KR20230115243A - A method for production of compound-K by using photosynthetic bacterium - Google Patents

Abstract

In the present invention, a recombinant enzyme obtained by translational fusion of squalene epoxidase and cytochrome P450 reductase, dammarenediol II synthase, and protopanaxadiol synthase A recombinant enzyme in which protopanaxadiol synthase and cytochrome P450 reductase are translated fused, and a recombinant strain obtained by introducing a UDP-glycosyltransferase enzyme gene into photosynthetic bacteria are prepared, and squalene is produced using the recombinant strain. It relates to a method for producing ginsenoside compound-K as a starting substrate. The method of the present invention can secure a yield of compound-K of 0.74 g/L or more per day, and can achieve maximum product yield efficiently and quickly in a short time compared to conventional methods.

Description

Method for producing compound-K using photosynthetic bacteria {A method for production of compound-K by using photosynthetic bacterium}

The present invention relates to a method for producing a useful material, ginsenoside compound-K, using squalene as a starting material using a recombinant strain of photosynthetic bacteria.

Ginsenoside compound-K (hereinafter referred to as CK) is a physiologically active substance derived from ginseng. Studies over the past few decades have verified the health-promoting effects of CK, such as cancer, anti-inflammation, anti-aging, prevention of diabetes, and brain/liver/skin protection (Sharma and Lee. 2020. Biomolecules. 10: 1028). CK is not detected in ginseng in its natural state, but is produced when ginsenosides (ginsenosides Rb1, Rb2, Rc) are decomposed by bacteria in the intestine when humans consume ginseng (Akao et al. 1998. J. Pharm. Pharmacol. 50: 1155-1160). Although ginsenosides, a natural ginseng ingredient, have very low intestinal absorption, when converted to CK, the intestinal absorption increases, so it is considered that CK is the substance that is actually absorbed into the body and exhibits effects when ginseng is consumed (Akao et al. 1998. J. Pharm. Pharmacol. 50: 1155-1160).

Industrially producing CK uses a method of using ginseng extract as a raw material and going through a post-process. Post-processing methods include adding hydrolase to ginseng extract (Yang et al. 2015. Fitoterapia 100: 208-220) and adding specific microorganisms capable of producing CK as metabolites (Oh and Kim, 2016 Food Funct. 7: 4506), conversion method through simple heat treatment (Kim. 2012. J. Ginseng Res. 36(1): 1-15), etc. However, these methods are difficult to control the conversion reaction itself, and various ginsenosides other than CK are mixed in the reaction product. Considering only the yield of CK itself, the CK yield relative to the dry weight of ginseng in the above methods is estimated to be about 0.001% (0.1 g of CK obtained per 10 kg of ginseng). Therefore, recently, studies on CK production through metabolic engineering fermentation of yeast have been conducted, unlike the method using ginseng extract. In this method, CK synthase is metabolically engineered into yeast, and CK is produced through an intermediate product of squalene synthesized by the yeast itself from glucose. The maximum CK yield of yeast fermentation reported to date is 5.0 g/L (9-day fermentation) (Shi et al. 2021. Metab. Eng. 67: 104-111). Since the production method using yeast uses squalene, a metabolite of yeast itself, the production of CK, the final product, may be affected by physiological changes in the cells, and there is a problem that a long culture time of about a week is required on average.

The matters described as the background art are only for explanation of the background of the present invention, and should not be taken as an admission that they correspond to prior art already known to those skilled in the art.

The present invention aimed to produce CK faster and more efficiently than conventional methods. To this end, we tried to develop a method for synthesizing CK from squalene using photosynthetic bacteria into which CK synthase was introduced through metabolic engineering.

The present inventors utilized photosynthetic bacteria to solve the problems of existing CK production methods. In an embodiment of the present invention, a recombinant strain was obtained by metabolically introducing enzyme genes capable of converting and synthesizing CK, a final material, using squalene as a starting material in the photosynthetic bacterium Rhodobacter sphaeroides (Fig. 1, RspCK). Unlike yeast, Rhodobacter spheroides does not have the process of synthesizing and metabolizing squalene, so a process of adding squalene to the medium is required. When culturing the CK-producing recombinant strain (RspCK), squalene added from the outside is metabolized and converted to CK only by CK synthetases introduced into the recombinant strain. Compared to the conventional method using yeast, where squalene is its own metabolite, this method has the advantage of high substrate-product conversion efficiency because it can independently produce target products regardless of the metabolic control of the cells themselves. In particular, the initial three enzymes among the six introduced CK synthase are enzymes present in the cell membrane, and the content of the cell membrane during photosynthetic growth of Rhodobacter spheroides is increased by about three times compared to aerobic conditions, so the final expression of the introduced gene volume can be increased. In addition, due to the nature of bacteria having a fast growth time, the maximum product yield can be secured in a much shorter culture time (about 1-2 days) than yeast.

