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Biological characterization of bacterial strain MT15 – A polyhydroxyalkanoate producer bacterium isolated

from noodle waste water Ha Noi city Tran Huu Phong1, Bui Thi Thanh Nga2, Phan Due Thanh1, Duong Van Hop3, Doan Van Thuoc1

1Department of Microbiology and Biotechnology, Faculty of Biology, Hanoi National University of Education, Vietnam 2VanNoi High School, Dong Anh, HaNoi 3Institute of Microbiology and Biotechnology, Vietnam National University - Hanoi, Vietnam

Abstract

144 bacterial strains (named MT1 to MT144) were isolated from waste water collected at Mach Trang village, DongAnh district, HaNoi city (Vietnam). By using Nile blue A staining method and gas chromatography analysis, 20 bacterial strains which are capable of polyhydroxyalkanoate (PHA) production were determined. The strain MT15 – a potential PHA producer was chosen for further study. Strain MT15 is a rod-shaped baterium, gram-positive, slightly acidic, mesophilic, and spore formation. The bacterium is a heterotroph, able to utilize various nitrogen and carbon sources. Strain MT15 was able to produce extracellular enzymes such as amylase, CMCase, and xylanase. The accumulated PHA from this strain is a copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV), the 3HV fraction (up to 42 mol%) was produced when sodium propionate at concentration of 1 g/L was added into cultivation medium. Highest CDW and PHBV content of 5,1 g/L and 66% were respectively obtained by this strain after 32 to 36 hours of cultivation at pH 5,5, 35oC, 20 g/L of glucose and 3,5 g/L of yeast extract were respectively used as carbon and nitrogen sources. Key words: polyhydroxyalkanoate (PHA), poly(3hydroxybutyrate-co-3hydroxyvalerate) (PHBV), waste water

Introduction

Polyhydroxyalkanoates (PHAs) are polymers, belong to polyester, composed by various hydroxyalkanoic acid. In imbalanced condition, excess of carbon source but limite of others nutrient as nitrogen, sulfur, phosphor, oxygen, …, PHAs were accumulated by various bacteria as reserved carbon and energy (Anderson and Dawes, 1990)[1]. When extracting from cell, PHAs showed similar properties as synthetic plastic but more biocompatible and biodegradable. So PHAs were applied in range of application such as medicine, new material, agriculture, ect. (Reddy et al., 2009) [2].

Received 26 September 2014 / Accepted 20 October 2014Duong Van Hop: Participant of the 15th UM, 1987-1988.

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Bacillus megaterium was the first reported about polyhydroxybutyrate (PHB) by Lemoigne (1926) [3]. Today thousands of microorganism were reported in PHA accumulation. And there are more than 30% of soil bacteria could produced PHA with more 150 diffenrent PHA were discovered (Wu et al., 2000)[4]. Polyhydroxybutyrate (PHB) is the most studied PHA and accumulated commonly in many microbacteria. PHB is homopolymer with brittle and hard properties, so limited in applications. Finding copolymer such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) or poly(3-hydroxybutyrate-co-4-hycroxybutyrate) [P(3-HB-co-4-HB)] which are more elastic and different properties to enhance ability in application is interest research nowaday (Philip et al., 2007) [5].

Recently, some studies have mentioned about PHA in Vietnam. Mai et al,. have isolated strain Bacillus cereus V23-X1 from mango fruit with capable of PHB accumulation [7]. Eight of total 100 potential bacterial strains, which isolated from mangrove soil, with high PHA accumulation were reported by Thuoc et al. [8]. Mach Trang village, DongAnh district, HaNoi city (Vietnam) is known as noodle production areas for Hanoi community. We conducted this study to find good PHA producer isolated from the noodle wastewater.

