Halobacteria as producers of polyhydroxyalkanoates

FEMS Microbiology Reviews 103 (1992) 181-186 © 1992 Federation of European Microbiological Societies 0168-6445/92/$15.00 Published by Elsevier

181

FEMSRE 00239

Halobacteria as producers of polyhydroxyalkanoates Francisco Rodriguez-Valera and Jos6 A.G. Lillo Departamento de Gen~tica Molecular y Microbiolog[a, Universidad de Alicante, Alicante, Spain

Key words: Polyhydroxyalkanoate; Halobacteria; Haloferax; Halophile; Extremophile

1. INTRODUCTION

1.1. General characteristics of halobacteria Halobacteria are the extremely halophilic branch of the archaea (previously, archaebacteria). As most other members of the archaea, the halobacteria are obligate extremophiles, not only tolerating, but requiring high NaCI concentrations (2-5 M) and considerably high magnesium salt concentrations as well, particularly some genera [1]. They form large populations in the salt precipitation ponds of solar salterns giving them their typical red hue. Certainly the most typical and peculiar feature of halobacteria is their halophilic osmoregulation, counteracting the near-saturating extracellular salinity by accumulation of intracellular potassium ion (5 M K ÷ has been described in the cytoplasm of optimally growing halobacteria) [2]. As a consequence of their hypersaline cytoplasm, all the building blocks of the halobacterial cell (proteins and lipids are particularly affected by high ionic strength in their environment) are irreversibly adapted to these conditions [3,4]. Other halophilic organisms, eubacterial or eukaryotic, have their cyto-

Correspondence to: F. Rodriguez-Valera, Departamento de Gen~tica Molecular y Microbiologla, Universidad de Alicante, Apartado 374, 03080 Alicante, Spain.

plasms protected by organic compatible solutes and exclude salts from their intracellular space [5]. Another remarkable characteristic of halobacteria is their extreme sensitivity to exposure to low-salinity conditions: most species of halobacteria lyse within seconds of being exposed to distilled water [1]. Although the halobacteria have been universally associated with the peculiar bacteriorhodopsin-based photosynthesis, this phototrophic metabolism is found only in a few species, and under special conditions [1]. Normally they behave like aerobic organotrophs with a metabolism closely similar to that of the aerobic eubacteria (including respiratory chains that contain cytochrome c) [6]. This is an important point because the archaea are generally associated with extravagant metabolisms that are little suited to classical industrial microbiology. The halobacteria are a heterogeneous group with six genera described up to now, including neutrophilic and alkaliphilic organisms and showing three clearly distinct morphologies: cocci with thick heteropolysaccharidic cell walls, rods and pleomorphic cells that possess glycoprotein Slayers as major cell envelope [1]. The genus Haloferax has a number of interesting characteristics that differentiate it from the more classical halobacteria belonging to the genus Halobacterium [7]. It does not have bacteriorhodopsin

182

with a metabolism that is strictly organotrophic; it grows fast compared to other halobacteria, with generation times in the range of 3 - 4 h under optimal conditions [8]. This has considerable merit bearing in mind the low water activity of the environment in which they grow. Haloferax also has a wide range of substrates that can be used as sole carbon and energy source, including several sugars and polysaccharides (e.g. starch). Ribulose bisphosphate carboxylase (RuBP) activity (the key enzyme of the Calvin cycle) and RuBP-dependent CO 2 fixation have been detected in cell-free extracts in Haloferax mediterranei and other species that do not have bacteriorhodopsin [9]. Therefore, photoautotropic growth could not occur in those organisms. Instead, some evidence indicates that the reduction power could be derived from chemolithotrophic reactions (H 2 oxidation). Interestingly, there is a clear correlation between

the RuBP activity detected and the poly-3-hydroxybutyrate (PHB) accumulated by the cell [9]. 1.2. Halobacteria as producers of poly-jg-hydroxyal-

kanoates Some years ago we showed that some species of halobacteria produced considerable amounts of PHB when growing on sugars. The species Haloferax mediterranei was the one that produced the largest amounts [10]. In batch cultures we found that the key factors determining the amount of PHB accumulated by H. mediterranei were the concentration and nature of the carbon source and the concentration of the phosphorus source (phosphate). Neither the nature or concentration of the nitrogen source nor the oxygen concentration or any other factor assayed gave significant changes in the amount of polymer [11].

