Production of twoand three-component polyhydroxyalkanoates by luminous bacteria of the Photobacterium genus Текст научной статьи по специальности «Биологические науки»

Journal of Siberian Federal University. Biology 2 (2016 9) 162-168

УДК 579.222

Production of Two- and Three-Component Polyhydroxyalkanoates by Luminous Bacteria of the Photobacterium Genus

Anatoly N. Boyandin*

Institute of Biophysics of SB RAS 50/50 Akademgorodok, Krasnoyarsk, 660036, Russia

Siberian Federal University 79 Svobodny Ave., Krasnoyarsk, 660041, Russia

Received 00.12.2015, received in revised form 00.02.2016, accepted 00.06.2016

The study addresses the ability of luminous bacteria Photobacterium leiognathi Boisvert et al. and Photobacterium phosphoreum (Cohn) Beijerinck to synthesize polyesters of hydroxycarbon acids (polyhydroxyalkanoates, PHAs) as storage macromolecules. The screened strains widely vary in their PHA productivity. Ph. leiognathi (but not Ph. phosphoreum) produces PHAs containing monomers of 3-hydroxyvaleric acid and, in some cases, 3-hydroxyhexanoic acid, in addition to common monomers of3-hydroxybutyric acid. All studied strains of Ph. phosphoreum produce pure poly-3-hydroxybutyrate only. In the case of Ph. leiognathi, addition of valeric acid as substrate can increase the amounts of medium-chain-length hydroxy acids contained in the produced polymers. The results suggest a conclusion that luminous microorganisms of Photobacterium genus can be considered as producers of multi-component PHAs.

Keywords: luminous microorganisms, Photobacterium, polyhydroxyalkanoates, polyhydroxybutyrate.

DOI:

© Siberian Federal University. All rights reserved Corresponding author E-mail address: [email protected]

Продукция двух- и трехкомпонентных полигидроксиалканоатов

светящимися бактериями рода Рко1оЬас1ег1пт

А.Н. Бояндин

Институт биофизики СО РАН Россия, 660036, Красноярск, Академгородок, 50/50 Сибирский федеральный университет Россия, 660041, Красноярск, пр. Свободный, 79

В работе исследована способность светящихся бактерий видов Photobacterium leiognathi Boisvert et al. и Photob acter ium phosphoreum (Cohn) Beijerinck к синтезу полиэфиров гидроксикарбоновых кислот (полигидроксиалканоатов, ПГА) в качестве резервных макромолекул. Исследованные штаммы значительно различались по их способности к синтезу ПГА. Ph. leiognathi (но не Ph. phosphoreum) синтезировал ПГА, которые помимо мономеров поли-3-гидроксибутирата содержали мономеры 3-гидроксивалериановой и иногда, 3-гидроксикапроновой кислот. Все изученные штаммы Ph. phosphoreum образуют только гомополимерный поли-3-гидроксибутират. В случае Ph. leiognathi добавление валериановой кислоты в качестве субстрата может увеличивать содержание 3-гидроксивалериановой и 3-гидроксикапроновой кислот в полимере. Результаты позволяют рассматривать светящиеся микроорганизмы рода Photobacterium в качестве продуцентов многокомпонентных ПГА.

Ключевые слова: светящиеся микроорганизмы, Photobacterium, полигидроксиалканоаты, поли-3-гидроксибутират.

Introduction

Polyhydroxyalkanoates (PHAs) -microbial reserve polymers of hydroxy fatty acids - are synthesized by microorganisms under unbalanced growth (e.g., in the presence of carbon source and under deficiency of nitrogen or oxygen). PHAs serve as energy store and help microorganisms survive under unfavorable conditions. These biopolymers have received much attention recently for potential applications in various spheres. The greatest advantage of PHAs is that biosynthesis can yield polymers of various chemical structures, exhibiting different properties - from high-crystallinity thermoplastic polymers to rubber-

like elastomers (Steinbüchel, 2003; Sudesh et al., 2000). To increase PHA production and to create new types of PHAs, scientists isolate new PHA producers, modify culture conditions, and construct genetically modified strains (Madison, Huisman, 1999; Volova, 2004).

Poly hydroxybuty rate is the most common of PHAs but the chemical compositions of PHAs vary widely. Physiological features of bacterial strains and cultivation conditions affect the composition of monomer units, which is caused by the substrate specificity of PHA-synthases (key enzymes of polymer biosynthesis) of different microorganisms. As PHAs can vary in their composition, it would be expedient to search

for PHA producers among different bacterial strains.

Although there are more than 300 known PHA producers described in a number of fundamental reviews, no mention of luminous bacteria as potential producers of these macromolecules has been made. However, as it has been reported by some researchers, luminous bacteria can synthesize some amounts of polyhydroxybutyrate, and biochemical processes of light generation and PHA accumulation involve the use of common metabolites (Miyamoto et al., 1998; Sun et al., 1994). Thus, the purpose of this study was to investigate luminous bacteria as a novel potential PHA producer.

