JP3169927B2 - Isolation of polyhydroxyalkanoic acids (PHAs) -deficient strains of Rhodobacter sphaeroides RV with improved hydrogen photoproduction yield - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to Rhodobacter sphaeroides RV CBS 100467, which cannot accumulate polyhydroxyalkanoates (PHAs).
(Rhodobacter sphaeroides RV CBS 100467) mutant,
And a method for producing hydrogen using the mutant strain.

[0002]

BACKGROUND OF THE INVENTION Hydrogen is the most abundant element in the sun and plays an important role in generating energy.
However, hydrogen on earth, along with oxygen, is a fundamental compound in the biological energy cycle.

[0003] The amount of hydrogen contained in the lowest layer of the atmosphere is estimated to be about 2.2 × 10 ⁹ tons which equals a concentration of about 0.55ppm in the volume of air. O ₂ is 1.1 × 1
In comparison with the 0 ¹⁵ of a ton, hydrogen in the earth can certainly regarded as rare gas. However, hydrogen is distinct from inert gases and is constantly produced and used by a wide range of microorganisms in both aerobic, aerobic and anaerobic conditions, in the presence or absence of light.

[0004] Hydrogen is certainly an ideal energy source from an energy point of view, since it has a higher heat generating capability than any other energy source. In addition, water, which is easy to store, does not involve environmental pollution, and is the end product of oxidation, is not only toxic, but also an essential compound for sustaining life on earth.

[0005] The ability of microorganisms to produce hydrogen, along with the ongoing depletion of conventional energy resources and the severe environmental problems associated therewith, has been a significant source of biological access to this energy source for many years. Has inspired his research efforts.

[0006] Numerous microorganisms capable of spontaneously producing hydrogen exist in the natural environment.

Among these organisms, non-oxygen generating photosynthetic bacteria, such as purple non-sulfur bacteria [particularly Rhodopseudomonas sp., Rhodobacterium]
ter) and Rhodospirillum] are particularly suitable for producing hydrogen. In fact, they not only use light, a zero-cost energy source, but also use waste material as a substrate for growth, so that the simultaneous disposal of waste and the production of energy, which are significant environmental problems, occur simultaneously. be able to.

[0008] Red non-sulfur bacteria However, these bacteria have drawbacks resulting from their low hydrogen production. As a result, as long as these microorganisms are used, it is difficult to develop a method for producing biological hydrogen, albeit interesting from both practical and economic viewpoints.

In fact, in the literature, red non-sulfur bacteria are:
It is known that it is also characterized by its ability to synthesize large amounts of accumulating substances [generally indicated by the term polyhydroxyalkanoate (PHA)] under unbalanced growth conditions. PHAs accumulate in the form of cytoplasmic inclusions and account for a significant proportion (up to 70%) of cell weight. In addition, strain R.
In Spheroides RV, between the accumulation of polyhydroxyalkanoates (PHAs) and the production of hydrogen light,
It has also been demonstrated that there is an antagonistic relationship depending on the growth conditions.

[0010] In fact, the generation of gaseous hydrogen and the synthesis of PHAs correspond to alternative metabolic pathways that consume the reducing power of cells.

[0011] The duration of the _{H 2} production, and therefore in terms of the total production of _{H 2,} R. In order to improve the hydrogen-producing ability of Spheroides RV, a mutant strain having no ability to accumulate polyhydroxyalkanoates (PHAs) was created.

This variant, called SMV071, was established on February 16, 1998, under the deposit number CBS100467, under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Proceedings, under the Central Bureau of Fungal Culture (CBD).
entraal Bureau voor Schimmelcultures).

[0013]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide polyhydroxyalkanoates (PHAs).
That do not have the ability to accumulate R. Spheroides RV
It relates to a variant of CBS 100467.

A further object of the present invention is to provide a mutant R. The present invention relates to a method for producing hydrogen using Spheroides RV CBS 100467.

[0015]

DETAILED DESCRIPTION OF THE INVENTION Most bacteria capable of accumulating PHAs, in particular R. cerevisiae. In Spheroides RV, poly (3-hydroxybutyrate) (PHB) represents the most widely produced polymer.

In the literature, Alcaligenes eutrophus, the most widely studied bacterium
The synthesis of PHB in N. eutrophus is achieved by a metabolic pathway that involves three different enzymes in succession: β-ketothiolate, acetoacetyl-CoA reductase, and synthase, which catalyzes the polymerization of monomers to give PHB polyester. Is known to be.

The present invention is based on the consideration that synthase is an important enzyme in the metabolic pathway. Indeed, the relatively low substrate specificity of this enzyme allows the use of various intermediates resulting from alternative metabolic pathways as substrates.

Therefore, we have found that the gene encoding this enzyme (phaC) is R. After confirming that the gene is present in the genome of Spheroides RV, the gene (phaC)
Was considered to eliminate the final step in the biological synthesis of PHB.

The gene can be inactivated by using conventional methods such as random mutagenesis (based on the use of transposons) or specific mutagenesis (using interposons). These components are inserted into the genome, causing disruption of the gene sequence (as revealed by means of phenotypic mutation) and conferring on the host resistance to certain antibiotics.

According to a preferred embodiment of the present invention, R.C. The inactivation of the spheroides RV wild-type synthase gene phaC was determined by synthesizing the previously isolated and characterized synthase gene (SEQ ID NOS: 1 and 2 with the first 30 bases and the last 60 bases without R). A gene cassette (interposon) encoding a kanamycin resistance gene inside the spheroides RV wild-type synthase gene phC (nucleotide sequence and amino acid sequence).
The mutants were then selected on medium supplemented with the antibiotic.

