CN111139203A - Method for improving pit mud at bottom of pool in layered mode through artificially cultured bacterial liquid - Google Patents
Method for improving pit mud at bottom of pool in layered mode through artificially cultured bacterial liquid Download PDFInfo
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Abstract
The invention discloses a method for improving pit mud at the bottom of a pool in a layering manner by artificially culturing bacterial liquid, which improves pit mud from different depths and different sites of the pit mud according to local conditions to promote the aging of pit mud flora. Because the essence of pit mud aging is the aging of pit mud flora, the pit mud flora is an executor of pit mud functions. The method can comprehensively maintain the bottom mud of the new pit pool from different positions at different depths according to local conditions, and promote the aging of pit mud flora.
Description
Technical Field
The invention relates to a method for improving pit mud at the bottom of a pool in a layered manner by artificially culturing bacterial liquid, and belongs to the technical field of wine brewing.
Background
The pit mud is an important material basis for fermenting the Luzhou-flavor liquor, and abundant anaerobic bacteria have an important effect on the formation of flavor substances of the Luzhou-flavor liquor. At present, it is acknowledged that pit mud quality and pit age have close relation, and the older the pit is, the better the pit mud is. Compared with the pool wall mud, the pool bottom mud has larger influence on wine making, and the closer to the pool bottom part, the better the quality of the brewed wine is. Therefore, the manual improvement of pit mud to promote the aging of pit mud, especially the aging of pit mud at the bottom of a pool, is the direction of the research of many strong aromatic wineries at present. After all, the number of old cellar pools is limited, so that the acceleration of the conversion from new cellar mud to old cellar mud is particularly important.
The prior method for improving pit mud relates to the following steps: the pit mud is cured by culturing and manufacturing various pit mud curing media such as artificial pit mud, pit mud curing liquid and the like, but the above pit mud modifying modes are limited to modification of surface pit mud. The essence of improving pit mud is to improve pit mud flora. The cellar mud is used as a carrier for bearing cellar mud floras, and has a three-dimensional structure, so that the floras do not exist in a homogeneous manner, particularly, between the surface of the cellar mud and the deep part of the cellar mud, the cellar mud floras are different in composition due to the permeability difference of nutrient substances in the fermentation process between the central point and the angular point. Therefore, the improvement of pit mud should be made according to the specific distribution of pit mud flora at different sites and different depths. In principle, the mature laojiao puree flora has formed a cooperative metabolic chain. Due to the fact that the surface of the pit mud is easy to contact with oxygen, facultative anaerobes (such as Lactobacillus) are more; while the deep pit mud contains more absolute anaerobic bacteria. Therefore, only the surface pit mud flora is maintained, but the deep pit mud flora is not maintained, the short-term effect can be achieved, and the aging of pit mud flora cannot be fundamentally promoted.
According to the metabolic function of the currently known bacterial genus, the metabolism of the aged pit mud flora is completed by the mutual cooperation of a plurality of bacterial genera, namely, a complete pit mud flora metabolic chain is formed. Caproic acid bacteria (Capriciperonucens) and Clostridium (Clostridium) can produce H by anaerobic fermentation2，CO2Butyric acid and caproic acid; c can be converted into C by the genus Syntrophomonas4-C8Degradation of fatty acids to propionic acid, acetic acid and H2(ii) a Amino acid bacteria (aminobacter) and sedimentary bacteria (settlementobacter) can metabolize amino acids to form acetic acid and butyric acid; the methanobacteria in the hydrogenotrophic form (such as Methanovulleus and Methanobrevibacter) can convert H2And CO2Is methane. In principle, the metabolism function of pit mud flora is the process of metabolizing organic substances and releasing gases such as hydrogen, methane and the like. In the process, the phenomenon of 'interspecies hydrogen transfer' between hydrogen producing bacteria (such as caproic acid bacteria) and methane bacteria is important for promoting the aging of pit mud, because hydrogen is a substrate of hydrogenotrophic methane bacteria, and the existence of methane bacteria is a mark for aging of pit mud. It is known at present that the concentration of hydrogen needs to reach a certain concentration to meet the requirement of methane bacteria for absorbing hydrogen for producing methane. Therefore, the aging of the pit mud needs to be promoted from two aspects, on one hand, the growth and the propagation of hydrogen-producing bacteria (such as caproic acid bacteria and clostridium) are promoted; on the other hand, the growth and the propagation of methane bacteria are promoted.
