Serviços Personalizados
Journal
Artigo
texto em 
Inglês (pdf)
Artigo em XML
Referências do artigo
Tradução automática
Enviar este artigo por emailIndicadores
-
Citado por SciELO -
Acessos
Links relacionados
-
Similares em
SciELO
Compartilhar
Permalink
Agrociencia
versão On-line ISSN 2521-9766versão impressa ISSN 1405-3195
Agrociencia vol.50 no.5 Texcoco Jul./Ago. 2016
Animal Science
Use of activated carbon to preserve lyophilized cellulolytic bacteria
1Unidad Académica de Medicina Veterinaria y Zootecnia No. 2, Universidad Autónoma de Guerrero. Km. 197 Carretera Acapulco-Pinotepa Nacional. 41940. Cuajinicuilapa, Guerrero, México (sanchezsantillanp@gmail.com).
2Programa de Ganadería, Colegio de Postgraduados, Km 36.5, Carretera México-Texcoco. 56230. Montecillo, Texcoco, Estado de México, México.
3Instituto de Agrobiotecnología del Litoral, Universidad Nacional del Litoral. Paraje el Pozo, Santa Fe S3000ZAA. Argentina.
4Programa de Suelos, Colegio de Postgraduados, Km 36.5, Carretera México-Texcoco. 56230. Montecillo, Texcoco, Estado de México, México.
Successful conservation of microorganisms is attained by preventing contamination during the process and optimizing high survival and genetic stability. The objective of this study was to evaluate the use of activated carbon as a preserver of cellulolytic bacteria during the process of lyophilization. The cellulolytic bacterial culture was obtained from four transfers of fresh ruminal liquid in culture media and Whatman paper. As a lyoprotectant, activated carbon (CA) was added before lyophilizing and compared with a control treatment without lyoprotectant (SL). The lyophilized bacteria were reactivated in culture media; characteristics of both media and bacteria were measured. The reactivated bacteria were the inoculum in the evaluation of in vitro dry matter degradation (%DMDEG) and production of volatile fatty acids (VFA). The experimental design was completely randomized repeated in time. The CA treatment degraded 83.3% of the Whatman paper during reactivation. The oxide-reduction potential and culture media pH were not different between treatments (p>0.05). The CA treatment showed higher concentration of bacteria (9.58×108 bacteria mL-1), greater degradation of substrates (32.75 %DMDEG), and higher production of acetic (54.50 mM L-1) and butyric (12.74 mM L-1) acids, compared to the SL treatment (p≤0.05). Propionic acid and total VFA showed significant treatment-time interaction. The CA treatment produced more total VFA (p≤0.05), but propionic acid was different only between treatments at the first measurement time (p≤0.05). Activated carbon has characteristics that preserve cellulolytic bacteria for lyophilization.
Key words: Lyoprotectant; ruminal bacteria; in vitro; lyophilization; degradation
El éxito de la conservación de microorganismos se logra al evitar contaminaciones en el proceso, y al optimizar la sobrevivencia alta y la estabilidad genética. El objetivo de este estudio fue evaluar el uso del carbón activado como preservador de bacterias celulolíticas en el proceso de liofilización. El cultivo de bacterias celulolíticas se obtuvo de cuatro trasferencias de fluido ruminal fresco en medios de cultivo y papel Whatman. Como preservador se adicionó carbón activado (CA), antes de liofilizar, y se comparó con un tratamiento testigo sin preservador (SL). Las bacterias liofilizadas se reactivaron en medios de cultivo, en los cuales se midieron sus características y las de las bacterias. Las bacterias reactivadas fueron el inóculo para evaluar la degradación in vitro de la materia seca (%DEGMS) y la producción de ácidos grasos volátiles (AGV). El diseño experimental fue completamente al azar repetido en el tiempo. El tratamiento CA degradó 83.3 % de papel Whatman en la reactivación. En la reactivación de las bacterias el potencial óxido-reducción y el pH de los medios de cultivo no fueron diferentes entre tratamientos (p>0.05). El tratamiento CA mostró concentración mayor de bacterias (9.58×108 bacterias mL-1), degradación mayor de sustratos (32.75 %DEGMS), producción mayor de ácido acético (54.50 mM L-1) y butírico (12.74 mM L-1), comparado con el tratamiento SL (p≤0.05). El ácido propiónico y AGV totales presentaron interacción significativa de tratamiento-tiempo. El tratamiento CA produjo más AGV totales (p≤0.05), pero el ácido propiónico sólo fue diferente entre tratamientos en el primer tiempo de medición (p≤0.05). El carbón activado tiene características de preservador de bacterias celulolíticas para la liofilización.