Materials and Methods

Isolation and dedium: Bacteria were isolated from waste water in MachTrang village (DongAnh district, HaNoi capital, VietNam). Basal medium for isolation contains (g/l): MgSO4.7H2O – 0,25; CaCl2.2H2O – 0,09; KCl – 0,5; KBr – 0,06; yeast extract – 10; peptone – 5; glucose – 1; pH – 7.0. Solid medium was made by adding agar with concentration at 2%. The medium for PHA accumulation was modified by basal medium with carbon source (20 g/l). Samples were prepared at serial exponential dilution and spreaded in basal medium, after 24 hours at 30OC, separate colonies were transferred to fresh medium for further studies [9].

Detecting PHA producers: The isolated strains were cultivated on production medium agar plate containing Nile Blue A with concentration at 0.5µg/ml. The incubation was carried out at 30oC for 24-48 hours, and exposed under UV radiation (λ254nm). Strains showed bright orange color under UV were selected (Spiekermann et al., 1999) [10].

Morphology and colour of bacterial colony was observed using optical microscope. Cell and polymer granule morphology were determined using electrical microscope. Extracellular enzymes were determined when cultivation bacteria on nutrient agar medium using substrate appropriately and stained by congo red or lugol solution [9].

Fermentation and cell harvesting bacterial cells.

Two loop activated bacterial strains on solid basal medium were transferred to liquid medium and incubated in 15 hours at 180 rpm/min to get OD = 8±0.5. Seed solution (5%) was inoculated into production medium (50ml) and incubated for 24 hours at 180 rpm/min. Cell were harvested

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by centrifuging at 13000 rpm/min in 5 mins. Pellets were washed two time by distilled water to remove medium components. Finally, the pellets were dried at 105oC to constant weight and cell dried weight (CDW, g/l) was determined.

Effect of nutrient conditions on growth and PHA accumulation..

Different carbon and nitrogen sources were used to measure growth and PHA accumulation of selected bacteria. Best initial concentration of carbon and nitrogen source were determined by supplying serial concentration (carbon or nitrogen) in production medium. 3hydroxyvalarate (3HV) fraction content (%mol) in polymer was studied by using GC (gas chromatograph).

Effect of cultivated conditions on growth and PHA accumulation .

The selected bacterial strain was cultivated at different temperature (25oC-35oC) and initial pH (4.0-8.0) to determine optimum condition for growth and PHA accumulation. While cultivated time profile was measured by collecting bacterial sample interval 4 hours in fermentation period.

PHA content analysis.

PHA content (%CDW) was determined by using gas chromatography (GC). Samples were prepared as Braunegg et al. method (1978) [11]. Hydroxyalkanoate methyl ester was analyzed on Agilent HP5890-II (Hewlett Packard CO,USA) system equipped with capillary HP-5 collumn (Hewlett Packard CO, USA) and flame ionization detector (FID) (Silva et al., 2000) [12].

Results and Discussion

Isolation and screening PHA accumulated bacteria.

From 10 samples of collected waste waters, 144 isolated strains were obtained with fast growth named from MT1 to MT144. Using nutrient agar contain Nile Blue A for screening, 20 strains showed clearly bright orange fluorescence indicating PHA accumulation were selected for further study (fig. 1). Among them, 17 strains are positive Gram and only 3 strains are negative bacteria (MT84, MT85, MT97). GC analysis showed that all selected strains could accumulate PHA (almost PHB and several strains accumulated PHBV, data not show). Interestingly, the best strain MT15 was selected for further study by fast growth (CDW at 3.81 g/l) and highest PHBV content (53%) with 2mol% of 3HV fraction.

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Figure 1. Flourescent Nile Blue A staining of isolated bacterial strains from traditional noodle production village MachTrang (DongAnh, HaNoi). Colonies showed bright orange fluorescence are PHA-positive colonies.

MT-33 MT-84 MT-15 MT85

Figure 2. Gram staining of PHA accumulated bacteria

Biological characterizations of strain MT15

When cultured in agar medium, the strain MT15 was found white , round, dry colonies. The cell is short rod-shape (1,25-1,35 µm x 4,1-6,94µm), positive gram, and endospore forming and PHA accumulation. In addition, this strain could produce hydrolyzed polysaccharide enzymes such as amylase, CMC-ase, xylanase.

