Directional domestication and algae mud anaerobic acidogenic strengthening method for cellulose degradation acidogenic flora

Abstract

The invention discloses a directional domestication method of cellulose degradation acid-producing flora and an algae mud anaerobic acid production strengthening method. The method comprises the following steps: by taking anaerobic sludge as a bacterium source, carrying out continuous passage domestication in an anaerobic culture medium containing inorganic salt, buffer salt and a reducing agent by taking sodium carboxymethyl cellulose as a restrictive substrate and taking glucose as an auxiliary starting carbon source; transferring to a fresh culture medium according to 5-20% (v / v) after each generation is finished, increasing the concentration of sodium carboxymethyl cellulose generation by generation, reducing the concentration of glucose generation by generation until the concentration is 0, and repeating for 4-8 generations to obtain the flora. And centrifugally washing the flora, externally adding the flora into an algae mud acidification system according to 1-20% (v / v) of the working volume of the fermentation system, adding 0.5-5g / L of 2-bromoethanesulfonic acid sodium salt, adjusting the initial pH value to 9-11, and carrying out anaerobic reaction at 30-40 DEG C for 5-15 days to obtain fermentation liquor enriched with volatile fatty acids.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of environmental biotechnology and biomass resource utilization technology, and specifically relates to a method for directional domestication of a cellulose-degrading acid-producing mixed bacterial community, and a method for strengthening the enrichment of volatile fatty acids in the process of anaerobic acid production of algal sludge (including blue-green algal sludge) by using the bacterial community. BACKGROUND

[0002] Eutrophic water bodies such as lakes and reservoirs are prone to blue-green algae blooms under suitable light and temperature conditions. During the process of emergency disposal and normal management of water blooms, algae bodies usually need to be salvaged, concentrated and dewatered to form algal sludge which needs to be further disposed. Algal sludge generally has a high water content and a high proportion of degradable organic matter. If it is directly stored or disposed of improperly, it is easy to cause environmental risks such as leachate and odor. Anaerobic treatment of algal sludge as wet biomass substrate, especially its conversion into volatile fatty acids (VFA) in the acidification stage of anaerobic digestion, can provide available carbon sources for subsequent biological conversion processes and has certain resource utilization potential.

[0003] However, in the process of anaerobic acidification of algal sludge, the dissolution and hydrolysis of the substrate often become the limiting link. Algal sludge usually contains extracellular polymeric substances (EPS) and structural components such as cell walls / sheath layers. The polysaccharide network and support structure formed by these structural components may enhance the density of the floc and reduce the release rate of soluble organic matter, thereby limiting the hydrolysis and conversion of macromolecular organic matter, resulting in an impact on the VFA generation rate and accumulation level in the acidification stage.

[0004] In order to strengthen the dissolution and hydrolysis of algal sludge, some existing solutions propose to use physical and chemical pretreatment methods such as heat treatment, alkali treatment, ultrasonic, high-pressure homogenization to destroy the cell and floc structure, promote the release of soluble components and hydrolysis reaction. However, in engineering applications, the above methods may bring about an increase in energy consumption or reagent consumption, an increase in equipment investment and maintenance cost, and a burden of pH adjustment, salt accumulation or byproduct disposal during operation. On the other hand, the use of exogenous addition of a single strain or a single enzyme preparation to promote the hydrolysis of specific components may improve the local reaction under certain conditions, but in a complex anaerobic microecological system, it is easily affected by factors such as substrate composition fluctuation, niche competition, tolerance difference and batch stability, and the strengthening effect is difficult to be stable for a long time.

[0005] Therefore, it is still necessary to develop a mixed bacterial community that can stably exhibit cellulose-like polysaccharide hydrolysis and acid production functions under mesophilic anaerobic conditions, and form a matching preparation and application method, so as to reduce the dependence on high-energy physical and chemical pretreatment while improving the dissolution, hydrolysis and VFA accumulation efficiency in the anaerobic acidification stage of algal sludge. SUMMARY

[0006] Technical problems to be solved In view of the problem that the substrate dissolution and hydrolysis are limited due to the structural components of algae (such as EPS and cell wall / sheath polysaccharide network) in the anaerobic acidification process of algae sludge, and the VFA generation and accumulation level is low, the present application aims to provide a method for constructing a mixed bacterial community with cellulose-like polysaccharide hydrolysis and acid production functions under anaerobic mesophilic conditions, and to provide a method for strengthening the bacterial community in the anaerobic acid production process of algae sludge, so as to improve the starting efficiency and functional stability and reduce the dependence on high-energy consumption physical and chemical pretreatment.