Palabras clave: Preservador; bacterias ruminales; in vitro; liofilización; degradación
Introduction
Successful conservation of microorganisms is attained when contamination is avoided during the process and when high survival and genetic stability is optimized. Conservation methods vary and a method should be selected specifically for the microorganisms to be conserved (García and Uruburu, 2000; Morales-García et al., 2010). No single lyoprotectant exists for the conservation of any type of bacteria and the best for each type must be identified (Morales-García et al., 2010).
One of method is lyophilization, in which the water content of the material to be conserved is frozen and eliminated by sublimation caused by a difference in pressure (Kumar et al., 2013). Lyophilization is the most used process for conserving biological products because it dehydrates when the frozen water is eliminated. The nature, time and expense of the process are dependent on the chemical and physical nature of the microorganism to be lyophilized (Ramírez, 2006). It is an effective process for conserving cells in a viable state and organisms such as eukaryotes and bacteria in a latent state. To increase survival of cells subjected to lyophilization, substances are used that act as protectors.
Conservation of bacteria relevant to animal nutrition by lyophilization is documented as part of the research process (Safronova and Novikova, 1996; Jung et al., 2004; Cobos et al., 2007; Cobos et al., 2011), but there is no information about the process of conservation of microorganisms. Morgan et al. (2006) indicated that conservation of microorganisms by lyophilization is an empirical method without including proven theories. In the specialized literature, we found no information about the use of lyoprotectants with ruminal anaerobic cellulolytic bacteria. The objective of this study was to evaluate the effectiveness of activated carbon as a lyoprotectant of a culture of cellulolytic bacteria by observing their capacity to degrade cellulolytic substrates after having been lyophilized.
Materials and Methods
The study was carried out in the laboratory of Ruminal Microbiology and Microbial Genetics at the Colegio de Postgraduados, Campus Montecillo. Besides, it was developed in a biosafety bell (Labconco®), with a class II purifier, and provided with ultraviolet rays.
Ruminal fluid culture medium (FR)
The culture medium was composed of 30 mL ruminal fluid (RF) clarified with 5 mL mineral solution I [6 g K2HPO4, (Sigma) in 1000 mL distilled H2O], 5 mL mineral solution II [6 g KH2PO4 (Sigma), 6 g (NH4)2SO4 (Merck), 12 g NaCl (Sigma-Aldrich), 2.45 g MgSO4 (Sigma) and 1.6 g CaCl-2H2O (Sigma) in 1000 mL distilled H2O], 0.1 mL 0.1 % resarzurin (Sigma-Aldrich), 0.2 g soy peptone (Merck), 0.1 g yeast extract (Sigma), 2 mL sulfide-cysteine solution [2.5 g L-cysteine (Sigma) in 15 mL 2N NaOH (Meyer), 2.5 g Na2S-9H2O (Meyer) gauged to 100 mL distilled H2O], 5 mL 8 % solution Na2CO3 (Baker) and 52.6 mL distilled H2O. The medium was sterilized for 15 min in an autoclave (Tuttnauver® 2540F, Israel) at 121 °C and 15 psi).
Cellulolytic bacterial culture
The ruminal fluid was obtained from a Jersey cow with a ruminal cannula. The fluid was centrifuged at 1157 g in a centrifuge (Eppendorf® 5804, Germany) for 3 min at 25 °C. The supernatant was recovered and used as the inoculum. Nine milliliters of sterile FR medium were added to sterile tubes (18×150 mm) containing a strip of Whatman paper (3×30 mm) and 0.05 g crystalline cellulose (Sigma) under a flow of CO2. Tubes were kept in an incubator (Riossa® EO-71, México) at 39 °C until the Whatman paper degraded. One milliliter of the inoculated medium was transferred to another sterile tube and incubated at 39 °C until the Whatman paper was degraded. Four transfers were performed to obtain a culture of cellulolytic bacteria (CCB) capable of degrading Whatman paper. In sterile serological vials (50 mL) containing a strip of Whatman paper (3×30 mm) and 0.1 g crystalline cellulose, 27 mL sterile FR medium were deposited under constant flow of CO2 and kept at 39 °C for 72 h to detect sterility. The vials were inoculated with 3 mL of the product obtained from the four transfer and incubated at 39 °C until the Whatman paper degraded (10 d).