Accordingly, the present invention provides a recombinant microorganism for producing ginsenoside compound-K (CK) containing the following genes:

a) A gene encoding a recombinant enzyme in which squalene epoxidase (SE) and cytochrome P450 reductase (CPR) are translated fused,

b) a gene encoding dammarenediol II synthase (DDS);

c) a gene encoding a recombinant enzyme in which protopanaxadiol synthase (PPDS) and cytochrome P450 reductase are translated fused, and

d) a gene encoding UDP-glycosyltransferase (UGT).

The recombinant microorganism may further include genes encoding the following two coenzymes in addition to the four CK synthetase genes:

e) glucose-6-phosphate dehydrogenase (ZWF)

f) sucrose synthase (SUS).

A recombinant photosynthetic strain for CK production was prepared by cloning the genes of the four CK synthase enzymes (a, b, c, d) and two coenzymes (e, f) into an expression vector and then introducing them into a photosynthetic strain ( RspCK).

The photosynthetic strain is Rhodobacter sp., Rhodospirillum sp., Rhodopseudomonas sp., Rhodopseudomonas sp., Rhoseobacter sp., Bradyrhizobium sp. .) and rubrivivax ( Rubrivivax sp.) or Corynebacterium genus ( Corynebacterium sp.) It can be replaced with a selected bacterium and can be used for CK production by applying the method of the present invention. . In a specific embodiment, the present invention produced CK using Rhodobacter spheroides as a recombinant strain.

The “squalene epoxidase (SE)” uses squalene, NADPH, and oxygen (O ₂ ) as substrates to form 2,3-oxidosqualene, NADP ⁺ , and water (H ₂ O). It can be selected from the group of enzymes that mediate the reaction. SE requires CPR for enzymatic reactions. The "cytochrome P450 reductase (CPR)" is defined as a group of proteins that contain cytochrome P450 inside the enzyme and can mediate electron transfer to various substances. SE and CPR can be derived from various organisms, and the enzymatic activity of SE can vary greatly depending on their combination. Therefore, for a preferred embodiment of the present invention, SE derived from Methylococcus capsulatus and yeast ( Saccharomyces cerevisiae ) and CPR derived from Arabidopsis thaliana and yeast ( Saccharomyces cerevisiae ) were combined to obtain SE and After translational fusion of CPR, the enzymatic activity of the SE-CPR fusion protein in each combination was tested. As a result, it was confirmed that the maximum activity was shown when SE of Methylococcus capsulatus and CPR of yeast were combined. However, this is only one of the preferred embodiments of the present invention, and the types of SE and CPR that can be used are not limited thereto. In a specific embodiment, the SE-CPR fusion protein may be expressed from a gene having the nucleotide sequence represented by SEQ ID NO: 1.

The "dammarenediol II synthetase (DDS)" may be selected from a group of enzymes that mediate a reaction of forming dammarenediol II using 2,3-squalene oxide and water as reactants. In the embodiment of the present invention, DDS derived from ginseng ( Panax ginseng ) was used, but the range of DDS usable for the implementation of the present invention is not limited thereto. In a specific embodiment, the damarendiol II synthetase (DDS) may be expressed from a gene having the nucleotide sequence represented by SEQ ID NO: 2.

The "protopanaxadiol synthetase (PPDS)" may be selected from the group of enzymes that mediate a reaction of forming protopanaxadiol using damarendiol II and NADPH as reactants. CPR is required In a preferred embodiment of the present invention, PPDS derived from ginseng and CPR (ATR1) derived from Although this translational fusion recombinant enzyme (PPDS-ATR1) was used, the range of PPDS and CPR that can be selected for implementation of the present invention is not limited thereto. The recombinant enzyme (PPDS-ATR1) in which (ATR1) is translated fused can be expressed from a gene having the nucleotide sequence represented by SEQ ID NO: 3.