Figure 3. Morphology of cell (TEM) and PHBV granule (SEM) of strain MT15

Effect of nutrient conditions to PHA accumulated cultivation.

Effect of carbon source.

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The strain MT15 could use all tested carbon sources, that exist normally in waste water from traditional noodle production village where bacteria were isolated. High CDW (3.95 g/l) and PHA content (60.8 %CDW) were obtained when glucose was used as sole carbon source. The lower growth and PHA content (<30 %CDW) observed with other carbon sources such as fructose, saccharose, and sugarcane molasses. This result is similar with previous study of Thuoc and Quillaguamán on Bacillus sp. ND153 strain [13].

Figure 4. Effect of various carbon source to growth and PHBV accumulation of strain MT15

Figure 5. Effect of glucose concentration to growth and PHBV accumulation of strain MT15

Carbon source concentration is very important with growth and PHA accumulation of bacteria because it supplies energy and material for cell construction. In addition almost bacteria only accumulate PHA at high carbon source concentration in medium. Data on figure 5 showed that the CDW of strain MT15 increased from 1.74 to 4.14 g/l when glucose concentration increased from 2.5 to 20 g/l, PHA content was higher 20% when glucose concentration at 10 g/l and higher, while at low glucose concentration (2.5 and 5 g/l) PHBV accumulation very low (about 2% CDW). Efficiency of conversion glucose into PHBV (gram PHBV per gram glucose) increase 10-folds from 0.0132 g/g glucose (at concentration 2.5 g/l) to 0.125 g/g glucose (at concentration 20 g/l). This result once indicates that PHBV accumulation performed stronger when excess of carbon source occurs in medium.

Effect of nitrogen source.

Seven nitrogen sources were tested to find appropriate nitrogen for growth and PHA accumulation of strain MT15 (fig.6). Yeast extract showed best efficiency with CDW and PHBV content were 4.15 g/l and 59%, respectively. Also, peptone and sodium nitrate showed positive effect to strain MT15 but lower than using yeast extract. Low results (CDW< 1 g/l; PHBV content lower 10%) were gotten when using others nitrogen source such as meat extract, mono sodium glutamate, ammonium chloride, ammonium sulfate.

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Figure 6. Effect of various nitrogen sources to growth and PHBV accumulation of strain MT15

Figure 7. Effect of yeast extract concentration to growth and PHBV accumulation of strain MT15

Similarly, nitrogen source is important factor that supplies material for cell construction and enzyme system. Especially with PHA producer, nitrogen concentration effect not only to growth but also to PHA synthesis. Fig. 7 showed that CDW increased from 0.72 g/l to 5.53 g/l when yeast extract concentration increased from 0.5 g/l to 4.5 g/l. Highest PHBV accumulation obtained 69% CDW at 3.5 yeast extract concentration. Although CDW tend to increase when increasing of nitrogen concentration, but PHBV content slight decreased at high concentration. PHA accumulation is an adaptation of bacteria when nutrition conditions change. This process is stronger at unbalanced medium, but extremely nutrient lack will effect negative to growth and metabolized reactions in cell cause to PHA accumulation. In contrast, excess of nutrient cause low demand for PHBV accumulation, polymer content suddenly decreased from 69% to 46% when yeast extract concentration increased from 3.5 to 4.5 g/l.

Effect of sodium valerate supply to 3HV fraction.

P(3HB) has brittle and hard properties with high crystalline and melting temperature about 175-180oC (Anderson and Dawes, 1990). Combine 3HB with others hydroxyalkanoate monomer (HA) including 3-hydroxyvalerate (3HV) or 3-hydroxyhexanoate (3HHx) into polymer chain enhance physical properties of PHA. Many studies indicated that 3HV fraction increasing caused by supply of precursor such as propionic acid or valeric acid to fermented medium. So effect of sodium valerate supplying to amount of 3HV fraction was studied on strain MT15. Highest 3HV fraction (42.4 mol%) was obtained at 1 g/l of sodium concentration supplied to medium. Morever, increasing of sodium propionate concentration resulted slight decreasing of PHBV accumulation of strain MT15 (Table 1). More amount of sodium propionate present in medium helped increase 3HV fraction (from 5.7 mol% to 42.4 mol%) in PHBV polymerization process of strain MT15. Inhibition of propionic precursor to PHA accumulation of Bacillus megaterium

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OU303A has studied by Reddy et al. (2009). Also, 3HV fraction increasing when precursor presented has demonstrated in this study [2].

