CN100413959C

CN100413959C - Method of separating sulfobacillus thermosulfidooxidans

Info

Publication number: CN100413959C
Application number: CNB2006100323376A
Authority: CN
Inventors: 邱冠周; 丁建南; 张成桂; 吴学玲; 康健; 高健
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2006-09-28
Filing date: 2006-09-28
Publication date: 2008-08-27
Anticipated expiration: 2026-09-28
Also published as: CN101012441A

Abstract

The invention discloses a separating method of Sulfobacillus thermosulfidooxidans, which comprises the following steps: predisposing samploe at 70-80 deg.c; switching in culture medium 1; stewing at 50-55 deg.c and 55-60 deg.c; transmitting into culture medium; vibrating continuously to enrich at 50-55 deg.c for three times; obtaining rich didymium 3; vibrating continuously for the forth time under the same temperature; diluting; separating; obtaining diluted material 4 as purified S.thermosulfidooxidans strain.

Description

A kind of method of separating sulfobacillus thermosulfidooxidans

Technical field:

The present invention relates to a kind of method of from environmental sample, separating the moderate sulfobacillus thermosulfidooxidans.

Background technology:

Moderate have a liking for temperature soak the ore deposit bacterium be a class optimum growth temperature at 45-60 ℃, optimum pH 1.3-2.0 can the oxidation elementary sulfur and/or ferrous bacterium or Archimycetes.Wherein the most typical, and soak the ore deposit effect best be sulfobacillus thermosulfidooxidans (Sulfobacillus thermosulfidooxidans), since this bacterium can iron protoxide again can the oxidation elementary sulfur, and happiness high temperature (optimum growth temperature 50-55 ℃), so it soaks ore deposit efficient far above mesophilic bacteria commonly used, as thiobacillus ferrooxidant (Acidithiobacillusferrooxidans, 25-35 ℃) and iron protoxide hook end spirobacteria (Leptospirillumferrooxidans, 35-40 ℃).But this bacteria growing environment is special, and separating difficulty is very big.

S.thermosulfidooxidans is a kind of chemoautotrophy or facultative heterotrophic bacteria, and with the spore adaptation adverse environment of dormant state, yeast extract can promote its growth.Commensalism also has optimum growth temperature same or similar with it, does not possess product spore that soaks the ore deposit function or the heterotrophic bacterium that does not produce spore.We once attempted using conventional flat band method to separate S.thermosulfidooxidans, but find to solidify agar very easily hydrolysis liquefaction under sour environment below the pH1.6 and the hot conditions more than 50 ℃, and under the uniform temp condition, when pH1.6 was above, the bacterial strain that is separated to flat band method all was not S.thermosulfidooxidans.

Use conventional isolation by dilution method instead, sample is placed under the optimum temperuture, directly containing ferrous or elementary sulfur, and be added with when carrying out enrichment in the perfect medium of yeast extract, because the spore of heterotrophic bacterium or the sprouting of nourishing body and the speed of growth are far away faster than soaking the ore deposit bacterium, so the dominant bacteria that enrichment is come out is not have the heterotrophic bacterium of soaking the ore deposit effect, the S.thermosulfidooxidans that tool soaks the ore deposit function is then suppressed by heterotrophic bacterium, thereby finally can not be separated to S.thermosulfidooxidans from sample.

Summary of the invention:

For from environmental sample, filtering out sulfobacillus thermosulfidooxidans (S.thermosulfidooxidans), leaching for the microorganism of sulphide ores provides high-efficiency strain, invent a kind of method of separating sulfobacillus thermosulfidooxidans, used this method successfully to be separated to S.thermosulfidooxidans.