In fact, in theory, the genomic DNA of the strain
Only clones with an integrated interposon can grow on this selection medium.

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

The colonies obtained after the transformation were subjected to restriction analysis and the isolated positive clones were identified with the synthase gene pha.
It was confirmed that the kanamycin resistance gene inserted into C was included. According to the nucleotide sequence of this clone,
It was revealed that the interposon was inserted in the direction opposite to the transcription direction of the synthase gene phaC.

Next, the disrupted synthase gene was cloned into a suicide vector (ie, a vector that could not self-replicate in R. spheroides) and the final construct was conjugated to confer resistance to the antibiotic rifampicin. E. coli strain S17-1 and wild-type strain R. Introduced to Spheroides RV.

R. Selection of mutant strains of Spheroides RV was performed in two steps. The first step involves mutagenized copies of the gene phaC retained in the suicide vector by selecting with kanamycin;
The second step is to confirm successful recombination between the functional copy on the genomic DNA and that the selection with gentamicin results in the loss of the suicide vector used to induce the mutation. Proving that you have done it.

The clones were characterized by Southern blot analysis to confirm the nature and location of this mutagenesis. Extract genomic DNAs and use restriction enzyme Eco
It was digested with RI and hybridized with a specific synthetic oligonucleotide of the gene phaC. Wild-type genomic DNA
Was prepared under similar conditions and used as a negative control.

One of these clones showed a hybridized band of the expected size. This clone was called SMV071 and was characterized physiologically by growth on different media. This clone is similar to the wild type strain, for example, aSy (J. Miyake, XY Mao and S. Kawamura, J. Ferment. Techn.
ol. , 62: 531-535, 1984) Similar growth potential was obtained in complete media such as media and substrates derived from municipal solid waste, and no growth was seen in media that promoted PHB synthesis. . The results indirectly confirmed that they did not have the ability to activate this metabolic pathway. In addition, gas chromatography analysis performed to determine the amount of PHB production did not show any peak for clone SMV071, demonstrating that the mutant did not have the ability to accumulate PHB. Was done.

As a result, the mutant R. Spheroides RV is particularly suitable for the development of fermentation methods for producing hydrogen.

The fermentation can be carried out in an aqueous medium containing assimilable carbon and nitrogen sources and various cations, anions and any trace vitamins or amino acids.

Assimilable carbon sources include pyruvate, malate, lactate, succinate or other conventional carbon sources. The nitrogen source may be selected, for example, from inorganic ammonium salts such as ammonium nitrate, ammonium sulfate, ammonium chloride or ammonium carbonate and urea, or substances containing organic nitrogen or inorganic nitrogen such as peptone, yeast extract or meat extract. It is possible.

Examples of cations and anions can be selected from, but not limited to, potassium, sodium, magnesium, iron, calcium, acidic phosphoric acid, sulfates, chlorides, manganese and nitrates. Absent.

According to a preferred embodiment of the process according to the invention, substances derived from the controlled acid fermentation of municipal solid wastes are used as substrates (ED'Addario et al.
(1992), Wat. Sci. Tech. 27, 18
3-192), the composition of which is shown in Example 7.

The fermentation has a luminous intensity of 6,000 under constructional conditions guaranteed by a continuous flow of argon.
Irradiate light in the range of 0 to 20,000 lux at 25 ° C
To 40 ° C., preferably 30 ° C. to 37 ° C.

The pH of the fermentation medium is from 6.50 to 8.
0, preferably in the range of 6.8 to 7.3. Adjustment of the pH can be effected, for example, by the addition of a basic aqueous solution, such as an aqueous solution of ammonia, potassium hydroxide, sodium hydroxide, sodium carbonate or potassium carbonate. A 1-5M aqueous sodium hydroxide solution is preferably used.

During the course of the fermentation, hydrogen is recovered by conventional techniques.

The method can be carried out continuously in an apparatus of the type shown in FIG. 5 [where 1 = substrate tank; 2 = argon gas (maintain anaerobic conditions by inflow); 3 = Peristaltic pump (regulates media draw into photobioreactor); 4 = photobioreactor; 5 = magnetic plate (maintains agitation inside photoreactor); 6 = halogen lamp (located inside photobioreactor). 7) common outlet for spent medium, biomass and biogas; 8 = trap; 9
= Digital meter of released biogas; 10 = biogas release; 11 = peristaltic pump (to drain spent media and biomass); 12 = release and sterilization of media and biomass].

[0037] When operated in the preferred conditions, the light generation H ₂ by strain, compared to lasted 16 days in the wild type strain, since the sustained further 10 days long in mutants (total of 26 days) the amount of H ₂ produced by the mutant strain, it was observed that the resulting about 26% greater relative to the wild type strain.

The following examples are merely intended to describe the invention in more detail and should not be construed as limiting the scope in any way.

[0039]

EXAMPLE 1 Extraction of Genomic DNA from Spheroides RV 100 ml of LB medium (10 g / l tryptone, 5 g
/ L yeast extract, 10 g / l NaCl) in a 500 ml flask containing strain R. Spheroides R
V [National Institute of Bioscience and Human Technology (NIB)
H: obtained from Tsukuba, Japan), and the resulting mixture was maintained at 30 ° C. for 48 hours under aerobic / dark conditions with stirring (220 rpm).