Table 1 Batch production of PHA by some producer strains Microorganism

Yield

Polymer composition ( % mol)

(g g - l )

3HB

5.08

0.25

100

51

4.23

0.21

25

75

Fructose

24

3.78

0.25

100

.

Starch

67

6.48

0.32

89

11

Glucose

60

4.16

0.21

.

Fructose

50

1.80

0.17

100

.

Octanoate

31

0.26

0.17

-

-

9.6

86.1

4.3

[15]

Decanoate

27.6

0.28

0.07

-

5.3

52.3

42.3

[16]

Butyrate

20.4

0.13

0.002

99.6

-

0.4

-

Acetate Glucose/ Butyrate Glucose/ Valerate

65 54

0.50 2.20

0.07

98

2

-

0.11

99

1

-

-

-

[18] [19]

40

1.00

0.05

88

12

-

-

-

[19]

Glucose

74

2.95

0.27

.

Substrate

content

Maximum production

(%DW)

(gl -l)

Glucose

54

Valerate

PHA

3HP

3HH

3HO

Reference 3HD

Alcaligenes eutrophus N C I B 11599

-

-

-

-

[12]

Alcaligenes eutrophus A T C C 17699

-

[12]

Alcaligenes eutrophus A T C C 17697

.

.

.

[13]

Haloferax mediterranei A T C C 33500

-

-

-

[11]

Haloferax mediterranei A T C C 33500

.

.

.

.

[11]

Pseudomonas cepacia A T C C 17697

.

.

.

[14]

Pseudomonas oleoL'orans A T C C 29347

Pseudomonas putida KT2442

Rhodospirillurn rubrum A T C C 25903

-

[17]

Rhodobacter sphaeroides A T C C 17023

Rhizobium meliloti 41

Rhizobiurn meliloti 41

Azotobacter beijerinckii N C I B 9067

.

.

.

.

[20]

183

Fig. 1. A. Electron micrograph showing cells of Haloferax mediterranei grown under optimal conditions for PHA accumulation. Large electron transparent granules are PHA. B. Individual granules, once released from lysed cells, showing a conspicuous envelope. Bar = 1/.tm. (Reproduced from [21] with permission.)

184

The optimal conditions for PHB production found in batch cultures were as follows: carbon source (glucose or starch), 2% (w/v); NHaC1, 0.2%; KH2PO 4, 0.00375% (w/v); mixture of marine salts, 25% (w/v); temperature 45°C; pH 7.2. Table 1 shows the results obtained under these conditions in comparison to similar parameters for other prospective PHA producers. The yield obtained from starch approaches the theoretical limit and the amount accumulated per cell dry weight is probably also close to the maximum (Fig. 1). Microscopic examination of samples from cultures under optimal PHB production conditions showed the accumulation of large amounts of granules similar in size and morphology to those described in eubacteria [22]. Figure 1 shows cells in which the cytoplasm is virtually filled with granules, in the same way as that occurring with Alcaligenes eutrophus and other highly efficient PHB-producing eubacteria. These micrographs also show that the proportion of PHB to total biomass must be reaching the maximum under these conditions, since the cytoplasm appears very reduced in relation to the volume occupied by the granules. Once released from the cell, the thin membrane covering them can easily be seen (Fig. 1B). The kinetics of accumulation closely resemble those found for eubacterial PHB producers [13] and consist of two clearly separate stages: one of total biomass growth and a second phase of PHB accumulation but no increase in other biomass components (e.g. protein), in which the phosphate concentration has to be lower than 10 mg !-l for the accumulation of the polymer to begin [11]. Production in single-staged continuous fermentation has also been studied under conditions which are optimal for batch production and at different dilution rates (D) [11]. Under these conditions, PHB production was always much lower than in batch culture, the highest concentration found was at a D of 0.04 h -l, resulting in 1.62 g l-I which represented about 46% of the dry weight of biomass and a yield of about 0.16 g g-~. However, in these experiments the concentrations of the carbon source and phosphate had not been optimized, and the continuous intake of phosphate in a single-stage chemostat caused a