Disclosure of Invention
The invention provides a method for improving pit mud at the bottom of a pool in a layered manner by artificially culturing bacterial liquid, aiming at the problem that the existing pit mud improvement method only improves surface pit mud, and improving pit mud according to local conditions from different depths and different sites of pit mud at the bottom of the pool to promote the aging of pit mud flora. Because the essence of pit mud aging is the aging of pit mud flora, the pit mud flora is an executor of pit mud functions. The method can comprehensively maintain the bottom mud of the new pit pool from different positions at different depths according to local conditions, and promote the aging of pit mud flora.
The invention relates to a method for improving pit mud at the bottom of a pool in a layered manner by artificially culturing bacterial liquid, which comprises the following steps:
step 1: dividing pit mud at the bottom of a mature old pit (the pit age is more than or equal to 50 years) and a new pit to be improved (the pit age is less than 10 years) into four layers of depths (the vertical depth is 0-1cm, 1-3cm, 3-5cm and 5-7cm), and sampling the pit mud at each layer of depth by three points (a central point, an angular point and a midpoint of a connecting line of the central point and the angular point, namely a quarter point). The sampling method is shown in FIG. 1.
Step 2: and (3) sampling pit mud samples of the old pit and the new pit obtained in the step (1) to detect the distribution of the flora, wherein the detection method comprises but is not limited to non-culture molecular biological methods such as high-throughput sequencing and the like, and a pure culture method is not adopted. Since the pure culture detection method is limited by the culture conditions, it cannot cover all culturable bacteria, and further cannot detect a large amount of uncultured bacteria. The real composition of the pit mud flora can be more accurately obtained by a culture-free molecular biological method such as high-throughput sequencing.
The composition ratio of the pit mud flora to be improved represents the maturity of pit mud flora, and particularly, the ratio of lactic acid bacteria in the immature pit mud is larger and is obviously larger than the relative contents of caproic acid bacteria (Capriciproducens), Clostridium (Clostridium) and methane bacteria (such as Methanovuleus); the ratio of caproic acid bacteria to clostridium in the pit mud with medium maturity is obviously greater than that of lactic acid bacteria, but the ratio of methane bacteria is still very low; caproic acid bacteria in the pit mud with high maturity are dominant bacteria, and the proportion of methane bacteria is as high as about 10%. According to the detection result of the pit mud flora, the maturity of the pit mud can be determined, and according to the maturity of the pit mud to be detected, the proportion of later surface pit mud maintenance liquid and deep pit mud maintenance liquid is determined. If the pit mud is at low maturity, the amount of the surface pit mud curing liquid is required to be larger than that of the deep pit mud curing liquid, and the caproic acid in the surface pit mud curing liquid has the function of inhibiting lactic acid bacteria; if the pit mud is at the medium maturity, the amount of the deep pit mud maintenance liquid is larger than that of the surface pit mud maintenance liquid, and the acetic acid in the deep pit mud maintenance liquid has the effect of promoting the growth of methane bacteria so as to promote the aging of the pit mud towards the direction of higher maturity.
And step 3: and (3) sampling pit mud of the old pit and the new pit obtained in the step (1) to detect physicochemical properties, wherein the physicochemical properties comprise a pH value, an oxidation-reduction potential and the like. And (3) uniformly mixing the pit mud sample of the new pit with deionized water according to the mass-volume ratio of 1g:5mL, and detecting the pH value (calculated by using FE20pH for example) and the oxidation-reduction potential (calculated by using an E-501 composite electrode for example) of the pit mud sample of the new pit. The pH value is detected because the influence of the pH value on the pit mud flora is generally accepted to be the largest, the purpose of detecting the oxidation-reduction potential is to conveniently predict the existence condition of methane bacteria, because the oxidation-reduction potential represents the reduction force in the environment, a positive value represents that the oxidation force ratio is larger, and a negative value represents that the reduction force ratio is larger. It is known that methanobacteria are suitable for growing in an environment of negative redox potential. The methane bacteria are mark bacteria for aging of pit mud, and the existence of the methane bacteria in suitable environment can be more conveniently known by detecting the oxidation-reduction potential.