Next, the culture broth was centrifuged (4 ° C., 500 ° C.) in an SS34 rotor model Sorvall RC-5B.
0 rpm for 10 minutes) to collect the cells,
Then, TE buffer (1 mM EDTA, 10 mM Tr
is-HCl, pH 7.4) (2 × 120 m
l). The obtained suspension was centrifuged again under the above conditions, and the cells were collected and suspended in 9.5 ml of TE buffer. 0.
After adding 5 ml of 10% SDS (sodium dodecyl sulfate) and proteinase K (20 mg / ml)
The suspension was incubated at 37 ° C. for 1 hour.

Next, 1.8 ml of 5M NaCl was added.
A solution containing 10% hexadecyltrimethylammonium bromide in 0.7M NaCl was added and the resulting solution was incubated at 65 ° C. for 20 minutes.

Subsequently, the solution was deproteinized with an equal volume of chloroform: isoamyl alcohol (24: 1, V / V), and DN was eluted with 0.6 volume of isopropanol.
A was precipitated.

The DNA was mixed with 1 ml of ethanol (70%)
And collected with a glass rod. Finally recovered DN
A was dissolved in 4 ml of TE and its concentration was determined spectrophotometrically at 260 nm.

The genomic DNA was centrifuged (55,000 rpm, 16 hours, Beckman V6) on a CsCl gradient (1%) containing 0.1 mg / ml ethidium bromide.
(5Ti rotor).

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

Example 2: Isolation of the gene synthase phaC Genomic DNA obtained as described in Example 1 was purified from Lung et al. (Leung DW, Chen E., Goedd).
el D. V. , Technique-a joura
nal of methods in cell an
d molecular biology, 1, (19
89): Amplified by the polymerase chain reaction (PCR) technique according to the instructions on pages 11-15) using the following set of oligonucleotides as primers: (1) 5 'CTGGACGCTC AGTTCGAGGC
G TCTGAAC 3 '(forward) (2) 5' CGGCGCGAGTC TCGGGATGT
T TCGAATC 3 '(Reverse) These oligonucleotides are based on the conserved amino acid sequence of bacterial synthase and are based on R.T. Spheroides
It was synthesized considering the selective use of codons in RV.

The amplification was carried out using a DNA thermal cycler 48.
0 device (Perkin-Elmer Cetus)
It was performed using a reaction mixture (100 [mu] l) containing the following ingredients: - 500 ng genomic DNA - 10nM Tris-HCl, pH8.3 - 2mM MgSO 4 - 25mM KCl - 5mM (NH 4) 2 SO 4 - 100 Picomolar 2 primers-200 μM dNTPs (dATP, dGTP, d
CTP, cTTP)-2.5 units of Pwo polymerase (Perki)
n Elmer).

After denaturation at 98 ° C. for 2 minutes, a cyclic program consisting of the following was started: 1 minute, 98 ° C. (denaturation) 1 minute, 60 ° C. (annealing) 2 minutes, 72 ° C. (extension) for a total of 25. Cycle, followed by 8 minutes at 72 ° C. (final extension).

The amplification product thus obtained was treated with phenol-chloroform (1: 1) to give NaCl (1
/ 10 vol / vol) and EtOH (2 vol) and resuspended in 20 μl H ₂ O. Next, about 17
The 00 bp fragment was purified on a 0.6% low melting point gel (Seaplaque, FMC Bioproducts), eluted from the gel and cloned into plasmid pUC18 (Boehringer) previously digested with the restriction enzyme SmaI.

In practice, 30 μl of the reaction mixture (DNA 5 μl / m
l), 2 μl of this mixture were used to transform electrocompetent E. coli TG1 cells (Dower WJ et al., Nucleic Acid Research, 16, 6127-6145, 1988). . The transformants were selected on culture dishes of LB agar medium containing 100 μg / ml ampicillin.

Plasmid DN extracted from positive clone
Analysis of A shows that the size is approximately 170, consistent with the expected size.
The presence of a 0 bp fragment was revealed. The plasmid contained in the positive clone is called pSM774 (FIG. 1).

Next, the plasmid was subjected to sequence analysis using a DNA sequencer ABI373 (Perkin-Elmer). The sequence consists of the first 30 bases and the last 6
The fragment was confirmed to be a 1738 bp fragment corresponding to the synthase gene phaC in which 0 base had been deleted (SEQ ID NO: 1).

Example 3 Inactivation of the Gene Synthase phaC Plasmid PSSUP2021 (R. Simon et al., 19) containing the gene (neo) encoding kanamycin resistance.
83, Bio / Technology, 1: 784-791)
(5 μg) with 5 units of restriction enzymes SalI and Hin
Digestion with dIII (Boehringer) at 37 ° C. for 1 hour. Next, 0.5 mM dNTPs and 8 units of Klenow polymerase (registered trademark) (Klenow Pol)
ymase) is added to the digestion mixture,
Incubated for 5 minutes.

The enzyme reaction was stopped at 75 ° C. for 10 minutes,
The digestion mixture was charged to a 0.6% low melting point agarose gel (Seaplaque) and run at 70 volts for 4 hours.

Subsequently, about 1500 base pairs (bp) of SalI-containing a structural gene encoding kanamycin resistance and a promoter region located upstream of this gene.
The HindIII fragment was eluted from the gel.

Under operating conditions equal to those described above,
Digestion with the enzyme StyI and treatment with Klenow polymerase (registered trademark) were performed in parallel on plasmid pSM774 (2 μg).