decreased yield and productivity. With this in mind, the above-mentioned yield and productivity appear really promising. An important condition for the use of an organism for production in continuous culture is the stability of the strain, since very often nonproducing mutants are favoured and displace the wild-type [23]. Moreover, some halobacteria are known for their high genomic instability [24]. To test whether that was the case with PHB production by Haloferax mediterranei, we set up a chemostat and maintained it in the steady state for 3 months with a D = 0.13 h -1, which roughly corresponds to 370 generations. Afterwards, the production of the strain isolated from the fermenter was evaluated in batch under optimal conditions. The total PHB production, yield, etc. were not significantly different from those of the original strain [11]. This remarkable stability makes the organism eminently suitable for use in continuous industrial production. Moreover, in a chemostat with alternating cycles of carbon starvation [11], which would theoretically favor overproducing strains, the strains tested at the end of the experiment also had the same productivity and yield.

2. HALOBACTERIAL GENES INVOLVED IN PHA BIOSYNTHESIS The cloned A. eutrophus PHA biosynthetic genes (polymerase, 3-ketothiolase and acetoacylCoA reductase) do not hybridize with H. mediterranei total DNA, even under low-stringency conditions. Non-producing and 'leaky' mutants of H. mediterranei have been obtained and we plan to use complementation tests to check the involvement of such sequences in PHA biosynthesis. Some preliminary evidence indicates that the genes could be located on a 300-kb megaplasmid typical of H. mediterranei.

3. ADVANTAGES OF H. MEDITERRANEI AS PHA PRODUCER

Haloferax mediterranei has a number of advantages as producer when compared with Alcaligenes eutrophus. The amounts of PHA accumu-

185

lated and the yield in relation to the carbon source are similar, but H. mediterranei can use starch as carbon source, which is a much cheaper substrate than glucose [25]. Moreover, this organism produces copolymers of HB and HV even if no precursor is added to the medium (J. Garcia Lillo and F. Rodriguez-Valera, unpublished data). With only starch as carbon source, a minimum of 9% of HV subunits are incorporated in the polymer, with melting points in the range 120-145°C, which roughly corresponds with the lower limit that allows a good thermal processing. Therefore, the cost of precursor can be reduced considerably. The M r values are considerably high, often ranging between 1 and 2 million, which is also convenient for processing. The fragility of halobacterial cells exposed to low salt concentrations allows the development of greatly simplified recovery processes. An unexpected finding has been the apparent inability of H. mediterranei, at least under the conditions used, to degrade intracellular PHA granules, cultures can be left for a long time in the stationary phase without noting a detectable decrease in the amount of PHA present in the culture. Furthermore, even if proper amounts of phosphate and ammonia are added to the culture there is neither growth nor degradation of the PHA accumulated. This could mean that either there is no depolymerase present in this organism, in which case the biological role of PHA accumulation would be other than carbon storage, or the depolymerase requires special conditions to be activated which that we have not yet been able to discover. At any rate, the absence of depolymerization allows considerable freedom when handling the cultures and could theoretically permit a higher yield. Finally, an advantage that requires special consideration is the ease of cultivation. It has been known for a long time that batches of up to 100 1 of halobacterial cultures can be grown under extremely simplified conditions [27]. In fact, working with Haloferax, the genus which includes the halobacteria with the fastest growth rates and widest physiological versatilities, it is feasible to devise conditions under which there is virtually no possible contamination. In our laboratory we have maintained continuous cultures of Haloferax

mediterranei running for 3 months, with minimal sterility precautions. This allows the design of production facilities of vast dimensions that would require relatively little investment, more resembling chemical reactors than classical fermenters, and the process can be carried out in a continuous form, optimizing production parameters far beyond the levels reached in batch processes. The idea of using extremophiles for contaminationfree industrial microbiology is not new, and was already contemplated in the case of thermophiles [28]. For standard thermophilic processes it does not seem to be feasible [29], but to use hyperextremophiles, such as archaebacteria often are, has never been attempted.