The pH value of the pit mud to be improved is the pH value of the growth environment of pit mud flora, and the oxidation-reduction potential of the pit mud to be improved reflects the living conditions of absolute anaerobic bacteria in the pit mud. The pH value and the oxidation-reduction potential of the pit mud to be improved comprehensively reflect the growth environment of pit mud floras, so that the quality of the pit mud can be judged according to the pH value and the oxidation-reduction potential of the pit mud. The pH value of the immature pit mud is lower than 5, the immature pit mud is acidic and is just suitable for the survival of lactic acid bacteria, so the content of the lactic acid bacteria is more abundant; the pH value of the pit mud with medium maturity is greater than 5 and close to 7, and the contents of caproic acid bacteria and clostridium are more abundant; the pH value of deep-layer aged pit mud with the highest maturity is 8-9, the deep-layer aged pit mud is alkalescent and is suitable for methane bacteria to live, and the methane bacteria are mark bacteria for aging of pit mud. The abundance of absolute anaerobic bacteria in the immature pit mud is low, and the oxidation-reduction potential is a positive value; the oxidation-reduction potential of the pit mud with the medium and high maturity is negative, and the higher the maturity of the pit mud is, the lower the oxidation-reduction potential is. Therefore, the maturity of the pit mud can be determined according to the pH value and the oxidation-reduction potential of the pit mud to be detected, and then the relative amount of the used surface pit mud oxidation liquid and deep pit mud maintenance liquid is determined. Generally, for curing pit mud with low aging degree, the required amount of surface pit mud curing liquid is more than that of deep pit mud curing liquid; however, for medium-maturity pit mud, the required amount of deep pit mud curing liquid is equal to or more than that of surface pit mud curing liquid. Because the ageing of cellar for storing things mud is along with cellar for storing things mud degree of depth is progressive, and the more ageing degree of past depths cellar for storing things mud is higher, to the cellar for storing things mud of medium maturity, main maintenance deep cellar for storing things mud, and the purpose that uses superficial layer cellar for storing things mud maintenance liquid then is mainly for protecting superficial layer cellar for storing things mud, avoids because the injury that deep cellar for storing things mud improvement brought.
And 4, step 4: expanded culture of artificially cultured bacterial liquid
And respectively carrying out group culture on the surface pit mud and the deep pit mud according to the detection result of the flora composition of the old pit mud.
4a, uniformly mixing the surface layer old pit mud and sterile deionized water according to a mass-volume ratio of 0.5-2g:5-10mL to obtain a mixed solution A; adding 5-20% of old cellar yellow water (as used in the following) and 5-20g/L of caproic acid into the obtained mixed solution A, adjusting pH to 6.0-8.0, and standing at 30-34 deg.C for 5-10 days for enriching caproic acid bacteria and inhibiting growth of lactic acid bacteria, because medium-long chain fatty acid such as caproic acid has the function of inhibiting lactic acid bacteria and is a component in yellow water. And obtaining the surface pit mud maintenance liquid for improving the surface pit mud of the new pit. The surface layer pit mud is pit mud of a pit with a vertical depth of 0-1 cm.
4B, uniformly mixing the deep aged pit mud and sterile deionized water according to the mass-volume ratio of 0.5-2g:5-10mL to obtain a mixed solution B; adding 25% of old pit yellow water and 5-7g/L of acetic acid into the obtained mixed solution B, adjusting the pH value to 7.0-9.0, removing oxygen by using dry ice, and standing and culturing at 30-34 ℃ for 5-10 days to obtain the deep pit mud maintenance liquid for improving the deep pit mud of the new pit. The addition of acetic acid is an essential component for the growth of methanogens, since acetic acid is required as a growth factor for the growth of the predominant methanogens (methanoculeus) in the bottom sediment of high quality old pit ponds. The deep layer aged pit mud is aged pit mud with the vertical depth of 1-5 cm.
The yellow water used in the preparation of the pit mud curing liquid is the yellow water of an old pit, and the yellow water can be directly used without sterilization. The yellow water has two functions in the formula, and on one hand, the yellow water is used as a culture medium to provide nutrient substances required by the growth of flora; on the other hand, the strain source of partial artificial culture liquid plays a role in inoculation. At present, the yellow water is known to contain rich strains, particularly the yellow water in the old cellar pool is rich in strains, and the enrichment culture is carried out under the proper growth condition, so that the maintenance of the cellar mud is greatly facilitated.
The aged pit mud used for the group culture in the step belongs to the high-maturity pit mud defined in the following table.