Subsequently, the digested plasmid pSM774
And the fragment SalI-HindIII were combined with 1 unit of T4 DNA using a plasmid: fragment = 1: 3 ratio.
Binding was performed in 20 μl ligase buffer containing ligase. The ligase reaction was performed at 23 ° C. for about 16 hours.

Next, E. coli TG1 electrocompetent cells were transformed using the ligase mixture. The transformants were selected on a culture plate of LB agar medium containing 20 μg / ml kanamycin and 100 μg / ml ampicillin.

Analysis of the positive clones revealed the presence of a clone (No. 56) in which the kanamycin gene was inserted into the synthase gene phaC as expected. Restriction analysis and nucleotide sequence analysis indicated that the gene neo was inserted in the opposite orientation with respect to the gene phaC.

Example 4: Clone No. 56 was replaced with the restriction enzymes Asp718 and Xb
Digested with aI at 37 ° C. Suicide vector pWKR1
02A (Colonna-Romano, S. et al., 19)
90, Mol. Gen. Genet. , 223,138
-147) was digested with the restriction enzyme HindIII. Next, the digestion products are mixed and treated with Klenow polymerase at 25 ° C. for 30 minutes in the presence of dNTPs,
Compatible ends were created.

Next, the sample was extracted with phenol / chloroform and precipitated with ethanol. The pellet,
Resuspended in ligase buffer containing 5 units / μg (DNA) of T4 DNA ligase. The reaction is
C. for 16 hours.

The ligase mixture was used to transform E. coli TG1 competent cells by electroporation. Transformants were selected on LB agar medium containing 20 μg / ml chloramphenicol and 20 μg / ml kanamycin. Among the positive clones, a plasmid having the correct insertion [pSM583
(FIG. 2) was isolated.

The plasmid pSM583 was transformed into E. coli strain S1.
7-1 (containing genes essential for conjugation)
A strain designated MC490 was obtained.

Example 5: E. coli and R. Spheroides
Conjugation of RV E. coli strain SMC490 was inoculated into 5 ml of LB medium containing 20 μg / ml chloramphenicol and 20 μg / ml kanamycin and cultured at 37 ° C. overnight.

The strain R. Spheroides RV wild type
Resistance to the antibiotic rifampicin (Rif) was conferred by applying selective pressure with the antibiotic up to 150 μg / ml.

The strain R. Spheroides RV (Rif
^R ) is inoculated into 40 ml of aSy medium having the following composition: (NH ₄ ) ₂ SO ₄ 1.25 g / l; sodium succinate 9.8 g / l; yeast extract 1 g / l; K ₂ H
_{PO 4 0.75g / l; KH 2} PO 4 0.85g /
_{l; EDTA 2mg / l; H} 3 BO 3 2.8mg /
_{l; Na 2 MoO 4 · 2H} 2 O 0.75mg / l; Z
nSO ₄ .7H ₂ O 0.24 mg / l; MnSO _{4.
4H 2 O 1.6mg / l; CuSO} 4 · 5H 2 O
0.041 mg / l; CaCl ₂ .2H ₂ O 0.75
_{mg / l; MgSO 4 · 7H} 2 O 0.2mg / l; F
_{eSO 4 · 7H 2 O 10mg /} l, pH7.0: 30
C. for 2 days in the dark under aerobic conditions.

3 ml of R. Spheroides RV (Ri
The culture of f ^R), in Falcon ^R tube, R. It was mixed with a volume of SMC490 corresponding to 1/10 of the optical density of the Spheroides RV culture.

The bacterium was 3,000 rpm at room temperature.
And resuspended in 100 μl of aSy medium, and aliquots of this suspension were distributed into similar agar culture dishes.

The culture dishes were incubated at 35 ° C. for 6 hours to allow exchange of genetic material between the two cells. The cells are collected from the culture dish and 400 μl of aSy
Resuspend in medium and rifampicin (150 μg / ml)
And kanamycin (20 μg / ml)
Dispensed into culture dishes with y medium.

6-7 in the dark under aerobic and aerobic conditions
After 40 days of culture, about 40-50 kanamycin resistance (K
m ^R ) colonies and rifampicin resistant (Rif ^R ) colonies were obtained.

Approximately 200 of these colonies were picked and 200 μl of aSy containing kanamycin (20 μl).
A 96-well plate (Corning) was inoculated in the medium. The plate was incubated at 30 ° C. in the dark under aerobic conditions.

After 2 days, colonies were picked and 5 μg / ml
ASy medium with gentamicin was added and diluted (10 μl of culture was added to 190 μl of fresh medium) and inoculated into three 96-well plates.

After one day of incubation at 30 ° C., it was observed that 50 out of 200 colonies did not grow. The phenotypes of the latter colonies (Km ^R and Gm ^R ) were consistent with the insertion of the interposon into the genome by double crossover and the resulting deletion of the suicide plasmid.

A test to confirm the site of mutagenesis was performed on genomic DNA isolated from these 10 colonies.
Was performed by Southern blot analysis.

The strain R. Spheroides RV wild type (R
if ^R ) and its mutants were inoculated into 5 ml LB containing 20 μg / ml kanamycin and incubated at 30 ° C. for 2 days in the dark under aerobic conditions.

Next, the genomic DNA was extracted and digested with a restriction enzyme EcoRI. This enzyme does not cleave the synthase gene (phaC) and kanamycin (neo).

The digestion products were analyzed by electrophoresis on an agarose gel. After separation by electrophoresis, Southern blot was performed by a standard method, and hybridized with a specific probe of the gene phaC using a membrane on which DNA was immobilized.

