with bovine neutrophils: Identification and partial characterization of a cytotoxin. Am J Vet Res 1981;42:1920-6.         [ Links ]

33. Czuprynski CJ, Noel EJ, Ortiz-Carranza O, Srikumaran S. Activation of bovine neutrophils by partially purified Pasteurella haemolytica leukotoxin. Infect Immun 1991;59:3126-33.         [ Links ]

34. Du Preez JC, Van Rensburg E, Kilian SG. Kinetics of growth and leukotoxin production by Mannheimia haemolytica in continuous culture. J Ind Microbiol Biotechnol 2008;35:611-8.         [ Links ]

35. Van Rensburg E, Du Preez JC. Effect of pH, temperature and nutrient limitations on growth and leukotoxin production by Mannheimia haemolytica in batch and continuous culture. J Appl Microbiol 2006;102:1273-82.         [ Links ]

36. Van Rensburg E, Du Preez JC, Kilian SG. Influence of the growth phase and culture medium on the survival of Mannheimia haemolytica during storage at different temperatures. J Appl Microbiol 2004;96:154-61.         [ Links ]

37. Czuprynski CJ, Noel EJ. Influence of Pasteurella haemolytica A1 crude leukotoxin on bovine neutrophil chemiluminescence. Infect Immun 1990;58:1485-7.         [ Links ] 

38. Clinkenbeard KD, Mosier DA, Confer AW. Transmembrane pore size and role of cell swelling in cytotoxicity caused by Pasteurella haemolytica leukotoxin. Infect Immun 1989;57:420-5.         [ Links ]

39. Repetto G, del Peso A, Zurita JL. Neutral red uptake assay for the estimation of cell viability/cytotoxicity. Nat Protoc 2008;3:1125-31.         [ Links ]

40. Slater K. Cytotoxicity tests for high-throughput drug discovery. Curr Opin Biotechnol 2001;12:70-4.         [ Links ]


Article history: Received 17 August 2016 Accepted 4 October 2016 Available online 20 October 2016

* Corresponding author at: Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen, Wiesenstrasse 14, 35390 Giessen, Germany. E-mail address: (P. Czermak). 

1 These authors contributed equally to this work. Peer review under responsibility of Pontificia Universidad Católica de Valparaíso.

Copyright © 2016 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of Pontificia Universidad Católica de Valparaíso.


Creative Commons License Todo el contenido de esta revista, excepto dónde está identificado, está bajo una Licencia Creative Commons