Optimization of Brucella abortus Fermenter Cultural Conditions and LPS Extraction Method for Antigen Production

Research Article
Optimization of Brucella abortus Fermenter Cultural Conditions and LPS Extraction Method for Antigen Production

Reza Shapouri, Ashraf Mohabati Mobarez, Hojat Ahmadi, Bahman Tabaraie, Reza Hosseini Doust, Dariush Norozian, Ahmad Zavaran Hosseini and Davar Siadat

Research Journal of Microbiology, 2008, 3(1), 1-8.

Abstract

We modified Brucella fermentation medium from FAO/WHO for enhancing of Brucella abortus S99 biomass. The modified media composed of 15 g L^-1 peptic digest animal tissue, 15 g L^-1 pancreatic digest of casein, 10 g L^-1 yeast extract and 0.10 g L^-1 sodium bisulphate. Glucose was added during the incubation by fed-batch method (1-30 g L^-1). Agitation speed and air flow rates were controlled at 300-500 rpm and 4-8 L min^-1, respectively. Cell density was 9-10% and viable count was 3-3.3x10¹¹ mL^-1. The modified conditions enhance the biomass production to more 2 times than the FAO/WHO method. Three methods were accomplished for LPS production: Extraction by butanol with enzymatic digestion, hot phenol extraction with enzymatic digestion, modified hot phenol with trichloroacetic acid (TCA) procedure. Yield of LPS extraction was 0.2, 0.8 and 1.3%, respectively. Method III results in a greater yield of LPS which is 6 and 1.5 times the yields of methods I and II, respectively. Protein contamination of LPS was <2, <2 and <2.9% and nucleic acid contamination of LPS was <1, <1 and <1.4%, respectively. The ketodeoxyoctonate content of LPS (in each of the three methods) was in agreement with ketodeoxyoctonate values obtained previously for highly purified LPS of B. abortus. According to present study, hot phenol with trichloroacetic acid (TCA) procedure is the most suitable procedure for large-scale LPS production from Brucellae, which can be employed for the production of Brucella biomass for vaccine and antigen preparations.

ASCI-ID: 83-236

Table 1.

Working with FAO/WHO protocol for biomass production from Brucella, usually gives unsatisfactory results and very low cell quantity. In present research, fed-batch addition of glucose and increasing or decreasing of air flow and agitation rate, on the biomass production of Brucella were investigated and optimized.

Table 1:	Comparison of the primary and modified Brucella fermentation medium ingredients

*: Glucose was added by fed-batch method

Table 2:	Comparison of the cell density and viable cells count between the primary and modified methods for Brucella cells fermentation culture

*: Indicates a significant difference comparing to the primary method (p<0.01)

Table 3:	Characteristics of B. abortus LPS that extracted with three methods

*: In method III enzymatic treatment is not used

In the modified method, fermentation was completed after 60 h and cell density (packed cell volume) was 9-10% and viable count was 3-3.3x10¹¹ mL^-1 and also cells exist in correct colonial phase.

Results of cell density and viable cell count and comparison of these for primary and modified method were shown in Table 2. The modified conditions enhance the biomass production to more than 2 times the FAO/WHO method. In Fig. 1, comparison of the variation in cell concentration of the medium with cultivation time for both FAO/WHO (primary method) and modified methods are shown. This comparison confirms the enhancement of biomass in the modified method.

LPS Extraction
The LPS of B. abortus S99 is extracted by three methods. In method III (modified hot phenol with trichloroacetic acid) we have the greatest yield of LPS (1.3%) which it is more than 6 and 1.5 times the yields of methods I and II, respectively. Ketodeoxyoctonate, which is a unique component of bacterial LPS, was found to be 0.9-1% by weight. Protein and nucleic acid contaminations of LPS extracted by three methods are given in Table 3. The protein and nucleic acid content of LPS extracted by methods I and II after enzymatic treatment were equal and low but the contaminations of LPS in method III without enzymatic digestion were slightly greater but consistently acceptable to use.

B. abortus LPS were analyzed with 14% SDS-PAGE gels with 4 M urea (Fig. 2) and stained by silver stain procedure, resulting in patterns similar to the patterns observed previously for the LPS of Brucella (Corbel et al., 1979; Hendry et al., 1985). Lanes 1 and 3 are pattern of 5 μg of B. abortus LPS extracted with methods I and II. Lane 4 is 10 μg of B. abortus LPS extracted with method III. Lane 2 is 5 μg of E. coli LPS.

In contrast to the LPS of B. abortus, the mature form of E. coli LPS migrates as a banded pattern rather than a smear, because the sugar units of E. coli LPS consist of four rather than one sugar molecule (Goldstein et al., 1992).