[0007] Technical solutions To achieve the above-mentioned purpose, the present application adopts the following technical solutions.

[0008] 1) Directional domestication method of cellulose-degrading acid-producing bacterial community The present application provides a domestication method for obtaining and maintaining a mixed bacterial community with cellulose-like polysaccharide hydrolysis and acid production functions under anaerobic mesophilic conditions, characterized by comprising the following steps: S1) Preparation of domestication medium An anaerobic enrichment domestication medium is prepared, which comprises inorganic salts, buffer salts, reducing agents and carbon sources; the carbon sources consist of restrictive substrates and auxiliary starting carbon sources, or at least include restrictive substrates and auxiliary starting carbon sources, wherein the restrictive substrates are cellulose and / or its derivatives, preferably carboxymethyl cellulose sodium (CMC-Na); the auxiliary starting carbon sources are fermentable sugars, preferably glucose. The fermentable sugars are preferably monosaccharides or disaccharides, which can be glucose, fructose, sucrose, maltose, lactose or xylose, etc. Preferably, no other fermentable carbon sources are added in the medium except the restrictive substrates and the auxiliary starting carbon sources, so that the selection pressure mainly comes from the difference in carbon source form.

[0009] The restrictive substrates are used to continuously exert the selection pressure of cellulose-like polysaccharide hydrolysis during the domestication process.

[0010] The auxiliary starting carbon sources are used to improve the starting efficiency at the initial stage of domestication, and are gradually decreased to zero in the continuous passage domestication process, so as to realize the smooth transition of carbon sources from easy degradation to cellulose-like polysaccharides. Wherein, the gradual decrease of auxiliary starting carbon sources can avoid weakening the selection pressure provided by the restrictive substrates due to the long-term existence of auxiliary starting carbon sources, and can also avoid the delay of starting or the lengthening of domestication period caused by the direct absence of starting carbon sources at the initial stage of domestication.

[0011] S2) Inoculation and culture Anaerobic sludge is used as the source of bacteria, and is inoculated into the medium of step S1) at 5% to 20% (v / v) of the volume of the medium; the culture is carried out under anaerobic conditions at 30 to 40℃ for 7 to 14 days to obtain the current generation culture solution.

[0012] S3) Continuous subculture and carbon source gradient transition The culture solution of the previous generation is transferred to fresh culture medium of step S1) at 5-20% (v / v) for the next generation acclimation; in each generation acclimation, the concentration of the limiting substrate is increased and the concentration of the auxiliary starting carbon source is decreased, until the concentration of the auxiliary starting carbon source is 0, to form a mixed bacterial flora with the core functions of cellulose-like polysaccharide hydrolysis and acid production. Preferably, during the continuous generation acclimation, the total amount of the added carbon source (the sum of the mass concentrations of the limiting substrate and the auxiliary starting carbon source) remains constant.

[0013] S4) Obtain bacterial flora Step S3) is repeated for 4-8 generations to obtain a cellulose-degrading acid-producing bacterial flora.

[0014] To improve the repeatability and comparability between generations of the acclimation process, "pH of the culture solution decreases and then stabilizes" can be used as the criterion for ending the culture of the generation and transferring; preferably, the pH change is not more than 0.1 within 12-24 hours; under engineering or laboratory conditions, the transfer time point can also be determined in combination with a fixed culture period (such as 7-14 days).

[0015] 2) Cellulose-degrading acid-producing bacterial flora The present application also provides a cellulose-degrading acid-producing bacterial flora, which is prepared by the above-mentioned directional acclimation method, and has the main functional phenotypes of cellulose-like polysaccharide hydrolysis and acid production, and can be used for strengthening the anaerobic acidification stage of algal sludge.

[0016] 3) Strengthening method of the bacterial flora in algal sludge anaerobic acid production The present application further provides a method for strengthening algal sludge anaerobic acid production, characterized in that it comprises the following steps: A1) Construction of algal sludge acidification system An anaerobic acidification fermentation system is constructed with algal sludge as the substrate; the system can contain inoculated sludge, and the mass ratio of the substrate to the inoculated sludge is 1:1-4:1 based on total solids (TS).

[0017] A2) Preparation and addition of exogenous bacterial flora The above-mentioned cellulose-degrading acid-producing bacterial flora is prepared into an exogenous bacterial liquid; before addition, the bacterial cells are preferably collected by centrifugation and washed 1-3 times (such as bicarbonate buffer solution or phosphate buffer solution) with a buffer solution to reduce the interference of residual soluble carbon sources in the acclimation medium on the acidification process and acid production evaluation; the bacterial liquid is added at 1-20% (v / v) of the working volume of the fermentation system.