And 5: improvement of pit mud at bottom of new pit
Firstly, inserting pit mud at the bottom of a new pit into a hole with the depth of 1-7cm by using a pit mud steel plate fork, uniformly spraying deep pit mud curing liquid into the hole, and trowelling the surface of the pit mud by using a tool (such as a trowel); and then, inserting the pit mud at the bottom of the new pit into a hole with the depth of 0-1cm by using a pit mud steel plate fork, uniformly spraying surface pit mud curing liquid into the hole, leveling by using a tool, and using the pit for entering the pit for fermentation after improvement. Fig. 2 is a schematic structural diagram of a pit mud steel plate fork used in the present invention, and the specific structure thereof is described in detail in CN 209537436U.
According to the different maturity of the cellar mud of the new cellar, the improved parameters in the step 5 are set as follows:
wherein, the low maturity cellar mud and the medium maturity cellar mud are divided according to the detection results of the steps 2 and 3, and the specific standards are as follows:
when the pit mud maturity is divided according to the above standards, the priority of evaluating the pit mud maturity by using three parameters of flora composition, pH value and oxidation-reduction potential is as follows: flora composition > pH > oxidation-reduction potential.
With regard to the above division criteria, the following are exemplified:
1. when a certain pit mud meets the requirements of the low-maturity pit mud on three parameters of flora composition, pH value and oxidation-reduction potential, the pit mud can be considered to belong to the low-maturity pit mud.
2. When the flora composition of a pit mud meets the parameter requirements of the pit mud with low maturity, but the pH value and/or the oxidation-reduction potential of the pit mud do not meet the parameter requirements of the pit mud with low maturity, the pit mud can be considered to belong to the pit mud with low maturity according to the evaluation standard of priority.
3. Taking the pit mud with low maturity as an example, the parameter requirements of the flora composition of the pit mud are two points ((1) and (2)), and the pit mud can be considered to meet the flora composition requirements of the pit mud with low maturity only by meeting the two requirements at the same time.
4. According to the evaluation standard of priority, the maturity of pit mud can be judged as long as the flora composition of certain pit mud is determined preferentially; when the bacterial composition of a pit mud does not belong to the requirements of the bacterial compositions of the three kinds of pit mud with maturity, the bacterial compositions cannot be divided according to the pH value or the oxidation-reduction potential value. Because the aging of pit mud is essentially the aging of pit mud flora, the pit mud flora is the true main body for performing the function of pit mud.
The detection results of the pit mud at the center point, the quarter point and the corner point respectively represent the quality change of the pit mud at the bottom of the pit from the center to the periphery. When the pit mud is improved, the density of the jacks and the addition of the maintenance liquid at each position of the bottom of the pit are directly influenced by the detection result. The lower the pH and the higher the oxidation-reduction potential, the higher the density of the required plug holes, and the more the amount of the maintenance liquid to be added.
The invention discloses a method for improving pit mud, which comprises the steps of firstly using a deep curing liquid to improve the deep pit mud and then using a surface curing liquid to improve the surface pit mud, wherein most of the deep curing liquid is absolute anaerobic bacteria, and most of the surface curing liquid is facultative anaerobic bacteria. For example, the specific implementation method comprises the following steps: firstly, improving pit mud at the deep position, inserting the pit mud at the bottom of the pool into a deep hole (1-7cm in depth) by using a steel plate fork, uniformly spraying deep pit mud maintenance liquid in the pit mud, and trowelling the surface of the pit mud by using a tool (such as a trowel); then, inserting shallow holes (0-1cm) in the bottom mud by using a steel plate fork, uniformly spraying surface layer pit mud curing liquid in the bottom mud, and finally, trowelling the surface of the pit mud by using a tool (such as a trowel). In addition, for some cellars, the maturity of different sites is different, and different maintenance methods can be adopted for different sites according to local conditions.
Compared with the prior art, the invention has the beneficial effects that: the existing pit mud improvement method generally comprises the steps of lifting pit mud, then improving the pit mud by using a curing liquid, and then flattening the pit mud. The pit mud improvement method is a pit mud improvement method of adding curing liquid into in-situ jacks. Compared with the existing method:
(1) the existing three-dimensional distribution structure of the pit mud flora is not damaged;
(2) the facultative/absolute anaerobic bacteria in the deep pit mud are protected from being damaged by improvement measures;
(3) considering the difference of the flora composition of the surface pit mud and the deep pit mud and the difference of the living environment (namely the corresponding difference of physicochemical properties of the pit mud) according to local conditions;
(4) considering the difference of aging degrees of pit mud at different positions at the bottom of the pit pool according to local conditions;
(5) the mode of adding the bacterial liquid through the jack enables the survival rate of anaerobic bacteria in the maintenance liquid to be improved, and the maintenance effect is improved.