Developing the autoradiography revealed the presence of a single band of about 1.6 kb (greater than that found in the wild type strain) in mutant number 10 (FIG. 3). This causes a double crossover, which results in the gene pha present on the genomic DNA.
It was demonstrated that C was inactivated by interposon mutagenesis.

On the other hand, other mutants showed a profile (ie, the mutated gene is next to the wild-type gene) that could be attributed to a single crossover resulting in gene duplication in genomic DNA.

Mutant number 10 is indicated by the abbreviation SMV071.

Example 6: Growth of mutant strain SMV071 Mutant strain SMV071 was cultured in various media and its growth was compared to that of a wild-type strain.

The medium used had the following composition: (A): (NH₄)₂SO₄ 1.25 g / l; succinic acid
Na 9.8 g / l; yeast extract 1 g / l; K₂HP
O₄ 0.75 g / l; KH₂PO₄ 0.85g /
l; EDTA 2mg / L; H₃BO₃ 2.8mg /
l; Na₂MoO ₄・ 2H₂O 0.75 mg / l; Z
nSO₄・ 7H₂O 0.24 mg / l; MnSO₄・
4H₂O 1.6 mg / l; CuSO₄・ 5H₂O
0.041 mg / l; CaCl₂・ 2H₂O 0.75
mg / l; MgSO₄・ 7H₂O 0.2 mg / l; F
eSO₄・ 7H₂O 10 mg / l.

(B): NH as a nitrogen source₄Cl 0.2
g / l; sodium acetate 50 mM as a carbon source; K₂
HPO₄ 0.75 g / l; KH₂PO₄ 0.85g
/ L; EDTA 2mg / l; H₃BO₃ 2.8mg
/ L; Na₂MoO₄・ 2H ₂O 0.75 mg / l;
ZnSO₄・ 7H₂O 0.24 mg / l; MnSO ₄
・ H₂O 1.6 mg / l; CuSO₄・ 5H₂O
0.041 mg / l; CaCl₂・ 2H₂O 0.75
mg / l; MgSO₄・ 7H₂O 0.2 mg / l; F
eSO₄・ 7H₂O 10 mg / l.

(C): Same as B, but further using Na succinate (9.8 g / l) as a carbon source.

(D): 98% Na lactate as a carbon source
7.4 ml; Na-L-glutamic acid as a nitrogen source
1.88 g / l; HaHCO ₃ 1.5 g / l; K ₂ H
_{PO 4 0.75g / l; KH 2} PO 4 0.85g /
1; EDTA 2 mg / L; H ₃ BO ₃ 2.8 mg / L
_{l; NaMoO 4 · 2H 2 O} 0.75mg / l; Zn
_{SO 4 · 7H 2 O 0.24mg /} l; MnSO 4 · 4
_{H 2 O 1.6mg / l; CuSO} 4 · 5H 2 O 0.
041 mg / l; CaCl ₂ .2H ₂ O 0.75 mg
/ L; MgSO ₄ .7H ₂ O 0.2 mg / l; FeS
_{O 4 · 7H 2 O 10mg /} l.

The medium was sterilized by filtration through a 0.2 μ Nalgene filter, removing small amounts of total oxygen to 3
Argon was removed by aeration for 0 min.

The culture was incubated in a test tube under anaerobic conditions containing 24 ml of medium at 30 ° C. for 3 days under light irradiation with a tungsten lamp of 9,500 lux intensity. At the end of the growth, the optical density was measured at 600 nm. The results are shown in Table 1.

[0088]

[Table 1]

From the values shown in Table 1, it can be seen that the mutation in the gene phaC was only found in medium B (the only available carbon source was sodium acetate, which is characteristic of a medium that induces PHA production). It was observed that the growth of the mutant strain was negatively affected.

Example 7: Photoproduction of hydrogen The photoproduction of hydrogen by the mutant strain SMV071 was determined using the wild-type strain R.V. Evaluated by fermentation tests performed in parallel with Spheroides RV.

The two strains were subjected to a magnetic stirrer, a module for adjusting the pH, two 100 watt halogen lamps installed inside to assure the illuminance of the culture, and a cooling to maintain the temperature at 30 ° C. The culture was performed in two 1-liter chemostats equipped with the system (FIG. 5).

The pre-inoculation for the fermentation was prepared by two sequential methods described below: 1.25 ml of glycerate in 1.25 ml of aSy medium was added at 30 ° C., aerobic / dark for 2 hours. 1. days incubation; A second inoculation was made in an anaerobic environment in a test tube containing 60 ml of the same medium used for growth in the chemostat, using a 1:10 dilution of the previous culture. The tubes were incubated in an anaerobic environment at 30 ° C. in the presence of light (tungsten lamp, 9,500 lux) for 2 days.

Two chemostats were inoculated with a dilution of the second inoculum (2), whose initial optical density was calculated to be 0.2 at 600 nm.

