ITMI980810A1 - RHODOBACTER SPHAEROIDES RV MUTANT AND ITS USE IN HYDROGEN PRODUCTION - Google Patents

Description

"Rhodobacter sphaeroides RV mutant and its use in hydrogen production".

Description

The invention relates to the Rhodobacter sphaeroides RV CBS 100467 mutant unable to accumulate polyhydroxyalkanoates (PHAs) and a process for the production of hydrogen which uses said mutant.

Hydrogen is the most abundant element in the sun and plays a key role in energy generation. On earth, however, together with oxygen it is the fundamental compound for the biological cycle of energy.

The quantity of hydrogen contained in the lower area of the atmosphere can be estimated at about 2.2xl0 <9> tons, equal to about 0.55 ppm per volume of air. Compared to 1.1 x IO <15> tons of 02, hydrogen is certainly to be considered a rare gas on earth. However, unlike noble gases, hydrogen is continuously produced and used by a large number of microorganisms both in aerobic and anaerobic conditions, in the presence or absence of light.

From an energy point of view, hydrogen is certainly an ideal source as it has a higher calorific value than any other. Furthermore, it can be easily preserved, it is not polluting and the final product of its oxidation, water, is not only non-toxic, but is an indispensable compound to guarantee life on earth.

The ability of microorganisms to develop hydrogen, together with the progressive depletion of traditional energy sources and the serious environmental problems associated with them, has long stimulated a considerable research effort to obtain this energy source biologically.

In the natural environment there are numerous organisms that can spontaneously produce hydrogen.

These organisms include anoxygenic photosynthetic bacteria, such as red non-sulphurous bacteria which include, among others, the genera Rhodooseudomonas. Rhodobacter and Rhodospirillum.

they are particularly suitable for the production of hydrogen. In fact, they are not only capable of using a source of energy at no cost, light, but can use waste substances as a substrate for their growth, thus combining the production of energy with the disposal of waste that represent a serious environmental problem. .

However, these bacteria have the drawbacks of low hydrogen production yields. It follows that the use of these microorganisms makes it difficult to develop processes for the bioproduction of hydrogen which are interesting from a practical and economic point of view.

It is known in the literature, in fact, that red non-sulphurous bacteria are also characterized by the ability to synthesize, under unbalanced growth conditions, high quantities of reserve substances, generally referred to as polyhydroxyalkanoates (PHA). PHAs are accumulated in the form of cytoplasmic inclusion bodies and represent a significant percentage (up to 70%) of the cell weight. Also for the strain R.sohaeroides RV, which overproduces hydrogen, a competition has been demonstrated between the accumulation of polyhydroxyalkanoates (PHAs) and the photoproduction of hydrogen as a function of the growth conditions.

In fact, the evolution of hydrogen gas and the synthesis of PHAs represent two alternative metabolic pathways to consume the cellular reducing power.

To improve the hydrogen production capacity of R.sohaeroides RV in terms of duration and therefore of the total amount of H2 produced, a mutant unable to accumulate polyhydroxyalkanoates (PHAs) has now been constructed.

This mutant, called SMV071, was deposited on 16.02.1998 at the Centraalbureau Voor Schimmelcultures where it received the CBS number 100467.

Therefore an object of the present invention is a mutant of R.sphaeroides RV CBS 100467 unable to accumulate polyhydroxyalkanoates (PHAs).

A further object of the present invention is a process for the production of hydrogen which uses the mutant of R.sphaeroides RV CBS 100467.

Brief description of the figures

Figure 1: the restriction map of the pSM774 plasmid is shown

Figure 2 (A-C): shows the nucleotide and amino acid sequence of the wild-type R.sphaeroides RV phaC synthase gene missing the first 30 bases and the last 60 bases.

Figure 3: the restriction map of the pSM583 plasmid is shown.

Figure 4: Southern Blot analysis on genomic DNA of mutant clones No 1-10 (lines 1-10) and wild-type strain R.sohaeroides RV digested with the restriction enzyme EcoRI and hybridized with a specific probe for the phaC gene .