The “UDP-glycosyltransferase (UGT1)” may be selected from a group of enzymes that mediate a reaction of forming CK and UDP using protopanaxadiol and UDP-glucose as substrates. In the examples of this study, ginseng-derived UGT1 was used, but the range of UGT1 that can be selected for the implementation of the present invention is not limited thereto. In a specific embodiment, the DP-glycosyltransferase (UGT1) may be expressed from a gene having the nucleotide sequence represented by SEQ ID NO: 4.

The "glucose-6-phosphate dehydrogenase (ZWF)" is 6-phosphate-D-glucono-1,5-lactone using glucose-6-phosphate, NADP ⁺ , and water as reaction substrates. (6-phospho-D-glucono-1,5-lactone) and NADPH. In addition, the "sugar synthase (SUS1)" uses UDP and sucorse as substrates. It can be selected from the group of enzymes that mediate reactions to form UDP-glucose and fructose. In the cell, ZWF and SUS1 help supply NADPH and UDP-glucose required for the activity of CK synthase (SE-CPR, DDS, PPDS-ATR1, UGT1). The CK-producing strain (RspCK) prepared in the examples of the present invention contains ZWF derived from Rhodobacter spheroides and SUS1 derived from Arabidopsis thaliana, but since this is not a necessary condition for the implementation of the present invention, the present invention The range is not limited by the presence or absence of the ZWF and SUS1 genes. In a specific embodiment, the glucose-6-phosphate dehydrogenase (ZWF) and sugar synthase (SUS1) may be expressed from genes having the nucleotide sequences represented by SEQ ID NOs: 5 and 6, respectively.

The recombinant microorganism of the present invention uses squalene as a starting substrate to synthesize CK by sequential reactions by exogenous enzymes SE-CPR, DDS, PPDS-ATR1, and UGT1 expressed in the strain. Squalene is converted to 2,3-squalene oxide by SE-CPR, which is converted to damarendiol II by DDS. Damarendiol II is converted to protopanaxadiol by PPDS-AT1, which is finally converted to CK by UGT1.

Accordingly, the present invention provides a biosynthetic method of ginsenoid compound K comprising culturing a recombinant microorganism into which a gene expressing the above enzymes has been introduced.

Descriptions of enzymes and recombinant microorganisms are the same as those described above, so they are omitted to prevent redundant description.

One feature of the CK production method using the recombinant microorganism of the present invention is that squalene, a starting substrate, is introduced from the outside. Rhodobacter spheroides used in the specific examples is unable to synthesize and metabolize squalene in a wild type. Therefore, squalene is externally added to the medium to induce CK synthesis by SE-CPR, DDS, PPDS-ATR1, and UGT1. Squalene introduced from the outside does not affect the original metabolic pathway of Rhodobacter spheroides and is metabolized only by recombinant CK synthase.

Here, since squalene as a substrate is an insoluble substance, it is preferable to increase the solubility of squalene in the medium by adding a small amount of a surfactant, an organic solvent, alcohol, etc. to promote absorption into the recombinant microorganism. However, in the present invention, the method of adding squalene is not limited.

In a specific embodiment, another characteristic of the CK production method using the recombinant microorganism of the present invention is to transfer the seed culture of photosynthetic bacteria cultured under anoxic photosynthetic conditions to aerobic conditions, and then add squalene to produce CK. .

The "anoxic photosynthetic conditions" to "photosynthetic conditions" refer to conditions in which cells are placed in a culture container isolated from the outside and cultured while irradiating light while blocking contact with the outside air. Preferably, a lamp that irradiates a lamp generating an emission wavelength in the near-infrared region (800-1,000 nm) at a light intensity of 15-100 W/m ² may be used as a light source. Rhodobacter spheroides, a photosynthetic bacterium used in a specific embodiment of the present invention, forms an intracytoplasmic membrane under photosynthetic conditions to increase the area of the cell membrane in which membrane proteins are distributed. Therefore, an environment in which the expression levels of SE-CPR, DDS, and PPDS-ATR1, which are expected to be membrane proteins, are maximized, can be created under photosynthetic conditions.