For the culture medium, a source of regulated acid-generating fermentation of municipal solid waste was used (ED'Ad).
dario et al. (1992), Wat. Sci. Tec
h. 27, 183-192). The average composition of this type of medium is as follows: total solids 3.7% volatile solids 72.0% lactic acid 36 g / l acetic acid 2.2 g / l total nitrogen 594 mg / l ammonia nitrogen 260 mg / l Total carbon 15.80 g / l TOC (total organic carbon) 14.90 g / l COD (chemical oxygen demand) 5.90 g / l Calcium 5.90 g / l Sodium 0.25 g / l Sulfate 0.25 g / 1 Phosphate 23.00 mg / l The medium for fermentation of the two strains was first diluted 1:10 with distilled water to obtain a lactic acid concentration of 3.6 g · l ⁻¹ and then: The following components were added: KH ₂ PO ₄ 0.4
_{g / l; H 3 BO 3} 2.8mg / l; Na 2 MoO 4
_{· 2H 2 O 0.75mg / l;} ZnSO 4 · 7H 2 O
_{0.24mg / l; MnSO 4 · 4H} 2 O 2.1m
g / l; Cu (NO ₃ ) ₂ .3H ₂ O 0.04 mg /
l; CaCl ₂ .2H ₂ O 0.75 mg / l; MgS
_{O 4 · 7H 2 O 0.1mg /} l; FeSO 4 · 7H 2
O 10 mg / l, pH 7.0.

The anaerobic environment was maintained by a continuous flow of argon. The light intensity used was adjusted to 6,000 lux and two fermentations were started in batch.

When hydrogen generation starts after about 24 hours,
The fermentation was continued.

The flow rate (F) of the culture medium in the chemostat was 31
ml (h) when fixed at ml / h
Is 930 ml, which corresponds to the equation D = F / V [where D corresponds to the dilution rate and the HRT value (hydraulic duration)
(1 / D) is equal to 30. That is, theoretically every 30 hours, the photobiological reactor will undergo a total change of culture medium.] Following the fermentation, the following analytical parameters will be evaluated: 1. Measure the optical density at 600 nm to reveal bacterial growth; 2. The pH was automatically maintained at a value of 7.0 by the addition of 1 M NaOH; 3. The total biogas produced was measured by means of a digital meter connected to one of the outlets of the chemostat; 4. The percentage of hydrogen contained in the biogas was determined by VARIAN 3400 gas chromatography equipped with a Carbosieve II column 80-100 mesh Supelco 200 × 3 mm; The amount of polyhydroxybutyrate accumulated by the cells was quantified by gas chromatography analysis.

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

Amount of hydrogen released / volume of reactor (liter) / days [ml H ₂ / l reactor / day]
Was calculated using the volume of biogas produced and the percentage of hydrogen.

Table 2 below shows the amount of total biogas produced by the two strains during fermentation.

[0101]

[Table 2]

Table 3 shows the amount of hydrogen production calculated based on the total amount of biogas in Table 2 and the percentage of hydrogen measured by gas chromatography.
Both strains were evaluated to be 80-90%.

[0103]

[Table 3]

If the production of hydrogen has not reached significant levels, the strain R. Fermentation of Spheroides RV wild type was stopped on day 17. Hydrogen production of the mutant strain SMV071 remained high until day 18 and was continued for 8 days (day 27) before reaching a minimum equivalent to the wild-type strain. Since the biogas produced contains about 90% hydrogen, the wild-type strain produces 16 liters of hydrogen in 16 days, whereas the mutant SMV071 produces 21 liters of hydrogen in 26 days. (Ie, 26% more than wild type).

Gas Chromatographic Analysis of Polyhydroxybutyrate (Brandl et al., 1988, A
ppl. Eviron. Microbiol. 54, 1
977-1982) according to the mutant strain SM
V071 revealed no peak characterizing β-hydroxybutyric acid methyl ester, whereas the wild-type strain did (FIG. 4). [Here, the respective analysis charts in FIG. 4 are as shown below; A: a standard product of β-hydroxybutyric acid methyl ester (peak at RT = 10.28);
B: R. after 14 days of fermentation. Extract from cell culture of Spheroides RV wild-type (peak at RT = 10.96); C: Extract from cell culture of mutant SMV071 after 15 days of fermentation (R indicating β-hydroxybutyrate)
There is no peak at T). ]

[0106]