Treatments
The treatments were: 1) CA, activated carbon as a lyoprotectant; one vial with 0.1 g activated carbon (Hycel) was incubated at 39 °C for 2 h and; 2) SL, no lyoprotectant; one vial as a control treatment. The vials were frozen in a roller freezer (Labconco® Shell Freezer, USA) up to ‒38 °C and then, in a lyophilizer (Labconco® Freezone 6 L, USA), they were lyophilized 24 h (‒50 °C and 13.5 Pa).
Reactivation of treatments
Tubes (18×150 mm) containing a strip of Whatman paper (3×30 mm) and 0.05 g crystalline cellulose were sterilized (15 min at 121 °C and 15 psi). To detect sterility, 9 mL sterile FR medium, under CO2 flow, was incubated at 39 °C for 72 h. Six tubes were inoculated with 0.05 g lyophilized CA and six with 0.05 g SL, under a flow of CO2. The tubes were incubated at 39 °C for 10 d. After 7 and 10 d, degradation of the paper was quantified. At the end of incubation the following measurements were taken: 1) pH with a potentiometer (Orion 250A, Brazil; calibration: pH 7 and 4); 2) oxide reduction potential with a potentiometer (Orion 710A, Brazil; calibration: solution +220 oxide-reduction); and 3) concentration of total bacteria by direct count in a Petroff-Hauser chamber (Hausser #39000, Electron Microscopy Sciences, USA) and the formula bacterial concentration = (average) (dilution factor (2×107).
In vitro dry matter degradation
Tubes (18×150 mm) containing 0.01 g Whatman paper and 0.05 g crystalline cellulose were sterilized 15 min at 121 °C and 15 psi. Under a CO2 flow, 9 mL of sterile FR medium was added and the tubes were incubated at 39 °C for 72 h to detect sterility. The tubes (12 independent replications) were inoculated with 1 mL CA or SL reactivated and incubated at 39 °C for 10 d. After 7 and 10 d of incubation, paper degradation was quantified. The in vitro capacity to degrade, %DMDEG, was calculated at 10 d with the formula %DMDEG=(initial sample ‒ undegraded sample / initial sample) ×100.
Concentration of volatile fatty acids (AGV)
One mL of culture medium with 10 d of incubation was mixed with 25 % metaphosphoric acid and centrifuged at 18 800 g for 10 min. The supernatant was placed in vials for chromatography (1.5 mL, Perkin Elmer®, USA). The concentration of AGV was determined in a gas chromatograph (Perkin Elmer®, model Claurus 500, USA) equipped with a flame ionization detector and a capillary column (Elite FFAP, Perkin-Elmer®) 15 m×0.32 mm. The carrier gas was nitrogen (flow 4 mL min-1) and H2 and O2 (flow 45 and 450 mL min-1) to produce the flame. The oven, injector and column temperatures were 120, 250 and 250 °C; 1 mL of the sample was injected. Three peaks were obtained during retention time 2.16, 2.59 and 3.11 for acetic, propionic and butyric acids.
Statistical design and analysis
The experimental design was completely randomized. The experiment was repeated once and the data on %DMDEG, AGV, pH, oxide reduction and concentration of total bacteria were analyzed as repeated measures with the MIXED procedure of SAS (SAS® Institute Inc., 2011). The averages were fit by minimum squares to compare them with the Tukey test (p≤0.05). The variables propionic acid and VFA were transformed to log10 and the square root of %DMDEG was calculated to comply with the homoscedasticity assumption of the data. The statistical model was
where: Y ijk is the response variable in observation k, repetition j, treatment i; 𝛍 is general mean; τ i is the effect of the i th treatment; δ j(i) is the random error associated with the j th repetition within the i th treatment; P k is the effect of the k th time; (τP) ik is the effect of the interaction treatment-time; ε ijk is the random error associated with the k th repeated measure within the j th repetition.