Materials and methods

The study microorganisms were strains of the species Photobacterium leiognathi Boisvert et al. and Photobacterium phosphoreum (Cohn) Beijerinck from Collection CCIBSO (WDCM836) (Rodicheva et al., 1997). Fifteen strains were screened for their ability to synthesize PHAs:

6 strains of Ph. leiognathi, and 9 strains of Ph. phosphoreum (Table 1).

Bacteria were stored on Egorova's Fish-peptone agar medium: water fish extract - 500 ml, NaCl - 30.0 g, peptone - 10.0 g, KH2PO4 -1.0 g, MgSO4 - 0.5 g, agar-agar - 18.0 g, H2O -up to 1 L. Water fish extract was prepared by boiling pike-perch (600 g) in water (1000 g) and filtering. In experiment bacteria were grown in batch suspension culture, using the medium of the following composition (g/L): NaCl - 30, MgSO4*7H2O - 0.2, KH2PO4 - 1, Na2HPO4x12H2O - 6, peptone - 5, glycerol - 3. The medium differed from the conventional one (Farghaly, 1950) by the absence of (NH4)2HPO4 (0.5 g/L). Valeric acid previously neutralized with potassium hydroxide was added when necessary at 1 g/L. Nitrogen contained in peptone was measured using a Flash EA 1112 CHN elemental analyzer ("Neolab", Italy) and amounted to 75 g/kg at 4.7% moisture. Before experiments two passages were made in batch culture. Bacteria were batch cultured in 500-ml flasks containing

Table 1. Screened strains of luminous bacteria

Species Strain Isolation source Isolation area

Photobacterium 208 Seawater The Pacific Ocean

leiognathi 231 Seawater The Pacific Ocean

543 Fish Sumbolophorus rufinus, stomach The Indian Ocean

544 N.D. The Indian Ocean

554 Seawater The Indian Ocean

683 Fish Diaphus lucidus, stomach The Indian Ocean

Photobacterium 1 Type strain (ATCC 11040; NCMB 1282)

phosphoreum 1694 Seawater The Indian Ocean

1699 Seawater The Indian Ocean

1812 Fish Chlorophthalmus sp., bowel The Indian Ocean

1856 Fish Opisthoproctus soleatus, the light organ The Indian Ocean

1883 Fish Coryphaenoides serrulatus, bowel The Indian Ocean

1909 Fish Coelorhynchus fasciatus, the light organ The Indian Ocean

1912 Fish Coelorhynchus fasciatus, bowel The Indian Ocean

1920 Fish Coelorhynchus fasciatus The Indian Ocean

250 ml culture at temperature 28°C on an incubator shaker. The biomass washed off agar cultures with a 3% NaCl solution was inoculated into the medium.

Optical density of bacterial suspension was measured using a KFK-2 photoelectric colorimeter at 540 nm, with optical path length 3 mm. Biomass yield (g/L) was determined using the standard curve of culture optical density versus cell concentration. The batch-grown biomass was centrifuged at 6000 rpm and washed twice with a 3%NaCl solution; then,biomass was dried at105°C for 24 h. The PHA concentration and composition were determined by chromatography of fatty acid methyl esters on a GCD-Plus gas chromatographmass spectrometer (GC-MS) (Hewlett Packard) after acid-catalyzed hydrolysis of a dry biomass sample (4 mg) and re-esterification of fatty acids. Benzoic acid was used as the internal standard (Brandl et al., 1988).

Results and discussion

It was earlier shown that polymer accumulation by luminous bacteria occurs during the stationary growth phase, after the decline in luminosity (which, as the culture grows, passes through a latency period, a rise, which occurs when the culture reaches high density levels, and subsequent decay), and remains almost unchanged for a long time (Boyandin, 2007). Thus, in studies aimed at finding efficient PHA producers among luminous bacteria, one can take samples of biomass in the stationary phase, after the luminosity decay, when polymer concentration is the highest.

We studied 15 strains of luminous bacteria grown in batch culture (Table 2). PHA yields varied widely, depending on the strain. Ph. leiognathi strains synthesized considerable amounts of the polymer (47% and more). In the case of Ph. leiognathi 208, PHA yield was higher

Table 2. PHA production by batch-cultured luminous bacteria

PHA content, % dry matter PHA yield, g/L PHA composition, mol%

Species Strain 3-hydroxy-butyrate 3-hydroxy-valerate 3-hydroxy-hexanoate

Photobacterium 208 59 1.0 99.9 0.1 n.i.**

leiognathi 231 57 0.6 100 traces n.i.

543* 47.1 0.62 99.8 0.2 traces

544 49 0.5 99.8 0.2 n.i.

554 63 0.3 99.9 0.1 n.i.

683* 71.0 1.36 99.3 0.5 0.2

Photobacterium 1 1 < 0.01 100 n.i. n.i.

phosphoreum 1694 12 0.08 100 n.i. n.i.

1699 5 0.01 100 n.i. n.i.

1812 4 0.01 100 n.i. n.i.

1856* 5.1 0.014 100 n.i. n.i.

1883* 1.3 < 0.002 100 n.i. n.i.

1909 < 0.1 < 0.001 100 n.i. n.i.

1912 n.i. 0 n.i. n.i. n.i.

1920 1 < 0.002 100 n.i. n.i.