Table 1.Effeciency of sodium propionate supply to amount of 3HV fraction

Sodium concen. (g/l) Content and composition of polymer PHBV content (% CDW) 3HB (mol%) 3HV (mol%)

Control 58.2 97.9 2.1 0.25 52.3 94.3 5.7 0.5 53.4 89.1 10.9

0.75 54.8 63.9 36.1 1.0 54.2 57.6 42.4

1.25 50.6 62.8 37.2 1.5 50.4 69.6 30.4

Effect of various cultivated conditions.

When transferring to fresh medium bacteria were affected by initial pH factor firstly. At favorable pH condition, the lag phase could be shorten and bacterial population may early come to exponential phase. The strain MT15 could grow and accumulate PHBV at wide range of pH (4.0-8.0) with CDW above 3g/l while PHBV content above 30% (fig. 8). Highest PHA content and CDW (68% and 4.85 g/l, respectively) were obtained at 5.5 initial pH of medium. It is found that pH of waste water from the noodle production village is around 4,5-5,5 (Loan, 2009) [6]. Bhuwal et al. (2013) used isolated bacteria from pulp, paper, and cardboard industry wastes for PHA producing using this wastes as sole nutrient [14]. Swine wastewater, paper mill wastewater, and active sludge wastewater were used for PHA production based on microbial system available in which [15,16,17]. This result indicated high adapptated characterization of strain MT15 to the environment, and rich nutrient wastewater from food industries are potential materials for PHA production by using this strain. Besides, almost PHA produced bacteria isolated recently in Vietnam were reported with pH optimum at 7.0 [6,18].

Figure 8. Effect of initial pH to growth and PHBV accumulation of strain MT15

Figure 9. Effect of cultivated temperature to growth and PHBV accumulation of strain MT15

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Cultivated temperature affect simultaneously to growth and PHBV accumulation of the strain MT15. The obtained result demonstrated that strain MT15 is mesophilic bacterium with optimum temperature for growth (4.8 g/l) and PHBV content (68%) at 32-35oC. The results decreased when cultivated temperature increased to 37oC (fig. 9). Mesophilic bacteria such as Cupriavidus necator, Bhurkolderia sp., Aeromonas hydrophila are the most producer was used for PHA production at industrial pilot [19].

Figure 10. Time profile of growth and PHA accumulation of strain MT15

Intracellular PHA granules were covered by protein coat which contain both PHA synthase and PHA depolymerase (Sudesh et al., 2000) [20]. In favorable condition (excess carbon), polymer synthesis was carried out by PHA synthase action. Conversely when depleption of carbon and other nutrient source occurred, polymer granules were degraded by depolymerase to supply nitrient for cell survival in adverse conditions. It causes PHA content reduction and lead to reduce of efficiency of fermentation. Therefore chosing of downstream time is very important to get high efficiency in batch fermentation. Time profile of growth and PHBV accumulation of strain MT15 was monitored for 40 hours of cultivation and the results were showed in figure 10. During 4 initial hours was lag phase, cell mass increased fastly until 28 hour of cultivation (log phase) with CDW from 1.18 to 5.18 g/l. PHBV accumulation in cell increased also from 7% to 65 %CDW. Stationary phase was maintained at hour of 28 to 36 with CDW about 5.15 to 5.22 g/l. Highest PHBV content was obtained at 32 hours of cultivation, and after 36 hours the bacterial population entered death phase with CDW and PHBV content values were 4.3 g/l and 50 %CDW respectively (40 hours of cultivation). This is important result for study of large production of strain MT15.

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