[0018] A3) Construction of acidification niche (methane production inhibition and initial pH regulation) A methanogenesis inhibitor, preferably sodium 2-bromoethane sulfonate (BES), is added to the fermentation system in an amount of 0.5-5 g / L to reduce the consumption tendency of carbon flow to the methanogenesis pathway, and the initial pH of the fermentation system is adjusted to 9-11 (e.g., using NaOH, KOH, Na2CO3 or Ca(OH)2) to promote the release, hydrolytic conversion and VFA accumulation of soluble organic matter. Among them, the methanogenesis inhibitor is used to inhibit the further conversion and consumption of VFA to the methanogenesis pathway; the alkaline initial pH is conducive to the dissolution and hydrolysis of the structural components of the algal sludge and forms a niche window conducive to acidification; the combination of the two can increase the accumulation level of VFA and enhance the process stability.

[0019] A4) Anaerobic mesophilic acidification reaction The fermentation system is subjected to inert gas replacement and sealing, and reacted under anaerobic conditions at 30-40°C for 5-15 days to obtain a fermentation broth rich in volatile fatty acids.

[0020] Advantages Compared with the prior art, the present application has at least the following advantages: (1) Clear path and directional selectivity of the microbial community: the transition acclimation strategy of "increasing the limiting substrate generation by generation + decreasing the auxiliary starting carbon source generation by generation until 0" provides sufficient starting drive in the early stage of acclimation, and gradually strengthens the selection pressure on the cellulose-like polysaccharide hydrolysis ability in the continuous passage process, so that the cellulose-like polysaccharide hydrolysis-acid-producing functional microbial community can be stably obtained and maintained under anaerobic mesophilic conditions.

[0021] (2) Strengthening the dissolution / hydrolysis of algal sludge and promoting VFA enrichment: the microbial community is added to the algal sludge anaerobic acidification system, and the acidification niche is constructed by combining the methanogenesis inhibitor (such as BES) and the alkaline initial pH regulation, which can promote the dissolution and hydrolytic conversion of the structural polysaccharide network, reduce the risk of further consumption of VFA by the methanogenesis pathway, increase the generation and accumulation level of VFA and enhance the process stability.

[0022] For the sake of understanding the technical solutions and beneficial effects of the present application, without constituting any limitation to the present application, the present application believes that: in the acclimation stage, if the auxiliary starting carbon source always exists, it may weaken the restrictive substrate selection pressure and lead to the dependence of the bacterial community on the easily degradable carbon source; if the auxiliary starting carbon source is not directly set at the beginning of acclimation, slow start, extended generation or reduced acclimation efficiency may occur. By setting and gradually withdrawing the auxiliary starting carbon source, a balance between "starting efficiency" and "selection pressure" can be achieved, thereby promoting the convergence of the community to the cellulose-like polysaccharide hydrolysis and acid production core functions. In the application stage, if the methanogenesis process is not inhibited, the generated VFA may be further converted and consumed; if the initial pH is in the non-alkaline interval, the algal sludge dissolution / hydrolysis rate may be low. By combining methanogenesis inhibition and alkaline initial pH, an acidification niche window that is conducive to VFA accumulation can be formed, and synergistic enhancement of the hydrolysis-acid production function of the bacterial community can be achieved.

[0023] (3) The evaluation of the added exogenous bacteria is more objective and has stronger comparability: before the addition of the exogenous bacteria, centrifugation and washing with a buffer solution can reduce the carrying and interference of residual soluble carbon sources in the acclimation medium, so that the acid production enhancement effect can better reflect the real contribution of the bacterial community to the algal sludge dissolution / hydrolysis and acid production functions.

[0024] (4) Technical effect example: under a group of embodiment conditions (see Figure 2 , Figure 4 and Table 2), the CMC-Na degradation rate of the 5th generation bacterial community increased from 30.1% to 78.2%, and the total cellulase activity increased from 3.5 U / mL to 5.8 U / mL; after adding the 5th generation bacterial community and BES in the algal sludge acidification system (see Figure 8 and Table 3), the SCOD increased from 1645 mg / L to 2103 mg / L (about 27.8%) after 2 days of reaction, the total VFA increased from 1.41 g / L to 1.90 g / L (about 34.8%) after 10 days of reaction, and the terminal TS decreased from 7.11% to 5.23% (about 26.4%), indicating that the bacterial community can effectively promote dissolution, hydrolysis and VFA accumulation.

