24 DETERMINATION OF PHB GROWTH QUANTITIES OF CERTAIN ...

Turkish Electronic Journal of Biotechnology Special Issue, p:24-30, 2002 © Biotechnology Association http://www.biyotekder.hacettepe.edu.tr/dergi.html

DETERMINATION OF PHB GROWTH QUANTITIES OF CERTAIN BACILLUS SPECIES ISOLATED FROM SOIL

Belma Aslim, Zehra Nur Yüksekdağ, Yavuz Beyatli Gazi University, Faculty of Arts and Science, Department of Biology, Ankara, TURKEY Özet Bu çalışmada Ankara tarlalarından alınan 6 farklı toprak örneğinden 40 adet bakteri izole edilmiştir. İdentifikasyon testlerinin sonuçlarına göre, 27 suş Bacillus subtilis, B. megaterium, B. firmus, B. sphaericus, B. thuringiensis ve B. pumilus olarak teşhis edilmiştir. Suşların hepsinde PHB tespit edilmiştir. B. megaterium Y6 (%48,13) ‘da en yüksek PHB verimi gözlenirken, B. subtilis K1 (%6,53) ‚de hücre kuru ağırlığına göre en düşük PHB verimi gözlenmiştir. Yüksek ve düşük PHB ‘ye sahip suşlardan izole edilen plazmidlerde farklı büyüklüklerde (3,4220,37 kb) bulunmuştur. Suşların üçünün plazmid DNA içermediği tespit edilmiştir. Anahtar Kelimeler: PHB, Bacillus, plazmid DNA, toprak. Abstract In this study, 40 bacteria were isolated from six different soil samples which were taken from grasslands of Ankara in Turkey. As a result of the identification tests, 27 strains were identified as Bacillus subtilis, B. megaterium, B. firmus, B. sphaericus, B. thuringiensis , B. pumilus. PHB was found in all strain. The higest percent yield of PHB was observed in B. megaterium Y6 (48.13%) whereas the lowest percent yield of PHB according to dry cell weight was determined in B. subtilis K1 (6.53%). The plasmids isolated from strains having low and high PHB were found in various size ( 3.42-20.37 kb), three of which did not contain plasmid DNA. Key words: PHB, Bacillus, Plasmid-DNA, Soil

Introduction Synthetic polymers obtained from petrol causes air pollution only because they are not dismantled in soil for a long time. For this reason, a microbial plastic poly-β-hydroxybutyrate (PHB) has gained importance since it can easily be dismantled in nature. PHB is a widely distributed intracellular reserve substance typical of prokaryotes. PHB exists in the cytoplasmic fluid in the form of crystalline granules about 0.5 µm in diameter and can be isolated as native granules or by solvent extraction (1-3). Various researchers have explained that soil bacteria generally produce PHB.PHB production increases if convenient condition is made available. Besides these biopolymers increase the resistance of bacteria (4). PHB has been identified in more than 20 bacterial genera, including Azotobacter, Bacillus, Beijerinckia, Alcaligenes, Pseudomonas, Rhizobium and Rhodospirillum (5). Lemoigne (1927) charactericed PHB chemically and observed that it was involved in the sporulation of Bacillus spp. This lipid inclusion is accumulated by many bacteria as they enter the stationary phase of growth to be used later as an internal reserved of carbon and energy (6). Bacillus spores wre produced during the stationary phase of the cultures and at a time when PHB was being produced and consumed (7). In one of the studies the soil bacteria were inoculated to the feeding conditions both including and excluding glucose as the source of carbon, and whereas PHB was determined 1.56-2.64 µg (crotonic

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acid)/g in a feeding condition excluding glucose, the produced PHB was reported to increase 20 fold in a feeding condition including 1% glucose (4). Some microorganisms may accumulate PHB to 80-90 % of dry cell weight (8). The controlled, large-scale production of PHB by bacteria has become a subject of increasing interest, with the hope that the use of biodegradable polymers to replace some petrochemical based polymers will help to reduce the highly visible pollution of land and sea environments by plastic products .Products made from PHB are environmentally significant since they biodegrade to CO2 and H2O. In this study the Bacillus genera in the soils taken from different areas were isolated and identified. Afterwards PHB productions of those bacteria were determined. Besides, the plasmid DNA profiles of some strains selected from the isolates were first determined. Then the molecular weights of the present plasmid DNA were identified. The aim of this study was to identify the Bacillus genera in different soil samples and determine the amount of PHB production.