The "aerobic conditions" preferably means culture conditions in which cells are placed in a culture vessel and stirred at a speed of 200 to 250 rpm. This is to effectively supply oxygen in the atmosphere into the medium to maximize the activity of SE. Oxygen supply through gas-sparging culture is also included in the scope of the aerobic conditions.

When using the CK-producing photosynthetic bacteria prepared in the present invention, CK can be produced with a yield of 0.74 g/L in one day. In terms of productivity, this is similar to 0.56 g/L (5.0 g/L for 9 days) per day, which is the maximum productivity of the existing method using yeast, but has the advantage of a short cell culture cycle. In general, the shorter the cell culture cycle, the easier it is to maintain the stability of the recombinant cells and control contamination. In addition, since squalene, which is not metabolized by the cell itself, is used as a substrate and it is metabolized only by recombinant CK synthase, the substrate-product yield is higher than that of the method using yeast.

1 is a schematic diagram of a recombinant strain (RspCK) expressing a CK synthase gene and a coenzyme in the photosynthetic bacterium Rhodobacter spheroides.
Figure 2 selects the optimal combination among various types of SE and CPR.
Figure 3 confirms the intracellular expression of CK synthase.
4 shows the determination of optimal growth conditions and production time to produce CK using RspCK.
5 shows the determination of the optimal input concentration of squalene as a substrate in CK production using RspCK.
Figure 6 confirms the CK production of RspCK under optimal conditions using HPLC.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. These examples are only for explaining the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited by these examples.

Selecting the optimal combination of SE and CPR

Four synthetases (SE, DDS, PPDS, and UGT1) are required to produce CK in the photosynthetic bacterium Rhodobacter spheroides (FIG. 1). Among these, SE requires CPR for enzymatic activity. In the present invention, an enzyme (SE-CPR) obtained by translating SE and CPR was prepared and used for CK production. To this end, SE and CPR of various origins were combined to select a combination that maximized enzymatic activity (FIG. 2). To produce a platform strain for screening, squalene synthase (SQS) gene (SEQ ID NO: 7) was expressed in the wild type of Rhodobacter spheroides KD131 strain (KCTC12085), and squalene was synthesized in the strain. The crtE gene (encoding geranylgeranyl pyrophosphate synthase) was deleted and the expression of its own ispA gene (encoding farnesyl diphosphate synthase) was additionally enhanced to increase the flow of metabolites for squalene synthesis. After introducing the gene expressing SE-CPR (SE1-6) of each combination into the platform strain thus completed, each recombinant strain was prepared with 100 mM glucose, 1% (w/v) yeast extract, and Shaking culture (50 rpm) was performed for 48 hours in Sistrom medium (Table 1) supplemented with 100 mM glycerol. For extraction of 2,3-oxidosqualene, a reaction product of SE, 0.5 mL of methylene chloride (dichloromethane) was added to 0.5 mL of the culture medium, mixed, and the supernatant was separated. Thereafter, the extract was analyzed using high performance liquid chromatography (HPLC) (Grievenson et al. 1997. Anal. Biochem. 252: 19-23). As a result, when SE-CPR (SE6), a recombinant protein combining yeast ( Saccharomyces cerevisiae ) SE and Methylococcus capsulatus ( Methlyococcus capsulatus ) CPR, was introduced, it was confirmed that the reaction product of SE within the cell was maximized. Through this, it was determined that the enzymatic activity of SE was maximized in the corresponding combination (FIG. 2). In subsequent examples, only the recombinant SE-CPR (SE6) of the corresponding combination was used.

The enzymatic activity of PPDS, like SE, also requires CPR. In the case of PPDS, based on a previous study (Zhao et al. 2016. Biotechnol. Bioeng. 113(8): 1787-1795), PPDS of ginseng and CPR (ATR1) of Arabidopsis thaliana were translated into a recombinant enzyme (PPDS-ATR1) was used.