4. DISADVANTAGES There are two major disadvantages inherent in using H. mediterranei for PHA production. This organism produces an exocellular polysaccharide [30] which is released into the medium producing highly viscous cultures and, possibly, reducing the maximum yield of PHA. This problem can easily be by-passed using E PS- mutants. In fact, such mutants are available and their PHA production parameters are similar or identical to those of the wild-type (Boan and Rodriguez-Valera, unpublished results). Another major problem with the use of an extremely halophilic organism in industrial processes is the necessity to use a considerable amount of salts (particularly NaCI and Mg salts) in the culture medium. In the optimal conditions described above, the cost of the inorganic salts (bulk prices) roughly equals that of the carbon source (industrial starch). Work is in progress to circumvent this problem.

ACKNOWLEDGEMENTS Our work related to PHA production by

Haloferax mediterranei has been supported by Grants PBT 86/0011, PTR 89/0003 and BIO 90/0475 of the Spanish Government and by a NATO Collaborative Research Grant CRG 910474. Secretarial assistence by K. Hernandez is gratefully acknowledged.

186 REFERENCES [1] Grant, W.D. and Larsen, H. (1989) In: Bergey's Manual of Systematic Bacteriology, Vol. 3. (Staley, J.T., Bryant, M.P., Pfennig, N. and Holt, J.G., Eds.), pp. 2216-2219. Williams and Wilkins, Baltimore, MD. [2] Christian, J.H.B. and Waltho, J.A. (1962) Solute concentrations within cells of halophilic and non-halophilic bacteria. Biochim. Biophys. Acta 65, 506-508. [3] Lanyi, J.K. (1974) Salt-dependent properties of proteins from extremely halophilic bacteria. Bacteriol. Rev. 38, 272-290. [4] Quinn, P.J. (1986) Models of haloadaptation in bacterial membranes. FEMS Microbiol. Rev. 39, 87-94. [5] Imhoff, J.F. (1986) Osmoregulation and compatible solutes in eubacteria. FEMS Microbiol. Rev. 39, 57-66. [6] Hochstein, LT (1988) The physiology and metabolism of the extremely halophilic bacteria. In: Halophilic Bacteria, Vol. II (Rodriguez-Valera, F., Ed.), pp. 67-83. CRC Press, Boca Raton, FL. [7] Torreblanca, M., Rodriguez-Valera, F., Juez, G., Ventosa, A., Kamekura, M. and Kates, M. (1986) Classifation of non-alkaliphilic halobacteria based on numerical taxonomy and polar lipid composition and description of Haloarcula, gen. nov. and Haloferax, gen nov. Syst. Appl. Microbiol. 8, 89-99. [8] Rodriguez-Vatera, F., Juez, G. and Kushner, D.J. (1983) Halobacterium mediterrei, spec. nov., a new carbohydrateutilizing extreme halophile. Syst. Appl. Microbiol. 4, 369-381. [9] Altekar, W. and Rajagopalan, R. (1990) Ribulose biphosphate carboxylase activity in halophilic archaebacteria. Arch. Microbiol. 153, 169-174. [10] Fernandez-Castillo, R., Rodriguez-Valera, F., GonzalezRamos, J. and Ruiz-Berraquero, F. (1986) Accumulation of Poly(/3-hydroxybutyrate) by halobacteria. Appl. Environ. Microbiol. 51,214-216. [11] Lillo, J.G. and Rodriguez-Valera, F. (1990) Effects of culture conditions on Poly(/3-hydroxybutyric acid) production by Haloferax mediterranei. Appl. Environ. Microbiol. 56, 2517-2521. [12] Doi, Y., Tamaki, A., Kunioka, M. and Soga, K. (1988) Production of copolyesters of 3-hydroxybutyrate and 3hydroxyvalerate by Alcaligenes eutrophus from butyric and pentanoic acids. Appl. Microbiol. Biotechnol. 28, 33O-334. [13] Mulchandani. A., Luong, J.H.T. and Groon, C. (1989) Substrate inhibition kinetics for microbial growth and synthesis of poly-/3-hydroxybutyric acid by Alcaligenes eutrophus ATCC 17697. Appl. Microbiol. Biotechnol. 30, 11-17. [14] Ramsay, B.A., Ramsay, J.A. and Cooper, D.G. (1989) Production of poly-/3-hydroxyalkanoic acid by Pseudomonas cepacia. Appl. Environ. Microbiol. 55, 584-589. [15] Brandl, H., Gross, R.A., Lenz, R.W. and Fuller, C. (1988) Pseudomonas olevorans as a source of poly-(/3-hy-