Materials and Methods Isolation and identification: Different soil samples were taken from different six regions of Ankara in Turkey. The grassland soil samples used in all experiments were collected from 0-15 cm layer. Each one gram of the sample was suspended in 99 ml of sterile distilled water and shaken vigorously for 2 min. The samples were heated at 60°C for 60 min in water bath. Than the liquid was serially diluted in sterile distilled water, and the dilution from 10-1 to 10-6 was plated on Nutrient agar medium. Plates were incubated at 28-37 °C for 24-48 h. The first isolated bacteria were identified. In the identification tests of strains, the spore morphology, gram stain and motile were examined. Respectively growth in Nutrient Broth 5% and 7% NaCl and the growth conditions in pH 5.7-6.8 and in 42°C, anaerobic growth conditions were examined. Besides the following identification tests were applied; utilization of citrate, VP test, gelatine liquefaction, esculin and starch hydrolysis, production acid from D-glucose, L-arabinose, D-xylose, D-mannitol, lactose, salicin, sucrose and maltose. Production gas from glucose, catalase, nitrate reduction, producing H2S and acid from TSI were tested. According to the results obtained from the tests above the Bacillus genera were determined (9-12). Analytic method: The cultures were inoculated with a 1% (v/v) inoculum, pregrown for 24 h in Nutrient Broth at the convenient temperatures (Table 1). Then active cultures were inoculated with a 2% (v/v) in Nutrient Broth. Shake flaks cultures (50ml/ 250 ml Erlen-mayer flask) were incubated at the convenient temperatures for each strain with shaking at 225 rpm for 48 h (stationary phase). At the end of this period, the samples were centrifuged for 15 min at 6000 rpm. The pellets were washed twice with sterile deionized water and dried for 24 h at 100 °C. Then total bacterial dry weight was determined. Dry cell materials were incubated at 60 °C for 1 h with sodium hypochlorite to break the cell walls of bacteria. Supernatant was obtained by centrifugation at 6000 rpm and transferred to a Soxhlet system. Cell lipids and other molecules (except PHB) were extracted by adding 5 ml 96% (1:1 v/v) ethanol and acetone. PHB was extracted by hot chloroform (adding 10 ml chloroform in a water bath). Then, chloroform was evaporated to obtain crystals of PHB. By adding 10 ml 98% sulfuric acid at 60 °C for 1 h, PHB crystals were converted into crotonic acid. The extract is hydrolysed to yield PHB, which undergoes dehydration to crotonic acid in concentrated sulfuric acid. Crotonic acid absorbs ultraviolet light. The absorbance of the solution was measured at 235 nm in a UV spectrophotometer against a sulphuric acid blank. The amount of PHB per gram dry weight of bacterial cells was determined using a standard curve of PHB (1315). All experiments were repeated two or three times. Their average values and standard deviations (SD) are given in the tables. In order to compare the PHB amounts produced by our isolates, two test bacteria were used (B. sphearicus ATCC 14577 and B. subtilis 128, Ankara University, Department of Biology).

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The isolation of plasmid DNA. The plasmid DNA profiles of nine strains selected from the isolates were obtained according to the plasmid mini-prep procedure (16,17). In the plasmid DNA study, the strains producing high and low PHB were selected. As a marker, super coiled DNA ladder was used (2.067-16.210 kb) and the molecular weights of the plasmids were calculated in kb (8). Statistical analyses: The correlation between bacterial cell dry weight (g 1-1) and PHB production (g 1-1) was determined according to Sperman’s ρ correlation coefficient test. The ρ value was estimated with the below formula and explained using Conver’s table (19).

ρ=1-

6∑ ( x i − y i ) 2 n (n 2 − 1)

Results and Discussion As a result of the identification test conducted by using 40 isolated strains from six different soils, 27 isolates were identified as Bacillus species; namely Bacillus subtilis (8 strains), B. megaterium (6 strains), B. firmus (5 strains), B. sphaericus (4 strains), B. thuringiensis (3 strains), B. pumilus (1 strain) (Table 1). Table 1. Distribution of identified Bacillus spp. bacteria and their growth temprature. Species

Number of Strains

Growth Temperature (°C)