Figure 5: gas chromatograms of wild-type R.sohaeroides RV strain and SMV071 mutant strain showing the presence and absence of the characteristic PHB peak, respectively.

A: methyl ester standard of β-hydroxybutyric acid (peak at RT = 10.28);

B: cell culture extract of R.sphaeroides RV wild-type after 14 days of fermentation (peak at RT = 10.96);

C: cell culture extract of the SMV071 mutant after 15 days of fermentation (no peak at RT characteristic of β-hydroxybutyric acid).

Figure 6: schematic representation of the bioreactor where: 1 = substrate reservoir; 2 = Argon gas fluxed to maintain anaerobic conditions; 3 = peristaltic pump that regulates the introduction of the soil into the photobioreactor; 4 = photobioreactor; 5 = magnetic plate to keep the agitation inside the photobioreactor; 6 = electrical connection for the halogen lamps located inside the photobioreactor; 7 = common output of exhausted soil, biomass and biogas; 8 = trap; 9 = digital meter for advanced biogas; 10 = biogas discharge; 11 = peristaltic pump for the elimination of exhausted soil and biomass; 12 = land discharge and biomass and sanitation. Detailed description of the invention

In most bacteria that are able to accumulate PHAs, including R.sphaeroides RV, poly (3-hydroxybutyrate) (PHB) is the most produced polymer.

It is known in the literature that in the most studied bacterium, Alcaligenes eutrophus, the synthesis of PHB is carried out through a metabolic pathway that involves three different enzymes in succession: a β-ketothiolase, an acetoacetyl-CoA reductase and a synthase that catalyzes the polymerization of monomer to give the PHB polyester.

The present invention is based on the consideration that synthase represents the key enzyme of the metabolic pathway. This enzyme, in fact, has a relative low substrate specificity, and therefore could use as intermediate substrates different from alternative metabolic pathways.

Therefore it was decided to eliminate the last stage of PHB biosynthesis, by inactivating the gene (phaC) that codes for this enzyme, after verifying its presence in the genome of R.sohaeroides RV.

For its inactivation, conventional methods such as random mutagenesis, which is based on the use of transposons, or targeted mutagenesis using interposons, can be used. These elements, after insertion into the genome, in addition to causing an interruption in the sequence of a gene, which manifests itself through a phenotypic mutation, give the host resistance to a particular antibiotic.

According to a preferred embodiment of the present invention, the inactivation of the wild-type R.sohaeroides RV phaC synthase gene was carried out by inserting a gene cassette into the previously isolated and characterized synthase gene (Figure 2). (interposon) coding for resistance to kanamycin and subsequent selection of the mutagenized strains on a medium supplemented with this antibiotic.

In fact, theoretically, only the clones in which the integration of the interposon has taken place in the genomic DNA of the strain can grow on this selective medium.

The construction of the interposon was carried out using first the E. coli TG1 strain as the host and pUC18 as the vector.

Colony restriction analysis obtained after transformation confirmed the isolation of a positive clone containing the kanamycin gene inserted in the phaC synthase gene. The nucleotide sequence of this clone highlighted the insertion of the interposon in the opposite orientation with respect to the transcription direction of the phaC synthase gene.

The disrupted synthase gene was then cloned into a suicide vector, i.e. a vector that is unable to replicate in R. sohaeroides, and the final construction was introduced by conjugation into the E. coli S17-1 strain and the wild-type R. sohaeroides RV made resistant to the antibiotic rifampicin.

The selection of R.sohaeroides RV mutants was carried out in two stages: the first stage consisted of selection on kanamycin to confirm the recombination between the mutagenized copy of the ohaC gene. carried by the suicide vector, and the functional copy found on the genomic DNA and the second stage in the selection on gentamicin to verify the loss of the suicide vector used for mutagenesis.

Clones were characterized by Southern blot analysis to confirm the nature and site of the mutagenesis event. Genomic DNAs were extracted, digested with the EcoRI restriction enzyme and hybridized with a synthetic oligonucleotide specific for the ohaC gene. Wild-type genomic DNA prepared under the same conditions was used as a negative control.

For one of these clones a hybridization band of the expected size was highlighted.