[16]

[17]

[18]

[19]

[20]

[21]

[22]

[23]

[24]

[25] [26] [27] [28]

[29]

[30]

droxyalkanoate) for potential applications as biodegradable. Appl. Environ. Microbiol. 54, 1977-1982. Huijberts, G.N.M., Eggink, G., de Waard, P. and Huisman, G.W. (1989) Pseudornonas putida KT2492 cultivated on glucose accumulates poly-(3-hydroxyalkanoates) consisting of saturated and unsaturated monomers. Appl. Environ. Microbiol. 58, 536-544. Brandl, H., Gross, R.A., Knee, E.J., Lenz, R.W. and Fuller, R.D. (1989) The ability of the phototrophic bacterium Rhodospirillum rubrum to produce various poly(/3-hydroxyalkanoates): potential sources for biodegradable polyesters. Int. J. Biol. Macromol. 11, 1. Brandl, H., Gross, R.A., Lenz, R.W., Lloyd, R. and Fuller, R.C. (1991) The accumulation of poly-(3-hydroxyalkanoates) in Rhodobacter sphaeroides. Arch. Microbiol. 155, 337-340. Tambolini, R. and Nati, M.-P. (1989) Poly-(/3-hydroxyalkanoate) biosynthesis and accumulation by different Rhizobium species. FEMS Microbiol. Lett. 60, 299-304. Senior, P.J., Beech, G.A., Ritchie, G.A.F. and Dawes, E.A. (1972) The role of oxygen limitation in the formation of poly-/3-hydroxybutyrate during batch and continuous culture of Azotobacter beijerinskii. Biochem. J. 125, 53-66. Rodriguez-Valera, F. (1991) Polyhydroxyalkanoates, a family of biodegradable plastics from bacteria. In: Progress in Membrane Biotechnology (Gomez-Fernandez, J.C., Chapmen, D., Packer, L., Eds.), pp. 266-278. Birkh~iuser Verlag, Basel, Switzerland. Lundgren, D.J. and Pfister, R.M. (1964) Structure of Poly-fl-hydroxybutyric acid granules. J. Gen. Microbiol. 34, 441-446. Stafford, K. (1986) Continuous fermentation. In: Manual of Industrial Microbiology and Biotechnology (Demain, A.L. and Solomon, N.A., EdS.), pp. 137-151. American Society for Microbiology. Washington, D.C. Huisman, G.W., Wonink, E., Meima, R., Kazemier, B., Terpstra, P. and Witholt, B. (1991) J. Biol. Chem. 266, 2191-2198. Keeler, R. (1991) Don't let food go to waste - - make plastic out of it. RandD Magazine 33, 52-57. Reference omitted. Kushner, D.J. (1966) Mass culture of red halophilic bacteria. Biotechnol. Bioeng. 8, 237-245. Cooney, C.L. and Wise, D.W. (1975) Thermophile anaerobic digestion of solid waste for fuel gas production. Biotechnol. Bioeng. 17, 1119-1124. Weimer, P.J. (1986) Use of thermophiles for the production of fuels and chemicals. In: General, Molecular and Applied Microbiology (Brock, T.D., Ed.), pp. 217-255. John Wiley and Sons, New York, NY. Anton, J., Meseguer, I. and Rodriguez-Valera, F. (1988) Production of an extracellular polysaccharide by Haloferax mediterranei. Appl. Environ. Microbiol. 54, 2381-2386.