[Sequence List] SEQUENCE LISTING <110> Eni Technologie <120> Mutant of Rhodobacter Sphaeroides RV and its Use in the Production of Hydrogen <130> B0098P0669 <140> <141> <150> IT / MI98A000810 <151> 1998-04- 17 <160> 4 <170> PatentIn Ver. 2.0 <210> 1 <211> 1738 <212> DNA <213> Rhodobacter sphaeroides <400> 1 ggccgtgacg ctcagttcga gcgtctgaac gcgaatctca cccgcatcga cgagctgtcg 60 aaacggctga cggccgccgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgcgccgcgcgcgcgcgcgcgcgcgcgcgccgcgcgccgcgccgccgccgcgccgcgcgcgcgcgcgcggcgcgcggcggcgcggcggcgcggcgcggcgcgcggcggg.com. gcctacatgg ccgagatgat gcagaacccg 180 gccaagatcc tcgagcatca gatcagtttc tggggcaaga gcctgaaaca ttacgttgag 240 gcgcagcatc agctggtgaa gggcgagctg aagccgccgc cggacgtgac gccgaaggac 300 cgccgcttct cgaacccgct ctggcagacg catccgttct tcaactatct caagcagcag 360 tatctgatga acgccgaggc ggttaatcag gccgtcgagg cgctggagca tatcgagcct 420 tccgacaaga agcgggtcga gtatttctcg cgccagatcg tcgatctttt ctcgcccacc 480 aacttcttcg gcaccaatcc cgacgcgctc gagcgcgcca ttgccaccga tggcgagagc 540 ctcgtgcagg ggctggagaa tctcgtgcgc gacatcgagg ccaacaaggg cgatctgct c 600 gtcacgctgg ccgatcccga ggccttccag gtgggccaga acctcgccac caccgaaggg 660 tcggtcgtct accgcaaccg catgttcgag ctgatccagt acaagcccac gaccgagacg 720 gtccacgaga cgccgctcct gatctttccg ccctggatca acaagttcta cattctcgat 780 ctcaagccgc agaactccct gctgaagtgg ctggtggatc agggcttcac ggtcttcgtc 840 gtctcctggg tgaaccccga caagagctat gccggcatcg gcatggacga ctacatccgc 900 gatggctaca tgcgcgccat ggccgaggtg cgcgcgatca cccggcagaa gcagatcaac 960 gcggtgggct actgcatcgc gggcaccacg ctgacgctga cgctggccca cctgcagaag 1020 gcgggcgatc cgtccgtccg ctcggccacc ttcttcacca cgctcaccga cttctcggat 1080 ccgggcgagg tgggggtgtt cctcaacgac gatttcgtcg acggtatcga gcggcaggtg 1140 gcggtggacg ggatcctcga caagaccttc atgtcgcgca ccttcagcta cctgcgctcg 1200 aacgacctga tctatcagcc cgccatcaag agctacatga tgggagaggc gccccccgcc 1260 ttcgatctgc tctactggaa cggagacggc accaacctgc cggcgcagat ggcggtcgaa 1320 tatctgcgcg gcctctgcca gcaggaccgg ctggcgggcg gcaccttccc ggtgctgggc 1380 tcgcccgtgg gcctgaagga tgtgacgctg ccggtctgcg ccatcgcctg cgagaccgac 1440 catatc gcgc cgtggaaaag cagcttcaac ggcttccgcc agttcggctc gaccgacaag 1500 accttcattc tttcggaatc gggccatgtg gcgggcatcg tgaacccgcc cagccgcaac 1560 aaatacggcc attacaccaa cgagggtcct ctcgacacac ccgccgcgtt ccgcgagggg 1620 gccgagttcc acgcaggctc ctggtggccg cgctggggcg cctggctggg cgagcggtcg 1680 ggcaagcagg tcccggcgcg ccagccgggc gattcgaaac atcccgaggt cgcgccgg 1738 <210> 2 <211> 579 <212> PRT <213> Rhodobacter sphaeroides <400> 2 Gly Arg Asp Ala Gln Phe Glu Arg Leu Asn Ala Asn Leu Thr Arg Ile 1 5 10 15 Asp Glu Leu Ser Lys Arg Leu Thr Ala Ala Leu Thr Lys Arg Lys Leu 20 25 30 Ser Asp Pro Ala Leu His Gly Pro Ser Ser Asp Val Phe Leu Lys Ala 35 40 45 Met Thr Ala Tyr Met Ala Glu Met Met Gln Asn Pro Ala Lys Ile Leu 50 55 60 Glu His Gln Ile Ser Phe Trp Gly Lys Ser Leu Lys His Tyr Val Glu 65 70 75 80 Ala Gln His Gln Leu Val Lys Gly Glu Leu Lys Pro Pro Pro Asp Val 85 90 95 Thr Pro Lys Asp Arg Arg Phe Ser Asn Pro Leu Trp Gln Thr His Pro 100 105 110 Phe Phe Asn Tyr Leu Lys Gln Gln Tyr Leu Met Asn Ala Glu Ala Val 115 120 125 Asn Gln Ala Val Glu Ala Leu Glu His Ile Glu Pro Ser Asp Lys Lys 130 135 140 Arg Val Glu Tyr Phe Ser Arg Gln Ile Val Asp Leu Phe Ser Pro Thr 145 150 155 160 Asn Phe Phe Gly Thr Asn Pro Asp Ala Leu Glu Arg Ala Ile Ala Thr 165 170 175 Asp Gly Glu Ser Leu Val Gln Gly Leu Glu Asn Leu Val Arg Asp Ile 180 185 190 Glu Ala Asn Lys Gly Asp Leu Leu Val Thr Leu Ala Asp Pro Glu Ala 195 200 205 Phe Gln Val Gly Gln Asn Leu Ala Thr Thr Glu Gly Ser Val Val Tyr 210 215 220 Arg Asn Arg Met Phe Glu Leu Ile Gln Tyr Lys Pro Thr Thr Glu Thr 225 230 235 240 Val His Glu Thr Pro Leu Leu Ile Phe Pro Pro Trp Ile Asn Lys Phe 245 250 255 Tyr Ile Leu Asp Leu Lys Pro Gln Asn Ser Leu Leu Lys Trp Leu Val 260 265 270 Asp Gln Gly Phe Thr Val Phe Val Val Ser Trp Val Asn Pro Asp Lys 275 280 285 Ser Tyr Ala Gly Ile Gly Met Asp Asp Tyr Ile Arg Asp Gly Tyr Met 290 295 300 Arg Ala Met Ala Glu Val Arg Ala Ile Thr Arg Gln Lys Gln Ile Asn 305 310 315 320 Ala Val Gly Tyr Cys Ile Ala Gly Thr Thr Leu Thr Leu Thr Leu Ala 325330 335 His Leu Gln Lys Ala Gly Asp Pro Ser Val Arg Ser Ala Thr Phe Phe 340 345 350 Thr Thr Leu Thr Asp Phe Ser Asp Pro Gly Glu Val Gly Val Phe Leu 355 360 365 Asn Asp Asp Phe Val Asp Gly Ile Glu Arg Gln Val Ala Val Asp Gly 370 375 380 Ile Leu Asp Lys Thr Phe Met Ser Arg Thr Phe Ser Tyr Leu Arg Ser 385 390 395 400 Asn Asp Leu Ile Tyr Gln Pro Ala Ile Lys Ser Tyr Met Met Gly Glu 405 410 415 Ala Pro Pro Ala Phe Asp Leu Leu Tyr Trp Asn Gly Asp Gly Thr Asn 420 425 430 Leu Pro Ala Gln Met Ala Val Glu Tyr Leu Arg Gly Leu Cys Gln Gln 435 440 445 Asp Arg Leu Ala Gly Gly Thr Phe Pro Val Leu Gly Ser Pro Val Gly 450 455 460 Leu Lys Asp Val Thr Leu Pro Val Cys Ala Ile Ala Cys Glu Thr Asp 465 470 475 480 His Ile Ala Pro Trp Lys Ser Ser Phe Asn Gly Phe Arg Gln Phe Gly 485 490 490 495 Ser Thr Asp Lys Thr Phe Ile Leu Ser Glu Ser Gly His Val Ala Gly 500 505 510 Ile Val Asn Pro Pro Ser Arg Asn Lys Tyr Gly His Tyr Thr Asn Glu 515 520 525 Gly Pro Leu Asp Thr Pro Ala Ala Phe Arg Glu Gly Ala Glu Phe His 530 535 540 Ala Gly Ser Trp Trp Pro Arg Trp Gly Ala Trp Leu Ala Glu Arg Ser 545 550 555 560 Gly Lys Gln Val Pro Ala Arg Gln Pro Gly Asp Ser Lys His Pro Glu 565 570 575 Leu Ala Pro <210> 3 <211> 27 <212> DNA <213> Rhodobacter sphaeroides <400> 3 cgtgacgctc agttcgagcg tctgaac 27 <210> 4 <211> 27 <212> DNA <213> Rhodobacter sphaeroides <400> 4 cggcgcgagc tcgggatgtt tcgaatc 27