Drawings
FIG. 1 is a schematic representation of a three-point and four-depth sampling of bottom mud. Wherein Cor: an angle; qua: a quarter point; cen: a center point; the suffixes "01", "13", "35" and "57" refer to pit mud depths respectively, for example, "01" refers to pit mud between 0-1 cm.
Fig. 2 is a pit mud steel plate fork used in the invention, wherein 1 handle, 2 rectangular supports, 3 steel plates and 4 steel nails are used.
FIG. 3 is a distribution diagram of the flora in pit mud at different positions and different depths (cm) at the bottom of the aged pit. Wherein Cor: an angle; qua: a quarter point; cen: a center point; the suffixes of 01, 13, 35 and 57 refer to pit mud depths respectively, for example, "01" refers to pit mud between 0 and 1 cm.
FIG. 4 is a plot of the population in pit mud at the bottom of the new pit at different sites and at different depths (cm). Wherein Cor: an angle; qua: a quarter point; cen: a center point; the suffixes of 01, 13, 35 and 57 refer to pit mud depths respectively, for example, "01" refers to pit mud between 0 and 1 cm.
FIG. 5 shows the pH values of pit mud at the bottom of the pit at different depths and different positions in the old pit. Wherein Cor: an angle; qua: a quarter point; cen: a center point; the suffixes of 01, 13, 35 and 57 refer to pit mud depths respectively, for example, "01" refers to pit mud between 0 and 1 cm.
FIG. 6 shows the pH values of pit mud at the bottom of the pit at different depths and different positions in the new pit. Wherein Cor: an angle; qua: a quarter point; cen: a center point; the suffixes of 01, 13, 35 and 57 refer to pit mud depths respectively, for example, "01" refers to pit mud between 0 and 1 cm.
FIG. 7 is the values of the redox potentials of pit mud at the bottom of the pit at different depths and different positions in the old pit. Wherein Cor: an angle; qua: a quarter point; cen: a center point; the suffixes of 01, 13, 35 and 57 refer to pit mud depths respectively, for example, "01" refers to pit mud between 0 and 1 cm.
FIG. 8 is the oxidation-reduction potential values of pit mud at the bottom of the pit at different depths and different positions in the new pit. Wherein Cor: an angle; qua: a quarter point; cen: a center point; the suffixes of 01, 13, 35 and 57 refer to pit mud depths respectively, for example, "01" refers to pit mud between 0 and 1 cm.
Fig. 9 is the distribution of prokaryotic flora in pond bottom mud after new pit improvement, where Cen — 01: the center point of the mud at the bottom of the pool is 0-1cm deep; cen _ 13: the center point of the bottom mud of the pool is 1-3cm deep; qua _ 01: a quarter point 0-1cm deep; qua _ 13: a quarter point is 1-3cm deep.
Detailed Description
Example 1:
the pit bottom pit mud of 3 new pits with the pit age of 6 years is improved by the embodiment, and the pit mud of the old pit with the pit age of 50 years and fresh yellow water are used for improving the pit bottom pit mud.
1. According to the sampling method shown in figure 1, two groups of pit mud at the bottom of the new pit and the old pit are respectively divided into 4 layers and 3 sites to obtain fresh pit mud samples. The samples were used directly for detection of their pH and redox potential, frozen at-80 ℃ for high throughput sequencing, and used for later genome extraction and culture-free molecular biology methods to detect microbial community composition (Illumina high-throughput sequencing method based on the 16S rDNA V4 region was used in this example).
2. The composition of the flora in the pit mud sample is detected by 16S rDNA high-throughput sequencing, and as shown in figure 3, the flora distribution diagram of different positions and different depths (cm) of the bottom mud of the old pit pool is shown, and the content of the dominant bacteria with the content of more than 1 percent is shown in the figure. In the transverse direction, the difference of pit mud flora compositions of three positions (a central point, an angular point and a quarter) of the bottom mud of the old pit is not large. In the longitudinal direction, the composition of the bacterial communities of pit mud at different depths is obviously different, and particularly the bacterial community difference between the surface pit mud of 0-1cm and the deep pit mud below 1cm is large. FIG. 4 shows the distribution of the flora at different sites and different depths of the bottom mud of the new pit. The graph shows the content of dominant bacteria with an average content > 1%. The compositions of the layered floras at the eccentric positions represented by the central point and the quarter point of the bottom mud of the new pit pool are similar, while the compositions of the floras at the corner positions of the new pit pool are obviously different from those at the central point and the quarter point.