Results and Discussion
Eighty-three percent of the repetitions of lyophilized bacteria reactivated with CA degraded the paper at 7 and 10 d of incubation. In contrast, the repetitions of SL did not degrade the paper in the same time. The SL treatment was 88.79 % less capable of degrading the cellulolytic substrates in vitro, relative to the CA treatment (Table 1). The CA treatment exhibited higher %DMDEG of crystalline cellulose and paper than the SL treatment (p≤0.05; Table 1). This is because cellulolytic bacteria are anaerobic, and the activated carbon reduces the presence of oxygen before lyophilization (Malik, 1990); lyoprotectants increase survival of lyophilized bacteria (Muñoz-Rojas et al., 2006; Morales-García et al., 2010). Activated carbon functions as a lyoprotectant in lyophilization processes because of its characteristics of reversible physical adsorption, adsorption in the liquid phase without elimination by simple desorption and porosity (Littrell et al., 2002; Roussak and Gesser, 2013). Activated carbon is used to support microbial growth in bioremediation (Gabr et al., 2009; Mercier et al., 2013) and to adsorb bacteria for their isolation (Yuan et al., 2012).
Table 1: Bacterial concentration [Bacteria], percentage of in vitro degradation of dry matter (%DMDEG), pH and acetic and butyric acid concentration after 10 d of incubation of lyophilized cellulolytic bacteria, with and without activated carbon as a lyoprotectant†.
♦ † The interaction treatment-time did not have a significant effect (p>0.05) on the variables. a, b: average values with different letters in the same column are statistically different (p≤0.05); EEM: standard error of the average value; CA: activated carbon as lyoprotectant; SL: without lyoprotectant.
The concentration of total bacteria in the CA treatment was 2.37×108 bacteria mL-1, more than with the SL treatment (p≤0.05). In its process of activation, the carbon increases in surface area (Elder, 2010; Villareal et al., 2015), which permits it to absorb bacteria into its porous structure and release them later (Roussak and Gesser, 2013; Villareal et al., 2015). The differences in bacterial concentration between treatments and %DMDEG (Table 1) are reflected in production of AGV (Table 2). The cellulolytic bacteria produce mostly acetic acid during carbohydrate fermentation (Zavaleta, 1976). The CA treatment produced 14.41 and 4.11 mM L-1 more acetic and propionic acid than treatment SL (p≤0.05).
Table 2: Oxide reduction potential and concentration of volatile fatty acids (AGV) and propionic acid† in media inoculated with lyophilized cellulolytic bacteria and preserved with activated carbon.
♦ † Variables affected by the interaction treatment-time (p≤0.05); a, b, c: values with different letter in a column are statistically different (p≤0.05); EEM: standard error of the average; CA: activated carbon as lyoprotectant; SL: without lyoprotectant, control.
pH showed neutral values with no differences between treatments (p>0.05; Table 1), facilitating cellulolytic bacterial growth since a decrease in pH interferes with their adhesion to the cellulosic material (Nag-Jin et al., 2005). Barboza et al. (2009) pointed out that cellulolytic bacteria require neutral pH (Table 1) for adequate activity. The negative oxide reduction potential of the culture media indicates that they are highly reducing. In our study, the culture media were not different (p>0.05) between treatments because activated carbon is an apolar compound (Mendonça et al., 2015) and does not affect growth. But, the interaction of the CA treatment at both measuring times was significant (Table 2).
Literatura Citada
Barboza, S. P., K. L. Parker, and I. D. Hume. 2009. Integrative Wildlife Nutrition. Springer-Verlag Berlin Heidelberg. Heidelberg, Germany. 342 p. [ Links ]
Cobos, M. A., A. Ley de Coss, N. D. Ramírez, S. S. González, and R. C. Ferrera. 2011. Pediococcus acidilactici isolated from the rumen of lambs with rumen acidosis, 16S rRNA identification and sensibility to monensin and lasalocid. Res. Vet. Sci. 90: 26-30. [ Links ]