[Brief description of the drawings]

FIG. 1. Restriction map of plasmid pSM774.

FIG. 2. Restriction map of plasmid pSM583.

FIG. 3. Digestion with restriction enzyme EcoRI, gene pha
Mutant Clone No. 1-10 (line 1-10) hybridized with the specific probe of C.
Southern blot analysis of genomic DNA of Spheroides RV.

FIG. Gas chromatography of Spheroides RV wild type and mutant SMV071 (P
The presence or absence of a peak indicating HB is shown).

FIG. 5 is a scheme diagram of a bioreactor.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI (C12N 1/21 C12R 1:01) (C12N 15/09 ZNA C12R 1:01) (C12P 3/00 C12R 1:01) ( 72) Inventor Elisabetta Franchi 2-8-11 Nishi-Shimbashi, Minato-ku, Tokyo 7th Oriental Maritime Building 8th Floor Global Environment Institute of Industrial Science and Technology (CO2) Project Room, etc. (72) Inventor Claudio Toge Tokyo 2-8-11 Nishi-Shimbashi, Minato-ku, Tokyo 7th Toyo Maritime Building 8th Floor, Institute for Global Environment, National Institute of Advanced Industrial Science and Technology (CO2) Project Room (72) Inventor Giuseppe Scolla 2--8 Nishi-Shimbashi, Minato-ku, Tokyo No.11 7th Oriental Maritime Building 8th Floor Global Environment Institute of Advanced Industrial Science and Technology Project Chamber (72) Inventor Francesco Rodriguez 2-8-11 Nishi-Shimbashi, Minato-ku, Tokyo 7th Oriental Maritime Building 8F Global Environment Industrial Technology Research Organization CO2 Fixation etc. Project Chamber (72) Inventor Paola・ Pedroni 8th floor, 7-8 Toyo Maritime Building, Nishi-Shimbashi, Minato-ku, Tokyo 7th floor of the Project Room, etc. (58) Field surveyed (Int. Cl. ⁷ , DB) Name) C12N 1/20-1/21 BIOSIS (DIALOG) WPI (DIALOG)

Claims (9)

(57) [Claims] Claims: 1. Deposited under the number CBS100477,
A mutant strain of Rhodobacter spheroides RV that cannot accumulate polyhydroxyalkanoates. 2. A continuous method of hydrogen photoproduction, comprising: a) 25 ° C. under anaerobic conditions in an aqueous medium containing assimilable nutrients of carbon and nitrogen and various cations and anions. From 40 to 40 ° C., pH from 6.5 to 8.0, and from 6,000 lux to 20,
In the presence of luminosity in the range of 000 lux, the mutant Rhodobacter sphaeroides RV CBS 1004
Culturing 67; and b) isolating and recovering the produced hydrogen. 3. The method of claim 2, wherein said assimilable carbon source is selected from the group consisting of pyruvate, malate, lactate, and succinate. 4. The method of claim 2, wherein said nitrogen source is ammonium nitrate, ammonium sulfate, ammonium chloride and ammonium carbonate, urea, peptone,
A method selected from a yeast extract and a meat extract. 5. The method according to claim 2, wherein said cations and anions are selected from potassium, sodium, magnesium, iron, calcium, acid phosphates, sulfates, chlorides, manganese and nitrates. How to be. 6. The method of claim 2, wherein the fermentation medium is a substance derived from a controlled acid-producing fermentation of a municipal solid waste. 7. A method according to claim 2, wherein said aqueous medium containing vitamins or amino acids. 8. The method of claim 2, wherein said p
A method of selecting H from 6.8 to 7.3. 9. The method according to claim 2, wherein said temperature is selected between 30 ° C. and 37 ° C.