3. And detecting the physical and chemical properties of the pit mud sample. As shown in figure 5, the pH values of the bottom mud of the old cellar pool at different positions and different depths are different. As is evident from fig. 5: on one hand, the pH value of pit mud is gradually increased from shallow to deep, the pit mud is weakly acidic at the depth of 0-1cm, the pit mud is quickly changed into neutral and weakly alkaline below 1cm, and the pH value below 3cm is as high as 9; on the other hand, the pH values of pit mud at 3 different positions have no significant difference, which indicates that the bottom mud of the old pit has no significant difference in horizontal dimension.
The pH value of the bottom mud of the new pit pool at different positions and different depths is different as shown in figure 6. As can be seen from FIG. 6, the bottom mud of the new pit at 3 sites increases from shallow and deep pH values; and comparing 3 positions, wherein the pH value of the pit mud at the outermost layer (0-1cm) is almost, and then sequentially increasing the pH value of the pit mud sample at the central point and the quarter (Qua) point to the deep position, the pit mud at the corner points is obviously different from the pit mud at the central point and the quarter (Qua) point, the pH value of the pit mud below 1cm is increased more quickly and is higher than the pH value of the pit mud at the front part and the quarter. By comparing fig. 5 and fig. 6, it can be seen that the pH of the old pit mud is significantly greater than that of the new pit mud. The difference value of the average pH value of 0-1cm on the surface layer of the new pit mud and the old pit mud exceeds 1. The difference of the pH value of pit mud in the deep layer (less than 1cm) of the new pit and the old pit is larger. Compared with the bottom mud sample of the old cellar, the pH value of the bottom mud corner point of the new cellar is 5-7cm closest to the deep pH value of the old cellar.
And determining the pH value of the new pit mud maintenance liquid and the using amount of the pit mud maintenance liquid according to the pH value of the old pit mud. The larger the difference of the pH values of the new pit mud and the old pit mud is, the more the pit mud improvement liquid is used, and the smaller the difference is, the less the pit mud improvement liquid is used.
FIG. 7 shows the oxidation-reduction potential of the bottom mud of the old cellar pool at different positions and different depths. As can be seen from fig. 7, the oxidation-reduction potential of the surface pit mud of the old pit is greater than 0; the oxidation-reduction potential of pit mud below 1cm is generally lower than-100 mv. The distribution rule of the oxidation-reduction potential of the pit mud at the three sampling sites is similar. FIG. 8 shows the composition differences of the redox potentials at different sites and at different depths of the bottom mud of the new pit. As can be seen from the figure, the oxidation-reduction potential of 3 sites of the bottom mud of the new pit gradually decreases from the surface to the depth. Compared with the bottom mud of the old pit, the oxidation-reduction potential of the bottom mud of the new pit is higher. Compared with the oxidation-reduction potential of the old pit mud below 1cm, the oxidation-reduction potential of the new pit mud below 1cm is negative only at one quarter of the new pit mud and in the depth of 5-7cm of the corner point, and the oxidation-reduction potentials of the pit mud at other positions are positive. The positive and negative values of the redox potential represent to some extent the strength contrast between the oxidation and reduction forces, with positive values representing stronger oxidation forces and negative values representing stronger reduction forces. From this, it is known that the deep aged pit mud has a relatively strong reducing power, while the surface aged pit mud (0-1cm) has a relatively strong oxidizing power. The new pit mud has stronger reducing force except corners and one quarter of 5-7cm pit mud, and the new pit mud at other sites has stronger oxidizing force. Based on the detection results, the new pit mud needs to be improved, and the physicochemical properties of the new pit mud are changed, so that the pit mud flora can be further improved. The oxidation-reduction potential of pit mud is directly related to the oxidation-reduction reaction capability of anaerobic flora, and the oxidation-reduction potential of mature deep old pit mud is generally negative because of strong reducing force (figure 7). As can be seen from FIG. 8, the oxidation-reduction potential of the new pit mud is generally high, which indicates that artificial improvement is necessary.