This clone, named SMV071, was physiologically characterized by growth in different media. This clone showed, similarly to the wild-type strain, the same growth capacity in complete media, for example aSy (J.Miyake, X.Y. Mao and S.Kawamura, J.Ferment. Technol., 62: 531-535, 1984 ) and substrates from market waste, and no growth in soil that induces PHB synthesis. This result indirectly confirmed the inability to activate this metabolic pathway. Furthermore, the determination of PHB production, carried out by gas chromatographic analysis on the SMV071 clone, did not show any peak, thus confirming the inability of the mutant strain to accumulate PHB.

The mutant of R, sohaeroides RV of the present invention is therefore particularly suitable in the development of fermentation processes for the production of hydrogen.

Fermentation can be carried out in an aqueous medium containing assimilable sources of carbon and nitrogen as well as various cations, anions and, possibly, traces of vitamins or amino acids.

Similar carbon sources include organic acids such as pyruvate, malate, lactate, succinate or other conventional carbon sources. Sources of nitrogen can be chosen, for example, from mineral ammonium salts, such as ammonium nitrate, ammonium sulfate, ammonium chloride or ammonium carbonate and urea or materials containing organic or inorganic nitrogen such as peptone, yeast extract or extract of meat .

Non-limiting examples of cations and anions can be selected from potassium, sodium, magnesium, iron, calcium, acid phosphates, sulphates, chlorides, manganese and nitrates.

According to a preferred embodiment of the process of the present invention, a material deriving from the controlled acidogenic fermentation of commercial waste is used as the substrate (E. D'Addario et al. (1992), Wat.Sci. Tech. 22, 183-192) having the composition reported in example 7.

Fermentation is carried out in anaerobiosis, guaranteed by the continuous flow of argon, in the presence of light with a light intensity between 6,000 and 20,000 lux, at a temperature between 25 ° C and 40 ° C, preferably between 30 ° C and 37 ° 0.

The pH of the fermentation medium is kept within values between 6.50 and 8.0 and preferably between 6.8 and 7.3. The pH adjustment can be carried out, for example, by adding a basic aqueous solution such as an aqueous solution of ammonia, potassium hydroxide, sodium hydroxide, sodium carbonate or potassium. A 1-5 M sodium hydroxide solution is preferably used.

During fermentation, the hydrogen is recovered using conventional techniques.

The process can be carried out continuously in equipment of the type shown in figure 6.

Operating under the preferred conditions, it was observed that the amount of H2 produced by the mutant was approximately 26% higher than in the wild-type strain, since the photoproduction of H2 by the mutant continued for another ten days (26 days in total). compared to 16 days for the wild-type strain.

The following examples, which have the sole purpose of describing the present invention in greater detail, must in no way be interpreted as a limitation to the purposes thereof.

Example 1

Genomic DNA extraction from R.sphaeroides RV A 500 ml flask containing 100 ml of LB medium (10 g / 1 tryptone, 5 g / 1 yeast extract, 10 g / 1 NaCl) was inoculated with the R .sphaeroides RV strain (provided by the National Institute of Bioscience and Human-technology (NIBH), Tsukuba, Japan) and the resulting mixture was stirred (220 rpm) at 30 ° C for 48 under aerobic / dark conditions.

Then, the cells were recovered by centrifugation of the broth culture in a Sorvall RC-5B model SS34 rotor (at 4 ° C and 5000 rpm for 10 minutes) and then washed (2x120 ml) with TE buffer solution (1 mM EDTA, 10 mM Tris-HCl, pH 7.4). The resulting suspension was centrifuged again as reported above and the cells were recovered and resuspended in 9.5 ml of TE buffer solution. After addition of 0.5 ml of 10% SDS (sodium dodecyl sulfate) and 50 µl of a solution of Proteinase K (20 mg / ml), the suspension was incubated at 37 ° C for 1 hour.

Then 1.8 ml of 5 M NaCl and 1.5 ml of a solution consisting of 10% hexadecyltrimethyl ammonium bromide (CTAB) in 0.7 M NaCl were added and the resulting solution was incubated at 65 ° C for 20 minutes .