Bacillus subtilis

8

37 °C

Bacillus megaterium

6

30 °C

Bacillus firmus

5

28 °C

Bacillus sphaericus

4

30 °C

Bacillus thuringiensis

3

37 °C

Bacillus pumilus

1

37 °C

TOTAL

27

Watanabe and Hayano (1993), identified B. subtilis, B. liceniformis, B. cereus, B. thuringiensis, B. megaterium, B. firmus and B. pumilus in the soil isolations (9). In another study Waksman (1961) identified 29 isolates B. megaterium and 24 isolates B. subtilis out of 306 samples isolated from soil (12). This shows that Bacillus genera were wide spread among the bacteria types in soil and agrees with the results of this study. Hydroxybutyric acid is synthesized as active polymer by many bacteria and stocked in granule form within cytoplasm as a source of carbon and energy. PHB, a thermoplastic crystal which can be reduced through biological process, its first production in industry started by using Alcaligenes latus and A. eutrophus (20). Reusch and Sadoff (1983) reported that D(-) poly-β-hydroxybutyrats were important molecules in Azotobacter vinelandii, B. subtilis and Haemophilus influenzae cytoplasm and cell membrane (21). 26

In this study, the productions of PHB by 27 Bacillus isolates and one strains of each B. sphearicus ATCC 14577 and B. subtilis 128 ranged between 0.04-0.207 (g l-1) with percentage productivity of 6.5348.13%. The production of PHB by B. subtilis 128 (0.09 g l-1, 31.03%) and B. sphearicus ATCC 14577 (0.18 g l-1, 22.50%) used as test bacteria and the productivity were similar to that of our isolates. The highest PHB production and productivity percentage were found in B. megaterium Y6 strain (0.207 g l-1, 48.13%) whereas the lowest percent yield of PHB according to dry cell weight was determined in B. subtilis K1 (6.53%) (Table 2). Table 2. PHB production by some Bacillus species. Bacterial species Bacillus subtilis K1 Bacillus subtilis K2 Bacillus subtilis K3 Bacillus subtilis K4 Bacillus subtilis K5 Bacillus subtilis K6 Bacillus subtilis K7 Bacillus subtilis K8 Bacillus megaterium Y1 Bacillus megaterium Y2 Bacillus megaterium Y3 Bacillus megaterium Y4 Bacillus megaterium Y5 Bacillus megaterium Y6 Bacillus firmus G1 Bacillus firmus G2 Bacillus firmus G3 Bacillus firmus G4 Bacillus firmus G5 Bacillus sphaericus X1 Bacillus sphaericus X2 Bacillus sphaericus X3 Bacillus sphaericus X4 Bacillus thuringiensis D1 Bacillus thuringiensis D2 Bacillus thuringiensis D3 Bacillus pumilus BI Bacillus sphaericus ATCC 14577 Bacillus subtilis 128 a Determined at cell dry weight. b According to cell dry weight.

Cell dry weight (g l-1) 1.04 ± 0.06 0.31 ± 0.03 0.94 ± 0.08 0.55 ± 0.02 0.98 ± 0.03 0.75 ± 0.07 0.33 ± 0.01 0.40 ± 0.02 0.41 ± 0.05 0.53 ± 0.02 0.33 ± 0.01 0.51 ± 0.02 0.36 ± 0.00 0.43 ± 0.03 0.73 ± 0.01 0.64 ± 0.03 0.43 ± 0.05 0.28 ± 0.02 0.65 ± 0.01 0.63 ± 0.04 0.32 ± 0.01 0.55 ± 0.02 0.40 ± 0.04 0.50 ± 0.01 1.02 ± 0.06 0.34 ± 0.05 0.51 ± 0.04 0.80 ± 0.07 0.29 ± 0.02

PHBa (g l-1) 0.068 ± 0.06 0.072 ± 0.00 0.091 ± 0.01 0.085 ± 0.00 0.204 ± 0.02 0.181 ± 0.04 0.100 ± 0.00 0.130 ± 0.01 0.136 ± 0.02 0.080 ± 0.00 0.080 ± 0.01 0.060 ± 0.03 0.090 ± 0.01 0.207 ± 0.02 0.120 ± 0.02 0.100 ± 0.01 0.130 ± 0.00 0.095 ± 0.00 0.195 ± 0.01 0.171 ± 0.01 0.080 ± 0.02 0.200 ± 0.03 0.049 ± 0.01 0.040 ± 0.02 0.077 ± 0.01 0.100 ± 0.01 0.186 ± 0.03 0.180 ± 0.002 0.090 ± 0.02

Yield of PHBb (%) 6.53 23.22 9.68 15.45 21.22 24.10 30.30 32.50 33.17 15.09 24.24 11.76 25.00 48.13 16.43 15.62 32.09 33.92 30.00 27.10 25.00 36.36 12.25 8.00 7.54 29.41 36.47 22.50 31.03