Comprehensively considering the flora composition, the pH value and the oxidation-reduction potential of pit mud, finding that the pit mud flora composition, the pH value and the oxidation-reduction potential of the new pit corner pit mud are relatively closer to those of old pit mud with higher maturity, and the corner pit mud maturity is medium maturity; whereas the rate of aging of the center point and quarter point is significantly lower. In consideration of the fact that the maturity of the center point/quarter point is inconsistent with that of the corner point, the pit mud in the center area including the center point and the quarter point is improved, and the pit mud in the corner point is not improved temporarily. Therefore, the aging of the pit mud floras at the eccentric position is accelerated, and the pit mud floras at the corner are not damaged.
4. And (4) preparing the manmade pit mud maintenance liquid. Preparing a surface pit mud maintenance liquid and a deep pit mud maintenance liquid which are respectively used for improving 0-1cm pit mud and 1-3cm pit mud of bottom mud of a new pit pool. The preparation method of the surface pit mud curing liquid comprises the following steps: mixing 0-1cm of pit mud on the surface layer of pit bottom mud of the old pit with deionized water (1g:10mL mass-volume ratio) uniformly to obtain a mixed solution A, adding 10% of old pit yellow water and 7g/L of caproic acid in the total volume of the mixed solution A, adjusting the pH value to 6.0-7.0, standing, sealing and culturing for 5-7 days to obtain an improved bacterial solution of the pit mud on the surface layer. The preparation method of the deep pit mud maintenance liquid comprises the following steps: mixing old cellar mud 1-3cm deep with sterile deionized water (1g:10mL mass volume ratio) to obtain mixed solution B, adding old cellar yellow water 5% of the total volume of the mixed solution B and 7g/L acetic acid, adjusting pH value to 7.0-8.0, removing oxygen with dry ice (solid carbon dioxide), and standing at 32 deg.C for 5-7 days to obtain deep cellar mud maintenance liquid for improving surface layer new cellar mud.
5. And (5) primarily improving the new pit mud. Improving the surface layer new pit mud by using a surface layer pit mud culture bacterial liquid; and (4) improving the deep new pit mud by using the deep pit mud culture bacterial liquid. This example is only for new cellarThe center and quarter points of the pool were modified with new pit mud. In view of the lower maturity of pit mud in the center of the new pit, the amount of the surface pit mud curing liquid used by the improved pit mud of the embodiment is 3 times that of the deep pit mud curing liquid. The improvement of pit mud is that firstly, a pit mud steel plate fork is used to insert a hole with the depth of 1-3cm into an eccentric zone (including a central point and a quarter point) of pit bottom mud, and deep pit mud maintenance liquid is added according to the proportion of 1.5L/m2Uniformly spraying the mixture into the holes, and troweling the surface of the pit mud; then, modifying surface layer pit mud, directly using surface layer pit mud curing liquid, using pit mud steel plate fork to insert 0-1cm shallow hole from eccentric zone (including central point and quarter point) of bottom mud of pool, and using surface layer pit mud curing liquid according to 0.5L/m2The pit mud is spread in the holes and trowelled. And the pit after improving the pit bottom mud is immediately used for fermentation production. After one period of fermentation production (about 70 days), after one row of fermentation production (which is the term of wine making industry, the row refers to one time of fermentation and distillation), sampling is carried out at the depth of 0-1cm and 1-3cm of the central point and the quarter point respectively, the composition of the flora in the sample is detected through high-throughput sequencing, and the improvement effect of the pit mud is verified, as shown in figure 9.
Comparing fig. 9 with fig. 3 and fig. 4, it can be seen that the improved pit mud has 0-1cm pit mud on the surface layer and 1-3cm pit mud on the deep layer, compared with the two, the change of the floras of the 1-3cm deep pit mud is most obvious, the content of caproic acid bacteria (caproic acid bacteria) is obviously increased, the content of lactic acid bacteria is reduced, the compositions of the floras of the pit mud at the corners of 1-3cm and 3-5cm are more similar to those of the pit mud at the corners of the spontaneous maturation state, and the compositions of the floras at the surface layer of 0-1cm old pit mud are also very similar to those of the pit mud (fig. 3). In addition, the content of amino acid bacteria (Aminobacterium) is increased, and the amino acid bacteria have the function of degrading amino acid, are important for aging of cellar mud and are bacteria rich in the cellar mud. The proportion of lactobacillus in the flora 0-1cm on the surface layer of the new pit mud is slightly reduced, and the proportion of caproic acid bacteria is slightly increased but is not obvious. In general, 0-1cm pit mud surface still maintains the condition that lactobacillus is dominant, and the yellow water deposition in the white spirit fermentation process is probably related after improvement. Since 95% of the flora in yellow water is currently known to be lactobacilli. However, 1-3cm pit mud flora is obviously improved, which shows that the improved scheme of the invention has the effect. In order to accelerate the aging of the pit mud in the new pit, the bottom mud in the new pit needs to be improved later after each row of fermentation is finished, and the pit mud is continuously improved.