Subsequently the solution was deproteinized with equal volume of chloroform: isoamyl alcohol (24: 1, v / v) and the DNA was precipitated with 0.6 volumes of isopropanol.

The DNA was washed with 1 ml of ethanol (70%) and recovered with a glass rod. Finally, the collected DNA was dissolved in 4 ml of TE and its concentration was determined by spectrophotometric reading at 260 nm.

The genomic DNA was further purified by CsCl gradient centrifugation (1%) containing 0.1 mg / ml of ethidium bromide (55,000 rpm for 16 hours in a Beckman V65Ti rotor).

The DNA band was visualized under UV light and the ethidium bromide was removed by extraction with butanol saturated in water. After dialysis against TE buffer, the DNA was precipitated with ethanol and resuspended in the desired volume.

Example 2

Isolation of the phaC synthase gene

The genomic DNA obtained as reported in example 1 was amplified by the Polymerase Chain Reaction (PCR) technique, according to the indications reported by Leung et al. (Leung D.W., Chen E., Goeddel D.V., Technique - a journal of methods in celi and molecular biology, 1, n ° 1 (1989): pp. 11-15), using the following pair of oligonucleotides as primers:

(1) 5'CGTGACGCTC AGTTCGAGCG TCTGAAC 3 '(FORWARD) (2) 5'CGGCGCGAGC TCGGGATGTT TCGAATC3' (REVERSE) These oligonucleotides have been synthesized on the basis of amino acid sequences preserved in bacterial synthase .sohaeroides RV.

The amplification was carried out in a DNA Thermal Cycler 480 apparatus (Perkin -Elmer Cetus) using a reaction mixture (100 μΐ) containing:

- 500 ng of genomic DNA

- 10 mM Tris HC1 pH 8.3

- 2 mM MgS04

- 25 mM KC1

- 5 mM (NHJ2S04

- 100 picomoles of the two primers

- 200 pM of dNTPS (dATP, dGTP, dCTP, dTTP)

- 2.5 Units of Pwo polymerase (Perkin Elmer).

After denaturation for 2 minutes at 98 ° C, the cyclic program was started, which included:

1 minute at 98 ° C (denaturation)

1 minute at 60'c (annealing)

2 minutes at 72 ° C (elongation)

for a total of 25 cycles, followed by 8 minutes at 72 ° C (final extension).

The resulting amplification product was treated with phenol-chloroform (1: 1), precipitated with NaCl (1/10 vol / vol) and EtOH (2 vol.) And resuspended in 20 µl of H20O. Then a fragment of about 1700 bp was purified on low-melting gel (SeaPlaque, FMC BioProducts) at 0.6% and after elution from the gel it was cloned in the plasmid PUC18 (Boheringer) previously digested with the restriction enzyme. Smal.

In practice, the pUC18 plasmid and the fragment were ligated in 30 µl of reaction mixture (DNA 5 pg / ml) and 2 µl of said mixture were used to transform E.coli TG1 cells made electrocompetent (Dower W.J. et al. , Nucleic Acids Research, 16, 6127-6145, 1988) The transformants were selected on plates of LB agar medium containing 100 pg / ml of Ampicillin.

The analysis of the plasmid DNA extracted from the positive clones revealed the presence of a fragment of about 1700 bp corresponding to the expected size. The plasmid contained in the positive clone was named pSM774 (figure 1).

The plasmid was then subjected to sequence analysis using the ABI 373 DNA sequencer (Perkin Elmer). The sequence confirmed that the 1738 bp fragment corresponded to the phaC synthase gene missing from the first 30 bases and the last 60 bases (Figure 2).

Example 3

Inactivation of the phaC synthase gene

The plasmid pSUP2021 (R. Simon et al., (1983), Bio / Technology, 1: 784-791) (5 μg) containing the gene encoding kanamycin resistance (neo), was digested with 5 units of the Sali and HindII (Boehringer) restriction enzymes at 37 ° C for 1 hour. The digestion mixture was then added with 0.5 mM dNTPs and 8 units of Klenow Polymerase <, s>> incubating at 30'C for 15 minutes.