[0025] (5) Engineering application potential: compared with high-energy consumption physical and chemical pretreatment methods, the present application mainly uses biological enhancement, which can reduce energy consumption, drug consumption and equipment requirements to a certain extent, and reduce the operating burden that may be brought by strong pretreatment. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Schematic diagram of the passage and carbon source gradient setting for the directional acclimation of the bacterial community; Figure 2 Schematic diagram of the total cellulase activity (a) and CMC-Na degradation rate (b) changes of the bacterial community at different acclimation generations; Figure 3 Figures for the changes of acetic acid acidification rate (a) and VFA composition ratio (b) of different acclimated generations of bacterial flora; Figure 4 Figure for the changes of reducing sugar content and pH with time during the acclimation of the 5th generation of bacterial flora; Figure 5 Figure for the OTU wein plot of different samples, wherein A0 is the original bacterial source sample, and A1-A5 are the 1st to 5th acclimated bacterial flora samples, respectively; Figure 6 Figure for the relative abundance at the phylum level of different samples, wherein Y0-Y5 correspond to A0-A5 in Figure 5 , respectively; Figure 7 Figure for the relative abundance heat map at the genus level of different samples, wherein the meanings of A0-A5 are the same as Figure 5 ; Figure 8 Figure for the effects of different generations of bacterial flora on the anaerobic acid production of algal sludge, wherein (a) is the change of SCOD, (b) is the change of VFA composition and total amount, and (c) is the change of TS. DETAILED DESCRIPTION

[0027] 1. Terms and detection methods In the following examples, unless otherwise specified, the operations are carried out under anaerobic conditions, which can be achieved by introducing nitrogen gas or nitrogen gas / carbon dioxide mixed gas into the headspace of a reactor or a serum bottle for 3-10 minutes and sealing.

[0028] (1) Algal sludge: refers to the wet biomass obtained by salvaging or enriching a cyanobacterial bloom, which can contain algal cells, extracellular polymers, and associated microorganisms and organic debris, etc.; wherein the cyanobacterial sludge is a preferred embodiment.

[0029] (2) VFA (volatile fatty acid): includes short-chain fatty acids such as acetic acid, propionic acid, butyric acid, valeric acid, etc. After the sample is centrifuged at 8000-12000 r / min for 5-15 minutes to remove the solid phase, the supernatant is treated with acidification and internal standard, and the concentrations of each component are determined by gas chromatography and summed to obtain the total VFA.

[0030] (3) Acetic acid acidification rate: preferably in terms of mole fraction, acetic acid acidification rate (%) = n(acetic acid) / ∑n(each VFA) x 100%.

[0031] (4) SCOD (soluble chemical oxygen demand): the COD value measured after centrifugation or filtration (e.g. 0.45 μm filter) of the sample to remove suspended solids; COD determination can use potassium dichromate digestion method or equivalent standard method.

[0032] (5) TS / VS: total solid / volatile solid, TS can be measured by drying at 105°C to constant weight, VS can be measured by ignition at 550°C.

[0033] (6) Reducing sugar concentration: DNS method can be used.

[0034] (7) CMC-Na degradation rate: the initial CMC-Na mass concentration Co and the residual mass concentration C after cultivation are measured, and the degradation rate (%) is calculated. t CMC-Na degradation rate (%) = (Co - C) / Co x 100%. t t The residual polysaccharide concentration in the supernatant can be measured by the phenol-sulfuric acid method and converted according to the standard curve.

[0035] (8) Total cellulase activity: filter paper method (FPA) or equivalent method can be used. As an example, the supernatant is taken as the crude enzyme solution, reacted with filter paper substrate at 50°C, and then colored with DNS, the absorbance is measured at 540 nm and converted to enzyme activity; the enzyme activity is defined as the amount of enzyme that produces 1 μmol of glucose equivalent reducing sugar per minute per 1 mL of crude enzyme solution.

[0036] (9) Microbial community analysis (optional): the microbial liquid at the end of fermentation is sampled, centrifuged to collect the precipitate, and stored at -80°C, the 16S rRNA gene V3-V4 region is amplified and sequenced for analysis.

[0037] Example 1: Directional domestication of cellulose-degrading acidogenic bacteria (1) Source of bacteria: anaerobic granular sludge or digested sludge is used as the source of bacteria. As an example, the anaerobic granular sludge is taken from the anaerobic reactor of a domestic waste or sewage treatment system, the sludge pH is 7.4±0.1, the total solid TS is 140.3±2.70 g / kg, and the volatile solid VS is 90.1±2.28 g / kg.