Claims (6)
1. A method for improving pit mud at the bottom of a pool in a layered manner by artificially culturing bacterial liquid is characterized by comprising the following steps:
step 1: dividing pit mud at the bottom of a mature old pit and a new pit to be improved into four layers of depths, and sampling pit mud in each layer of depth by three points, namely a central point, an angular point and a midpoint of a connecting line of the central point and the angular point;
step 2: sampling pit mud samples of the old pit and the new pit obtained in the step 1 to detect the distribution of floras, wherein the detection method comprises but is not limited to non-culture molecular biological methods such as high-throughput sequencing and the like to obtain the real composition of the pit mud floras;
and step 3: sampling pit mud of the old pit and the new pit obtained in the step 1, and detecting physicochemical properties, wherein the physicochemical properties comprise a pH value, an oxidation-reduction potential and the like;
and 4, step 4: expanded culture of artificially cultured bacterial liquid
And respectively carrying out group culture on the surface pit mud and the deep pit mud according to the detection result of the flora composition of the old pit mud.
4a, uniformly mixing the surface layer old pit mud and sterile deionized water according to a mass-volume ratio of 0.5-2g:5-10mL to obtain a mixed solution A; adding 5-20% of old pit yellow water and 5-20g/L of caproic acid into the obtained mixed solution A, finally adjusting the pH value to 6.0-8.0, and standing and culturing at 30-34 ℃ for 5-10 days to obtain surface pit mud maintenance liquid for improving the surface pit mud of a new pit;
4B, uniformly mixing the deep aged pit mud and sterile deionized water according to the mass-volume ratio of 0.5-2g:5-10mL to obtain a mixed solution B; adding 25% of old pit yellow water and 5-7g/L of acetic acid into the obtained mixed solution B, adjusting the pH value to 7.0-9.0, removing oxygen by using dry ice, and standing and culturing at 30-34 ℃ for 5-10 days to obtain deep pit mud maintenance liquid for improving the deep pit mud in the new pit;
and 5: improvement of pit mud at bottom of new pit
Firstly, inserting pit mud at the bottom of a new pit into a hole with the depth of 1-7cm by using a pit mud steel plate fork, uniformly spraying deep pit mud curing liquid into the hole, and smoothing the surface of the pit mud by using a tool; and then, inserting the pit mud at the bottom of the new pit into a hole with the depth of 0-1cm by using a pit mud steel plate fork, uniformly spraying surface pit mud curing liquid into the hole, leveling by using a tool, and using the pit for entering the pit for fermentation after improvement.
2. The method of claim 1, wherein:
in the step 1, the age of the old pit is more than or equal to 50 years; the age of the new pit is less than 10 years; the depth of the four layers is 0-1cm, 1-3cm, 3-5cm and 5-7cm in vertical depth.
3. The method of claim 1, wherein:
in the step 4, the surface layer old pit mud is old pit mud with the vertical depth of 0-1 cm; the deep layer aged pit mud is aged pit mud with the vertical depth of 1-5 cm.
4. The method of claim 1, wherein:
according to the different maturity of the cellar mud of the new cellar, the improved parameters in the step 5 are set as follows:
5. the method of claim 1, wherein:
the low-maturity pit mud and the medium-maturity pit mud are divided according to the detection results of the steps 2 and 3, and the specific standards are as follows:
6. the method according to claim 1 or 5, characterized in that:
the aged pit mud for group culture in the step 4 belongs to high-maturity pit mud.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112159061A (en) * | 2020-09-07 | 2021-01-01 | 中国环境科学研究院 | Method for accurately positioning and in-situ treating pollutants on mud-water interface |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112159061A (en) * | 2020-09-07 | 2021-01-01 | 中国环境科学研究院 | Method for accurately positioning and in-situ treating pollutants on mud-water interface |
| US11753317B2 (en) | 2020-09-07 | 2023-09-12 | Chinese Research Academy Of Environmental Sciences | Method for accurate positioning and in-situ treatment of pollutants at sediment-water interface |
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