After blocking the enzymatic reaction at 75 ° C for 10 minutes, the digestion mixture was loaded onto 0.6% low melting (Seaplaque) agarose gel and run at 70 volts for 4 hours.

Then the Sall-HindlII fragment of about 1500 base pairs (bp), comprising the structural gene encoding kanamycin resistance and the promoter region upstream of this gene, was eluted from the gel.

At the same time, the plasmid pSM774 (2 µg) was digested with the Styl enzyme and treated with Klenow Polymerase <®> under the same operating conditions as described above.

Then the digested pSM774 plasmid and the Sall-HindlII fragment were ligated in 20 µl of buffer ligase in the presence of 1 unit of T4 DNA ligase using a plasmid: fragment ratio of 1: 3. The ligase reaction was carried out at 23'C for about 16 hours.

The ligase mixture was then used to transform electrocompetent E. coli TG1 cells. The selection of transformants was carried out on plates of LB agarized medium containing 20 μg / ml of kanamycin and 100 pg / ml of ampicillin.

The analysis of the positive clones revealed the presence of a clone (# 56) with the expected insertion of the kanamycin gene in the phaC synthase gene. Restriction analysis and nucleotide sequence analysis revealed that the neo gene was inserted in the opposite orientation to the phaC gene.

Example 4

Clone # 56 was digested with restriction enzymes Asp718 and XbaI at 37 ° C for 1 hour. The suicide vector pWKR102A (Colonna-Romano, S. et al., 1990, Mol. Gen. Genet., 223, 138-147) was digested with the restriction enzyme HindlII. Then the digestion products were mixed and treated with Klenow Polymerase in the presence of dNTPs at 25'C for 30 minutes to create compatible end groups.

The sample was then extracted with phenol / chloroform and precipitated with ethanol. The pellet was resuspended in ligase buffer containing 5 DNA units / pg of T4 DNA ligase. The reaction was carried out at 23 ° C for 16 hours.

The ligase mixture was used to transform competent E. coli TG1 cells by electroporation. The selection of transformants was carried out on LB agar plates containing 20 ng / ml of chloramphenicol and 20 pg / ml of kanamycin. A clone containing a plasmid, designated pSM583 (Figure 3), with the correct insert was isolated from the positive clones.

The pSM583 plasmid was transferred to the E .coli S17-1 strain which possesses the genes necessary for conjugation, obtaining the strain called SMC490. Example 5

Conjugation of E.coli with R.sphaeroides RV The E.coli SMC490 strain was inoculated in 5 ml of LB medium containing 20 μg / ml of chloramphenicol and 20 ^ ig / ml of kanamycin and cultured at 37 ° C overnight.

The wild-type R.sphaeroides RV strain was made resistant to the antibiotic rifampicin (Rif) by selective pressure up to a maximum of 150 μg / ml of antibiotic.

Strain R. sphaeroides RV (Ref <R>) was: inoculated in 40 ml of aSy medium having the following composition:

under aerobic conditions in the dark for two days.

Then, 3 ml of the R.sphaeroides RV culture (Ref <R>) were mixed in Falcon <R> tubes with a volume of SMC490 corresponding to 1/10 of the optical density of the R.sohaeroides RV culture.

The bacteria were recovered by centrifugation at 3000 rpm, at room temperature for 10 minutes, resuspended in 100 μΐ of aSy medium and aliquots of this suspension were distributed on plates of the same agar medium.

The plates were incubated at 35'C for 6 hours in order to allow the exchange of genetic material between the two types of cells. Then, the cells were recovered from the plates, resuspended in 400 μΐ of aSy medium and aliquoted on plates of aSy medium containing rifampicin (150 μg / ml) and kanamycin (20 μg / ml).

After 6-7 days of growth in the dark under aerobic conditions, approximately 40-50 kanamycin resistant (Km <R>), rifampicin resistant (Ref <R>) colonies were obtained on each plate.

Approximately 200 of these colonies were collected and inoculated in three 96-well plates (Corning) in 200 μl of aSy medium containing kanamycin (20 μg / ml). The plates were incubated at 30 ° C in the dark under aerobic conditions.