A kind of method of separating sulfobacillus thermosulfidooxidans, this method is at first with in an amount of sample transfer to the sterilized triangular flask, this triangular flask is left standstill in the water-bath, progressively be warmed up to 70～80 ℃, kept this temperature 50～72 hours, this sample is added in the substratum 1 of pH1.5～1.8 by 6% inoculum size then, inoculum moves into 50～55 ℃ of water-baths, leaves standstill 24～48 hours, forwards 55～60 ℃ of water-baths again to, continued to leave standstill 12～24 hours, in through the inoculum that leaves standstill processing continuously, add various nutritive substances by the prescription of substratum 2, keep pH1.5～1.8,50～55 ℃ shake cultivation 150～168 hours down, obtain enriched substance 1; Enriched substance 1 by 2% inoculum size, is transferred in the substratum 2, and concussion was under the same conditions cultivated about 100～120 hours, and this is an enriched substance 2; By same procedure, enrichment is about 90 hours once more, obtains enriched substance 3; Enriched substance 3 is pressed 10 ^-8, 10 ^-9, 10 ^-10Extent of dilution inserts respectively in the substratum 3, shakes dilution and separate under 50～55 ℃, and with potassium dichromate process titration ferrous content, direct-counting method living cell counting number, when the ferrous oxidation rate reaches more than 90%, the bacterium number reaches 1 * 10 ⁷During/ml, stop to cultivate, select the highest culture of extension rate as dilution 1; Dilution separates four times so repeatedly, and obtaining dilution 4 at last is the S.thermosulfidooxidans bacterial strain of purifying.

Described substratum 1 is: (NH ₄) ₂SO ₄0.1g/l, MgSO ₄7H ₂O 0.3g/l, KH ₂PO ₄0.1g/l, KCl 0.05g/l, FeSO ₄7H ₂O 10mM, pH1.5～1.8;

Described substratum 2 is: (NH ₄) ₂SO ₄0.4g/l, MgSO ₄7H ₂O 0.5g/l, K ₂HPO ₄0.2g/l, KCl 0.1g/l, FeSO ₄7H ₂O 50mM, yeast extract (yeast extract) 0.1g/l, pH1.5～1.8;

Described substratum 3 is: (NH ₄) ₂SO ₄1.0g/l, MgSO ₄7H ₂O 1.0g/l, K ₂HPO ₄0.2g/l, FeSO ₄7H ₂O 100mM, yeast extract (yeast extract) 0.2g/l, ZnSO ₄7H ₂O ₃100mg/l, CoSO ₄7H ₄O 30mg/l, CuSO ₄5H ₂O 300mg/l, MnSO ₄4H ₂O 60mg/l, pH1.5～1.8.

The present invention had both broken through the restriction of plate isolation method, had overcome general dilution method again effectively and had been difficult to separate moderate and has a liking for the difficult point that temperature is soaked the ore deposit bacterium, for an approach has fast and effectively been found in the separation and purification of this type of bacterium.This method mainly is applicable to the gram-positive microorganism that can produce gemma, and emphasis is at sulfobacillus thermosulfidooxidans (S.thermosulfidooxidans).

Description of drawings:

Fig. 1: process flow diagram of the present invention;

Fig. 2. with the sulfobacillus thermosulfidooxidans (Sulfobacillusthermosulfidooxidans) of separation and purification of the present invention;

Fig. 3 .S.thermosulfidooxidans and A.ferrooxidans are to the leaching of chalcopyrite.

Embodiment:

Get pH4.5, the about 60 ℃ hot spring water sample 20ml of temperature is added in the aseptic triangular flask of 50ml, this triangular flask is left standstill in the water-bath, progressively be warmed up to 80 ℃, kept this temperature 72 hours, this sample is added in the substratum 1 of pH1.5 by 6% inoculum size then, inoculum moves into 50 ℃ of water-baths, leaves standstill 48 hours, forward 55 ℃ of water-baths again to, continued to leave standstill 24 hours.In through the inoculum that leaves standstill processing continuously, add various nutritive substances by the prescription of substratum 2, keep pH1.5,55 ℃ shake cultivation 168 hours down, obtain enriched substance 1.Enriched substance 1 by 2% inoculum size, is transferred in the substratum 2, and concussion was under the same conditions cultivated about 120 hours, and this is an enriched substance 2.By same procedure, enrichment is about 90 hours once more, obtains enriched substance 3.Enriched substance 3 is pressed 10 ^-9Extent of dilution inserts in the substratum 3, shakes dilution and separate under 55 ℃, and with potassium dichromate process titration ferrous content, direct-counting method living cell counting number, when the ferrous oxidation rate reaches more than 90%, the bacterium number reaches 1 * 10 ⁷During/ml, stop to cultivate, this is a dilution 1.Dilution separates four times so repeatedly, obtains dilution 4 at last, i.e. the S.thermosulfidooxidans bacterial strain of purifying.This bacterial strain is identified through morphocytology, molecular biology and physiological and biochemical index, is sulfobacillus thermosulfidooxidans (S.thermosulfidooxidans) that its 16SrDNA sequence accession number is the DQ650351 (see figure 2).

The chalcopyrite contrast of carrying out with the sulfobacillus thermosulfidooxidans (S.thermosulfidooxidans) of this method separation and purification and thiobacillus ferrooxidant (Acidithiobacillus ferrooxidans) is soaked the ore deposit test and is shown, under optimum condition separately, as inoculum size 15% (v/v), during pulp density 3% (w/v), the former reaches 43.3% at the leaching yield of copper in the time of the 26th day, is 1.47 times of the latter.(see figure 3).

Claims

1. method of separating sulfobacillus thermosulfidooxidans, it is characterized in that: at first sample is left standstill in the water-bath, progressively be warmed up to 70～80 ℃, kept this temperature 50～72 hours, this sample is added in the substratum 1 of pH 1.5～1.8 by 6% inoculum size then, inoculum moves into 50～55 ℃ of water-baths, left standstill 24～48 hours, forward 55～60 ℃ of water-baths again to, continued to leave standstill 12～24 hours, in through the inoculum that leaves standstill processing continuously, add various nutritive substances by the prescription of substratum 2, keep pH to shake down for 1.5～1.8,50～55 ℃ and cultivated 150～168 hours, obtain enriched substance 1; Enriched substance 1 by 2% inoculum size, is transferred in the substratum 2, and concussion was under the same conditions cultivated 100～120 hours, and this is an enriched substance 2; By same procedure, enrichment is 90 hours once more, obtains enriched substance 3; Enriched substance 3 is pressed 10 ^-8, 10 ^-9, 10 ^-10Extent of dilution inserts respectively in the substratum 3, shakes dilution and separate under 50～55 ℃, and with potassium dichromate process titration ferrous content, direct-counting method living cell counting number, when the ferrous oxidation rate reaches more than 90%, the bacterium number reaches 1 * 10 ⁷During/ml, stop to cultivate, select the highest culture of extension rate as dilution 1; Dilution separates four times so repeatedly, and obtaining dilution 4 at last is the S.thermosulfidooxidans bacterial strain of purifying;

Described substratum 1 is: (NH ₄) ₂SO ₄0.1g/l, MgSO ₄7H ₂O 0.3g/l, KH ₂PO ₄0.1g/l, KCl 0.05g/l, FeSO ₄7H ₂O 10mM, pH 1.5～1.8;

Described substratum 2 is: (NH ₄) ₂SO ₄0.4g/l, MgSO ₄7H ₂O 0.5g/l, K ₂HPO ₄0.2g/l, KCl 0.1g/l, FeSO ₄7H ₂O 50mM, yeast extract 0.1g/l, pH 1.5～1.8;

Described substratum 3 is: (NH ₄) ₂SO ₄1.0g/l, MgSO ₄7H ₂O 1.0g/l, K ₂HPO ₄0.2g/l, FeSO ₄7H ₂O 100mM, yeast extract 0.2g/l, ZnSO ₄7H ₂O ₃100mg/l, CoSO ₄7H ₄O 30mg/l, CuSO ₄5H ₂O 300mg/l, MnSO ₄4H ₂O 60mg/l, pH 1.5～1.8.