Fig. 1:	Comparison of the variation in cell concentration of the medium with cultivation time for both FAO/WHO method (primary method) and our modified method


Fig. 2:	SDS-PAGE of B. abortus LPS. Lane 1 and 3: Samples of 5 μg LPS extracted with methods I and II. Lane 4: 10 μg of LPS extracted with method III. Lane 2: 5 μg of E. coli LPS

DISCUSSION

Control and eradication campaigns against brucellosis have led to demands for large quantities of Brucella cells, both for vaccine and antigens production.

Two methods currently used are culture on a solid medium in Roux flasks and culture in a liquid medium (Hendry et al., 1985). Handling of large volumes of culture presents the following hazards: (a) the risks of contaminating the cells during inoculation, harvesting and bottling and (b) the potential risk to staff who may become infected or sensitized by contact with Brucella cells.

The choice of method for producing cells will depend on the quantity of cells required and on the facilities available; whichever method is used, great care should be exercised (a) in maintaining cultures for seed material, in the correct colonial phase (smoothness) and (b) in the choice of medium used.

When large volumes of cells are required for vaccine or antigen production, the use of liquid medium has some advantage over propagation in Roux flasks because the laborious and costly process of handling large numbers of individual flasks is eliminated and the attendant risk of contamination is reduced (Corbel et al., 1979; Hendry et al., 1985).

In this study, optimum media composition and conditions were developed for the overproduction of brucellae cells for antigen production. Present results indicated that modified media and conditions for batch fermenter increased cell quantity without incorrect colonial phase; also we found that incubation more than 60 h and agitation rate more than 500 rpm, decreased growth rate of the organisms.

In FAO/WHO protocol, the initial pH of medium is 6.6 but this tends to rise during the growth cycle and therefore it must be adjusted to pH 7.2-7.4 by the addition of sterile 0.1 M HCl. In modified media and conditions, the initial pH is 6.4 and rise to 7.42 at the end of fermentation, but this range of pH media is acceptable and no need to add HCl for pH adjustment.

A well known property of protein solutions is their propensity to foam when agitated. If the medium is allowed to foam it will occupy a larger volume, therefore the effective working volume of the fermenter is reduced. It may be necessary to reduce aeration and/or agitation rate, thus reducing the oxygen transfer rate with possible limitation to growth. If excessive foam formation occurs, it may enter the fermenter vent system thus posing a possible contamination risk. Addition of antifoam is necessary but antifoam compounds may inhibit growth and will de-gas the liquid and reduce oxygen availability (Celik and Calik, 2004).

In the FAO/WHO media antifoam is required but in our modified medium does not require antifoam, thus oxygen availability and growth rate of cells has not reduced.

Fed-batch addition of glucose after 2 h of inoculation of seed culture in the rate of 1 g L^-1 and increasing to 30 g L^-1, increased the cell concentration. In fed-batch addition of glucose, buffer system ingredients of the primary medium, sodium dihydrogen phosphate and disodium hydrogen phosphate were deleted. It is frequently observed that at high concentration of substrate, the substrate also expresses growth inhibiting properties. However, prior to reaching the maximum population phase there is a stage during which substrate depletion causes a restriction in the growth rate (Celik and Calik, 2004; Cheema et al., 2002; Dodge and Gerstner, 2002). In batch culture, as well as FAO/WHO protocol for Brucella fermenter culture, the microbial population are present in an environment in which initially all the substrates are present at excess concentrations, but in fed-batch method, the substrate (glucose) was added at low concentration and increased gradually, thus in this modified method we have not observed these undesirable effects. Compared to the primary method, these modified conditions, especially fed-batch addition of glucose increased the cell density to 9-10 from 4-5%.

The ketodeoxyoctonate content of LPS in this study is in agreement with ketodeoxyoctonate values obtained previously for highly purified LPS of B. abortus (Phillips et al., 1989). The yield of LPS extraction in phenol-water partition methods were more than butanol-water partition method. Also modified hot phenol procedure have excess product than original hot phenol procedure. The protein and nucleic acid content of LPS that extracted with three methods were acceptable to use. For large-scale production of LPS the use of enzymatic treatment and butanol-water partition method has disadvantages like laborious, costly process and low yield. In the present study, we modified the extraction method for large scale production of B. abortus LPS: In the method III we use 1/2 volume of cold methanol and TCA for precipitation of proteins and nucleic acids instead of enzymatic treatment. Present data (Table 3) clearly showed that this method is suitable for large scale LPS production from brucellae.

In conclusion the presented research can be applied easily for production of Brucella biomass for vaccine and antigen preparations.

ACKNOWLEDGMENT

This study was supported by research grant of Tarbiat Modares University and Baqhyatollah University (Tehran, Iran).

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