Sistrom badge additive final concentration KH ₂ PO ₄ 20 mM NaCl 8.5 mM (NH ₄ ) ₂ SO ₄ 3.78 mM L-Glutamic acid 0.67 mM L-Aspartic acid 0.25 mM Succinic acid 34 mM Nitrilotriacetic acid 1.05 mM MgCl ₂ ·6H ₂ O 1.2 mM CaCl ₂ 2H ₂ O 0.23 mM FeSO ₄ 7H ₂ O 7 µM (NH ₄ ) ₆ Mo ₇ O ₂₄ 0.16 µM EDTA 4.7 µM ZnSO ₄ 7H ₂ O 38 µM MnSO ₄ H ₂ O 9.1 µM CuSO ₄ 5H ₂ O 1.6 µM Co(NO ₃ ) ₂ 6H ₂ O 0.85 µM H ₃ B O ₃ 1.8 µM nicotinic acid 8.1 µM Thiamine hydrochloride 1.5 µM Biotin 41 nM

Confirmation of expression of CK synthase in photosynthetic bacteria

In order to confirm that the four CK synthase enzymes are well formed in Rhodobacter spheroides KD131, western blot was used (FIG. 3). Genes for each enzyme were cloned into the expression vector pBBRMCS2 and introduced into Rhodobacter spheroides KD131. At this time, each enzyme has a histidine-tag (His ₆ -tag) (SE-CPR, DDS, PPDS-ATR1) or strep-tag (UGT1) gene as a detection marker of the enzyme. Translational fusion was performed at the N and C termini, respectively (FIG. 3). After culturing each strain expressing the four enzymes under various growth conditions, the cells obtained by centrifugation at 6,000 g for 10 minutes were dissolved in physiological saline (phosphate-buffered saline, pH 7.4) and sonicated (model VCX130, Sonics & Materials), the cells were disrupted three times at intervals of 5 minutes, and then centrifuged at 6,000 g for 10 minutes to obtain a cell disruption solution. After loading the cell homogenate on SDS-polyacrylamide gel, Western blotting was performed using anti-histidine-tag and anti-strep-tag antibodies. For SE-CPR, DDS, and UGT1, the expression levels detected under aerobic (250 rpm shaking culture), semi-aerobic (50 rpm shaking culture), and anoxic photosynthetic (photoheterotrophic) culture conditions depended on the culture conditions 2 - There was a 3-fold difference. On the other hand, the expression level of PPDS-ATR1 was significantly higher in photosynthetic conditions than in aerobic and semi-aerobic conditions. In addition, in order to confirm the intracellular location of the enzyme, the cell lysate was subjected to ultracentrifugation at 100,000 g and 4 ° C for 1 hour to separate the cytosolic fraction and the membrane fraction. Western blotting was performed in the same manner as in the method. As a result, SE-CPR, DDS, and PPDS-ATR1, which are expected to be membrane proteins, were mostly detected in the plasma membrane fraction, and UGT1, which is expected to be a soluble protein, was detected in the cytoplasmic fraction.

Preparation of RspCK

Finally, the four CK synthase genes were simultaneously introduced into Rhodobacter spheroides KD131 (FIG. 1). At this time, genes for two coenzymes (ZWF, SUS1) were additionally introduced for smooth supply of cofactors necessary for the activity of CK synthase. ZWF supplies NADPH necessary for the reaction of SE-CPR and PPDS-ATR1, and SUS1 supplies UDP-glucose necessary for the reaction of UGT1. After cloning the four CK synthase enzymes and the two coenzymes into expression vectors pBBRMCS2 and pRK415, they were introduced into a wild strain of Rhodobacter spheroides KD131 to prepare a CK-producing strain (RspCK) (FIG. 1).

Determination of optimal conditions for CK production of RspCK

Since the enzymatic reaction of SE-CPR consumes oxygen as its substrate, RspCK must be cultured under aerobic or semi-aerobic conditions for CK production. However, from the results of Example 2, it was confirmed that SE-CPR, DDS, and UGT1 were expressed under all growth conditions, but the expression of PPDS-ATR1 was maximized under anoxic photosynthetic conditions. Therefore, for effective CK production, RspCK was cultured under anoxic photosynthetic conditions until just before the stationary phase to induce expression of CK synthase in cells, and then the cells were transferred to aerobic conditions to perform CK production (FIG. 4). As the medium under anoxic photosynthesis conditions, Sistrom medium containing 100 mM glucose, 1% (w/v) yeast extract, and 100 mM glycerol was added for high cell-density culture. When shifting to aerobic conditions after culture, squalene (1 g/L) and 100 mM glucose, which are CK-producing substrates, were added. While measuring growth under aerobic conditions, cell growth was measured at 0, 12, 24, and 48 h after the start of culture, and a portion of the culture medium was separated. For extraction and analysis of CK, n -butanol (0.5 mL) was added to the separated culture medium (0.5 mL), mixed, and the supernatant was obtained and analyzed using HPLC to quantify CK (Nan et al. 2020. Microb (Cell Fact. 19:41). As a result, the growth of the cells in aerobic conditions stopped after 12 hours after the start of culture, but the CK production of the cells continuously increased and reached a maximum at 24 hours. Therefore, in the following CK production analysis, a method of extracting CK from cells after anaerobic photosynthetic conditions and subsequent aerobic culture for 24 hours was used.