* Strains were studied earlier (Boyandin et al., 2007). ** not identified.

than obtained earlier (Boyandin, 2007) possibly because of additional passages of microbial cultures before experiments. Ph. phosphoreum strains produced much less polymer, and Ph. phosphoreum 1912 did not synthesize any polymer at all in our experiment.

Monomer composition of the synthesized polymers was analyzed. The polymers synthesized by five Ph. leiognathi strains contained quantifiable (0.1% to 0.5%) amounts on 3 -hydroxy valerate and the polymer synthesized by Strain 683 contained 0.2% of 3-hydroxycaproate (3-hydroxy hexanoate). Polymers synthesized by Ph. phosphoreum strains contained one component only - 3-hydroxybutyric acid.

PHA biosynthesis in cells of microorganisms occurs through the formation of acyl coenzymes A and can at this stage incorporate short-chain-length carboxylic acids through acyl CoA and then - 3-hydroxyacyl CoA. Of major significance is substrate specificity of PHA synthase, which either allows or does not allow monomers to be incorporatedintothe polymer chain (Volova,2004). In the studies involving other microorganisms it was shown that addition of carboxylic acids with

carbon chains containing five and more carbons as substrate can induce incorporation of the respective 3-hydroxy acids into the PHA (Volova et al., 2007). In our experiments luminous bacteria were grown on media supplemented with valeric acid. The supplements were added to the microbial culture at different growth stages. The lower the density of the culture, the more the addition of carboxylic acids suppressed the growth of microorganisms (Fig. 1).

Having analyzed the synthesized polymers, we found that the addition of valerate in the initial stage of bacterial culture induced incorporation of the largest amounts of 3-hydroxy valerate (Fig. 2).

To further screen the effect of valerate on the composition of the polymers produced by luminous bacteria, the substrate was added to the medium in the initial stage of the culture (during inoculation) (Table 3). In the medium supplemented with valerate, most of the study strains accumulated smaller amounts of the polymer and, as a result, their PHA yields were lower. In the Ph. phosphoreum 1920 culture supplemented with valerate no polymer was

2 1.8 1.6 >1.4

32 1 2

(D

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0.8

o

m 0.6 0.4 0.2 0

10

20

Time, h

30

-t=0

-t=9

-t=12

-t=15

-t=18

-t=21

-t=24

- Control

Fig. 1. Effect of valeric acid supplements on the growth of Ph. leiognathi 208. The legend shows the time since inoculation

a)

1.8 1.6 1.4 1.2

1

0.8 0.6

d 0.2 0

0 9 15 21 C

Time of valerate addition, h

b) 18

*16

<3 1.4 E

1.2

<u

Ü 1 ф

> 0.8

d 0.2 0

■f"

1

0 9 15 21 C

Time of valerate addition, h

Fig. 2. Effect of valeric acid supplements on the amounts of 3-hydroxyvalerate contained in the PHAs produced by Ph. leiognathi strains: a) 208, b) 231, depending on the time interval between inoculation and supplementation. C - control sample (without valerate)

£ 0.4

Id 0.4

Table 3. PHA production by batch-cultured luminous bacteria in the medium supplemented with valerate

PHA content, % dry matter PHA yield, g/L PHA composition, mol%

Species Strain 3-hydroxy-butyrate 3-hydroxy-valerate 3-hydroxy-hexanoate

Photobacterium 208 51 0.4 98.75 1.2 0.05

leiognathi 231 8 0.01 98.4 1.7 n.i.*

543 4 0.01 99.3 0.7 n.i.

544 13 0.03 99.5 0.5 n.i.

554 0.6 < 0.002 99.9 0.1 n.i.

683 32 0.1 99.3 0.5 0.2

Photobacterium 1 0.5 0.001 100 n.i. n.i.

phosphoreum 1694 0.2 0.001 100 n.i. n.i.

1699 traces < 0.001 100 n.i. n.i.

1812 0.6 0.001 100 n.i. n.i.

1856 0.4 0.002 100 n.i. n.i.

1883 0.6 < 0.001 100 n.i. n.i.

1909 traces < 0.001 100 n.i. n.i.

1912 n.i. 0 n.i. n.i. n.i.

1920 n.i. 0 n.i. n.i. n.i.

* not identified.

detected. Ph. leiognathi strains were the most stable PHA producers.

PHAs synthesized by all the study strains of Ph. leiognathi, except 554 and 683, in the culture supplemented with valerate contained significantly larger amounts of 3-hydroxyvaleric acid. No minor hydroxy acids were found in

the PHAs produced by Ph. phosphoreum in experiments both with and without the addition of valerate. The addition of valerate was found to induce incorporation of 3-hydroxyhexanoic acid in the polymer produced by Ph. leiognathi 208.

Thus, Ph. leiognathi cells grown in the medium supplemented with valerate, which is 167 -

hydroxyvalerate precursor substrate, can produce polymers containing increased amounts of this hydroxy acid. In a contrast, Ph. phosphoreum strains were found unable to synthesize multi-component PHAs.

Ackonowledgements

The study was supported by the State budget allocated to the fundamental research at the Russian Academy of Sciences (project No 01201351505).

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