Cobos, M. A., M. Pérez-Sato, J. Piloni-Martini, S. S. González, and J. R. Bárcena. 2007. Evaluation of diets containing shrimp shell waste and an inoculum of Streptococcus milleri on rumen bacteria and performance of lambs. Anim. Feed Sci. Technol. 132: 324-330. [ Links ]
Elder, G. M. 2010. Activated charcoal: to give or not to give. Int. Emerg. Nurs. 6: 76-80. [ Links ]
Gabr, R. M., S. M. F. Gad-Elrab, R. N. N. Abskharon, S. H. A. Hassan, and A. A. M. Shoreit. 2009. Biosorption of hexavalent chromium using biofilm of E. coli supported on granulated activated carbon. World J. Microbiol. Biotechnol. 25: 1695-1703. [ Links ]
García, L. M. D., y F. Uruburu F. 2000. La conservación de cepas microbianas. Actualidad SEM. 30: 12-16. [ Links ]
Jung H. G., F. M. Engels, and P. J. Weimer. 2004. Degradation of lucerne stem cell walls by five rumen bacterial species. NJAS - Wageningen J. Life Sci. 52: 11-28. [ Links ]
Kumar, S., P. L. Kashyap, R. Singh, and A. K. Srivastava. 2013. Preservation and maintenance of microbial cultures. In: Arora, D. K., S. Das, and M. Sukumar (eds). Analyzing Microbes. Manual of Molecular Biology Techniques. Berlin Heidelberg. Springer Protocols Handbooks. pp: 135-152. [ Links ]
Littrell, K. C., N. R. Khalili, M. Campbell, G. Sandí, and P. Thiyagarajan. 2002. Structural characterization of activated carbon adsorbents prepared from paper mill sludge. Appl. Phys. A 74 (Suppl): S1403-S1405. [ Links ]
Malik K. A. 1990. Use of activated charcoal for the preservation of anaerobic phototrophic and other sensitive bacteria by freeze-drying. J. Microbiol. Methods. 12: 117-124. [ Links ]
Mercier, A., G. Wille, C. Michel, J. Harris-Hellal, L. Amalric, C. Morlay, and F. Battaglia-Brunet. 2013. Biofilm formation vs. PCB adsorption on granular activated carbon in PCBcontaminated aquatic sediment. J. Soils. Sediments. 13: 793-800. [ Links ]
Mendonça, L. Z., Z. M. Magriotis, M. C. das Graças, W. S. Douglas, J. M. Guilherme, S. S. Viera, and D. N. Lee. 2015. Natural clay and commercial activated charcoal: properties and application for the removal of copper from cachaça. Food Control. 47: 536-544. [ Links ]
Morales-García, Y. E., E. Duque, O. Rodríguez-Andrade, J. de la Torre, R. D. Martínez-Contreras, R. Pérez-y-Terrón, y J. Muñoz-Rojas. 2010. Bacterias preservadas, una fuente importante de recursos biotecnológicos. BioTecnología. 14: 11-29. [ Links ]
Morgan, C. A., N. Herman, P. A. White, and G. Vesey. 2006. Preservation of micro-organisms by drying; a review. J. Microbiol. Methods. 66: 183-193. [ Links ]
Muñoz-Rojas, J., P. Bernal, E. Duque, P. Godoy, A. Segura, and J. L. Ramos. 2006. Involvement of cyclopropane fatty acids in the response of Pseudomonas putida KT2440 to freezedrying. Appl. Environ. Microbiol. 72: 472-477. [ Links ]
Nag-Jin, C., J. I. Y. I. Jee, O. Sejong, K. Byoung-Chul, H. Han-Joon, and J. K. Young. 2005. Effect of pH and oxygen on conjugated linoleic acid (CLA) production by mixed rumen bacteria from cows fed high concentrate and high forage diets. Anim. Feed Sci. Technol. 123-124: 643-653. [ Links ]
Ramírez N., J. S. 2006. Liofilización de Alimentos. ReCiTeIA. Cali, Colombia. 36 p. [ Links ]
Roussak, O. V., and H. D. Gesser. 2013. Carbon-based polymers, activated carbons. In: Springer US. Applied Chemistry: A Textbook for Engineers and Technologists. 2nd ed. Springer-Verlag New York Inc. pp: 279-290. [ Links ]
SAS. Institute Inc. 2011. Statistical Analysis System, SAS, User’s Guide: SAS Inst., Cary, NC. pp: 119-130. [ Links ]
Safronova V. I. and N. I. Novikova. 1996. Comparison of two methods for root nodule bacteria preservation: lyophilization and liquid nitrogen freezing. J. Microbiol. Methods. 24: 231-237. [ Links ]
Villarreal, J., C. A. Kahn, J. V. Dunford, E. Patel, and R. F. Clark. 2015. A retrospective review of the prehospital use of activated charcoal. Am. J. Emerg. Med. 33: 56-59. [ Links ]
Yuan, R., B. Zhou, C. Shi, L. Yu, C. Zhang, and J. Gu. 2012. Biodegradation of 2-methylisoborneol by bacteria enriched from biological activated carbon. Front. Environ. Sci. Eng. 6: 701-710. [ Links ]
Zavaleta E. de L. 1976. Los ácidos grasos volátiles, fuente de energía en los rumiantes. Ciencia Vet. 1: 223-240. [ Links ]
Received: June 2015; Accepted: November 2015
Este es un artículo publicado en acceso abierto bajo una licencia Creative Commons