To investigate whether any relationship between the dry-cell weight and PHB production existed Sperman's correlation test was used and it was found ρ = 0.244. When this value was compared to the table value, it was seen that the relationship was not significant (ρ = 0.244 < 0.311). In batch cultures glucose is degraded through the Emden-Mayerhoff-Parnas (EMP) pathway to yield pyruvate and acetate as the main products. Once glucose is exhausted, the acids are oxidized via tricarboxylic acid (TCA) cycle, this process being closely associated with the onset of sporulation. Acetat is partially converted into PHB, which is consumed during sporulation in Bacillus spp (7). Macrae and Wilkinson (1958), reported that PHB levels of up to 20-40 of cell dry weight could be obtained in B. megaterium grown in acetate (22). It was observed that PHB was 16% of dry cell weight (260 µg PHB ml-1) when the possible mutant strains of Bacillus megaterium was developed during 28 h (23). Kota et al. (1992), the dry-cell weight of B. megaterium B-124 strain was 20% of PHB (5). 27

According to the literature results of our isolations, isolated strains had higher PHB production. The strains Y6 (B. megaterium) its PHB production increased to 48.13%. Besides it was observed that the yield of PHB % of the strains X3 (B. sphaericus) and B1 (B. pumilus) were higher. When A. eutrophus NCIMB11599 was developed in different substrate, high concentrations of PHB 121 g l-1 and total cells 164 g l-1 were obtained (24). Another study reported that PHB was produced at arange 40% in A. latus culture (25). In our isolations, only one strain B. megaterium Y6 had a high PHB production (48.13%) close to PHB produced by Alcaligenes spp. In one study 32 Bacillus strains isolated from the soil, 21 had plasmid DNA [26]. In some research only one plasmid of 69 kb was identified in B. thuringiensis AF 101 and six plasmid DNA were observed respectively (20.1, 6.6, 5.6, 4.3, 3.6 Mda.) in B. thuringiensis sub sp. israelensis (27,28). The plasmid DNA profiles of nine strains selected from among the high and low PHB producing and two control strains (B. sphaericus ATCC 14577 and B. subtilis 128) were estimated. The number and molecular weight of plasmid DNA investigated in the strains were shown in Table 3. As seen in the table, the molecular sizes of the plasmids ranged between 3.42-20.37 kb. The plasmid profile of the B. subtilis K8 strain which had more plasmid DNA was shown in Figure 1. It was interesting that B. megaterium Y6 strain having the highest PHB production had the biggest plasmid DNA (20.37 kb). Although B. subtilis 128, B. firmus G4 and B. megaterium Y1 strains had PHB, their plasmid DNAs could not be determined. It might be said that in addition to plasmid DNA, genomic DNA could be effective in PHB production. This finding agrees with Steinbüchel and Schlegel’s review. In their review, the PHB biosynthetic genes in A. eutrophous are clustered and located on the chromosome rather than on the mega plasmid pHG1 (29). Table 3. Number of plasmid DNAs and their molecular size of Bacillus species. Bacterial species

Number of plasmid DNA

Molecular weight

Bacillus subtilis K5

3

10.62, 5.87, 3.42


4

13.42, 10.06, 5.77, 3.46

Bacillus megaterium Y6

2

20.37, 12.71

Bacillus thuringiensis D3

1

13.05


1

11.17

Bacillus megaterium Y1

a

-

Bacillus thuringiensis D1

3

12.17, 6.54, 4.71

Bacillus pumilus BI

2

8.04, 4.87

Bacillus firmus G4

a

-

Bacillus sphaericus ATCC 14577 Bacillus subtilis 128

3 a

16.22, 12.18, 4.47 -

a; absent plasmid

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Figure 1. a- Super coiled DNA ladder (16.210; 14.174; 12.138; 10.102; 8.066; 7.048; 6.030; 5.012; 3.990; 2.972; 2.067 kb). b- K8 strain (13.42; 10.06; 5.77; 3.46 kb). It was reported that P4A plasmid had ampicillin resistance gene and PHB biosynthesis gene. Besides it was estimated that the fragment in which the genes were related to PHB biosynthesis was about 6 kb. (30). In this study all the Bacillus strains produced PHB but the production amounts were variable in relation to the strains and types. These results may be attributable to the traditional genetic differences. References 1.

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