After 2 days the colonies were collected, diluted with aSy medium supplemented with 5 μg / ml of gentamicin (10 μΐ of the cultures in 190 μΐ of fresh medium) and inoculated in three 96-well plates.

After 1 day of incubation at 30 ° C it was observed that out of 200 colonies, 50 had not grown. The phenotype of these latter colonies (Km <R> and Gm <s>) was consistent with the insertion of the interposon into the genome by double crossover and the consequent loss of the suicidal plasmid.

To confirm the site of mutagenesis, Southern Blot analysis experiments were conducted with genomic DNA isolated from 10 of these colonies.

The strain R.sohaeroides RV wild-type (Ref <R>) and the mutants were inoculated in 5 ml of LB containing 20 μg / ml of kanamycin and incubated at 30 ° C for 2 days under aerobic conditions in the dark.

Then the genomic DNA was extracted and digested with the EcoRI restriction enzyme, which does not cut into the synthase (phaC) and kanamycin (neo) genes.

The digestion products were analyzed by agarose gel electrophoresis. After the electrophoretic separation, a Southern blot was performed according to standard procedures and the membrane on which the DNA was fixed was subjected to hybridization with a specific probe for the phaC gene.

The development of autoradiography showed the presence, in mutant # 10, of a single band approximately 1.6 kb higher than that of the wild-type strain (Figure 4). This demonstrated the double crossover and therefore the presence on the genomic DNA of the phaC gene inactivated by interposon mutagenesis.

The other mutants, on the other hand, showed a justifiable profile with a single crossover that had generated a gene duplication on the genomic DNA, that is, the presence of a mutated gene next to the wild-type gene.

Mutant # 10 was indicated with the initials SMV071.

Example 6

Growth of the SMV071 mutant strain

The SMV071 mutant strain was grown in different media and its growth was compared to that of the wild-type strain.

The soils used had the following compositions:

The media were sterilized by filtration with 0.2μ Nalgene filters and any trace of oxygen was eliminated by bubbling Argon for 30 minutes.

Cultures were incubated in light with tungsten lamps at an intensity of 9,500 lux, at 30 ° C for 3 days in anaerobic tubes containing 24 ml of medium. At the end of the growth, the optical densities at 600 nm were measured. The results are reported in Table 1.

From the values reported in the Table, it is observed that the mutation in the ohaC gene negatively influenced the growth of the mutant strain only in medium B, where the only available carbon source is sodium acetate, characteristic of the induction medium for production. by PHA.

Example 7

Photoproduction of hydrogen

The photoproduction of hydrogen for the SMV071 mutant strain was evaluated with a fermentation experiment performed in parallel with the wild type strain R.sohaeroides RV.

The two strains were grown in two 1 liter chemostats equipped with a magnetic stirrer, a pH control module, 2 100 Watt halogen lamps placed inside to ensure the illumination of the crop and a cooling system for keep the temperature at 30 “C (figure 6).

The preinoculum for fermentation was prepared in two successive steps described below:

1. 50 μΐ of glycerin in 25 ml of aSy medium were incubated at 30 ° C, in aerobic / dark conditions, for two days;

2. A 1:10 dilution of the previous culture was used for subsequent inoculation into anaerobic tubes containing 60 ml of the same medium used for growth in the chemostat. The tubes were incubated at 30 ° C anaerobically in light (tungsten lamps, 9,500 lux) for 2 days.

The two chemostats were inoculated with a dilution of the second inoculum (2) calculating an initial optical density at 600 nm of 0.2.

The culture medium used derives from the controlled acidogenic fermentation of commercial waste (E. D'Addario et al. (1992), Wat .Sci.Tech. 22, 183-192). The average composition of this type of soil is as follows:

For the fermentation of the two strains, the medium was first diluted 1:10 with distilled water to obtain a lactic acid concentration of 3.6 g-1 <"1> and then added with the following components:

The anerobiosis was maintained with a continuous flow of argon. The intensity of light used was adjusted to 6,000 lux and the two fermentations were started in batches.