The concentration of squalene, the starting substrate used when moving from photosynthetic conditions to aerobic conditions, is an important factor in CK production. Therefore, in order to determine the optimal concentration of squalene, squalene was added in the concentration range of 1 to 10 g/L in the same manner as the RspCK culturing method, and final CK production was measured (FIG. 5). As a result, it was confirmed that the CK production was maximized at a squalene concentration of about 5 g/L.

The yield of CK obtained by culturing RspCK under conditions optimized based on the above results was about 0.74 g/L (FIG. 6). As a result of HPLC analysis of CK in the culture solution collected over time, the peak (arrow in FIG. 6) with a retention time similar to that of the CK standard material (compound-K standard, Sigma-Aldrich) increased with time was observed, and the absorption spectrum (peak profile) of these peaks exactly matched the CK standard material (FIG. 6).

Claims (11)

a) a gene encoding a recombinant enzyme obtained by translational fusion of squalene epoxidase and cytochrome P450 reductase;
b) a gene encoding dammarenediol II synthase;
c) a gene encoding a recombinant enzyme in which protopanaxadiol synthase and cytochrome P450 reductase are translated fused, and
d) A recombinant microorganism for the biosynthesis of ginsenoside compound-K containing a gene encoding UDP-glycosyltransferase. According to claim 1,
e) a gene encoding glucose-6-phosphate dehydrogenase (ZWF), and
f) a recombinant microorganism further comprising at least one of genes encoding sucrose synthase (SUS1). The recombinant microorganism according to claim 1, wherein the squalene epoxidase in the recombinant enzyme of a) is derived from Methlyococcus capsulatus , and the cytochrome P450 reductase is derived from yeast ( Saccharomyces cerevisiae ). The recombinant microorganism according to claim 1, wherein the damarenediol II synthetase is derived from Panax ginseng . The recombinant microorganism according to claim 1, wherein in the recombinant enzyme of c), the protopanaxadiol synthetase is derived from Panax ginseng , and the cytochrome P450 reductase is derived from Arabidopsis thaliana . The recombinant microorganism according to claim 1, wherein the UDP-glycosyl transferase is derived from Panax ginseng . The recombinant microorganism according to claim 2, wherein the glucose-6-phosphate dehydrogenase is derived from Rhodobacter spheroides, and the sugar synthase is derived from Arabidopsis thaliana . According to claim 1,
a) The gene encoding the recombinant enzyme in which squalene epoxidase and cytochrome P450 reductase are translated fused is represented by SEQ ID NO: 1,
b) The gene encoding dammarenediol II synthase is represented by SEQ ID NO: 2,
c) a gene encoding a recombinant enzyme in which protopanaxadiol synthase and cytochrome P450 reductase are translated fused is represented by SEQ ID NO: 3, and
d) A recombinant microorganism whose gene encoding UDP-glycosyltransferase is represented by SEQ ID NO: 4. According to claim 1, wherein the microorganism is Rhodobacter sp., Rhodospirillum sp., Rhodopseudomonas sp., Rhoseobacter sp., Brady Leezo A recombinant microorganism, characterized in that selected from the group consisting of Bradyrhizobium sp. and Rubrivivax sp., or Corynebacterium sp. A method for producing Compound-K comprising the step of culturing the recombinant microorganism according to any one of claims 1 to 9 in a medium together with squalene as a substrate. 11. The method of claim 10, wherein the method comprises first culturing the recombinant microorganism under light-irradiated anoxic photosynthetic conditions, and then culturing the recombinant microorganism with squalene as a substrate after changing the culture conditions to aerobic conditions. method.