When the production of hydrogen started after about 24 hours, the fermentation was carried out continuously.

Since the flow rate (F) of the medium in the chemostat was set at a value of 31 ml / hour and the reactor volume (V) was 930 ml, according to the equation D = F / V, where D corresponds to dilution rate, the HRT value (hydraulic residence time) (1 / D) was equal to 30, ie theoretically every 30 hours the photobioreactor underwent a total change of soil.

Fermentation was followed by evaluating the following analytical parameters:

1. the optical density measured at 600 nm to follow bacterial growth;

2. the pH was automatically maintained at a value of 7.0 by adding 1 M NaOH;

3. the total biogas produced measured by means of a digital meter connected to one of the outputs of the chemostat;

4. the percentage of hydrogen contained in the biogas determined with a VARIAN 3400 gas chromatograph equipped with a Carbosieve II 80-100 mesh Supelco 200x3 mm column;

5. the quantity of polyhydroxybutyrates accumulated by the cell by gas-chromatographic analysis.

The intensity of the light was measured before inoculation on the external surface of the reactor using an LSI luxmeter.

The quantity of evolved hydrogen / liter of reactor / day (ml Hz / 1 reactor / day) was calculated from the values of the biogas produced and the percentage of hydrogen.

Table 2 below shows the total biogas values produced by the two strains during fermentation:

Table 3 shows the hydrogen production calculated on the basis of the total biogas values of Table 2 and the percentage of hydrogen measured by the gas-chromatograph and evaluated to be 80-90% for both strains.

Fermentation of the wild-type R.sohaeroides RV strain was stopped at day 17 when hydrogen production had reached insignificant levels. The hydrogen production of the SMV071 mutant strain remained high until the 18th day and continued for another 8 days (27th day), until the same minimum values of the wild-type strain were reached. Considering that the biogas produced contains about 90% hydrogen, the wild-type strain produced 16 liters of hydrogen in 16 days, while the mutant strain SMV071 produced 21 liters of hydrogen in 26 days, i.e. 26% more. compared to wild-type.

Gas chromatographic analysis of polyhydroxybutyrates (according to the method of Brandi et al., (1988), Appl. Environ. Microbiol. 54, 1977-1982) showed the absence of the characteristic peak of the ester in the SMV071 mutant strain methyl acid of β-hydroxybutyric acid, present instead in the wild-type strain (figure 5).

Claims (9)

CLAIMS 1. Mutant of Rhodobacter sohaeroides RV unable to accumulate polyhydroxyalkanoates deposited under CBS number 100467. 2. Continuous process for the photoproduction of hydrogen which includes: a) the cultivation of the mutant Rhodobacter sohaeroides RV CBS 100467 in an aqueous medium containing assimilable sources of carbon and nitrogen as well as various cations, anions, under anaerobic conditions, at a temperature between 25 ° C and 40 ° C, at a pH from 6.5 to 8.0, and under a light intensity of between 6,000 and 20,000 lux, b) separating and recovering evolved hydrogen. 3. The process of claim 2, where the assimilable sources of carbon are selected from organic acids such as pyruvate, malate, lactate, succinate or other conventional carbon sources. 4. The process of claim 2, wherein the nitrogen sources are selected from mineral ammonium salts, such as ammonium nitrate, ammonium sulfate, ammonium chloride or ammonium carbonate and urea or materials containing organic or inorganic nitrogen such as peptone, yeast extract or meat extract. 4. The process of claim 2, where the cations and anions are selected from potassium, sodium, magnesium, iron, calcium, acid phosphates, sulfates, chlorides, manganese and nitrates. The process of claim 2, where the fermentation medium is a material derived from the controlled acidogenic fermentation of commercial waste. The process according to claim 2, wherein the aqueous medium contains traces of vitamins or amino acids. 7. The process according to claim 2, where the luminous intensity is selected between 6,800 and 10,000 lux. 8. The process according to claim 2, where the pH is selected between 6.8 and 7.3. 9. The process according to claim 2, where the temperature is selected between 30 ° C and 37 ° C,