[0038] (2) Anaerobic enrichment domestication medium: the basic components of the medium are: NH4Cl 0.5 g / L, KH2PO4 0.2 g / L, MgSO4·7H2O 0.1 g / L, CaCl2 0.02 g / L, NaHCO3 3.0 g / L, L-cysteine 0.1 g / L, and trace element solution 1 mL / L and vitamin solution 1 mL / L are added respectively; the medium is prepared and used immediately, and sterilized by 121°C high pressure steam for 20 minutes before use.

[0039] ​The trace element solution (mother liquor) can comprise: FeSO4·7H2O 0.1 g / L, ZnSO4·7H2O 0.1 g / L, H3BO3 0.01 g / L, N(CH2COOH)3 1.5 g / L, Na2MoO4·2H2O 0.01 g / L, CoCl2·6H2O 0.1 g / L, NiCl2·6H2O 0.024 g / L, Na2WO4·2H2O 0.025 g / L, MnSO4·H2O 0.5 g / L, NaCl 1 g / L, CuSO4·5H2O 0.01 g / L, KAl(SO4)2·12H2O 0.01 g / L.

[0040] The vitamin solution (mother liquor) can comprise: cobalamin 0.01 g / L, vitamin C 0.025 g / L, riboflavin 0.025 g / L, citric acid 0.02 g / L, pyridoxal 0.05 g / L, p-aminobenzoic acid 0.01 g / L, creatine 0.025 g / L.

[0041] (3) Carbon source gradient setting: CMC-Na and glucose are added to the culture medium as carbon sources, wherein CMC-Na is a restrictive substrate and glucose is an auxiliary starting carbon source. The domestication adopts the strategy of increasing the concentration of CMC-Na and decreasing the concentration of glucose in each generation until 0, and the carbon source concentration of each generation is shown in Table 1.

[0042] (4) Cultivation and subculture: the culture medium is divided into serum bottles, and the working liquid volume is 100 mL. After nitrogen replacement for 5 minutes, it is sealed. The anaerobic sludge is inoculated at 10% (v / v), and cultured at 35±1℃ under the condition of 120 r / min shaking for 10 days; after each generation of culture, 10 mL of culture solution (about 10% (v / v)) is transferred to fresh culture medium for the next generation of domestication, and the domestication is repeated to the 5th generation to obtain a cellulose-degrading acid-producing bacterial community. Preferably, the pH decrease and tendency to stabilize at the end of the culture are used as the subculture endpoint criterion, preferably the pH change is not more than 0.1 within 12-24 consecutive hours.

[0043] Table 1 Carbon source concentration gradient of different generations (unit: g / L) Example 2: Performance characterization of domesticated bacterial community The CMC-Na degradation rate, total cellulase activity, and acid-producing capacity of the bacterial community of different domestication generations in Example 1 were characterized.

[0044] As Figure 2As shown, with the increase of acclimation generation, the cellulase activity and CMC-Na degradation rate of the bacterial flora showed an overall upward trend; the CMC-Na degradation rate of the first generation of bacterial flora was 30.1%, and the total cellulase activity was 3.5 U / mL, and the CMC-Na degradation rate of the fifth generation of bacterial flora increased to 78.2%, and the total cellulase activity increased to 5.8 U / mL.

[0045] As shown in Figure 3 , the acetic acid acidification rate of the fifth generation of bacterial flora was 62%, which was about 13 percentage points higher than that of the first generation, and the VFA composition changed, and the proportion of butyric acid increased with the increase of acclimation generation.

[0046] As shown in Figure 4 , during the culture of the fifth generation of bacterial flora with CMC-Na as the only carbon source, the reducing sugar concentration first increased and then decreased, with a maximum of 1.81 mg / L; the pH decreased during 2-6 days of culture and finally stabilized at about 5.58, indicating that the bacterial flora could stably produce acid under this condition and tend to be balanced.

[0047] Alternatively, 16S rRNA high-throughput sequencing can be used to analyze the changes in the structure of the bacterial flora. As an example of the results, at the end of the acclimation, the original bacterial source sample and the first to fifth generation of acclimated bacterial flora samples (A0-A5 in Figure 5 , Y0-Y5 in Figure 6 ) were collected for OTU and community composition analysis. Figure 5 As shown, the total (shared) OTU number of each sample was 32, the original bacterial source sample had 179 unique OTUs, and the unique OTU number gradually decreased with the increase of acclimation generation (A1 was 46, A2 was 16, A3 was 16, A4 was 13, and A5 was 3), indicating that the community gradually converged to the core functional group under the selective pressure of CMC-Na limited substrate. Figure 6 As shown in the door level analysis, the relative abundance of Bacteroidetes increased from 2.2% to 73%, becoming the dominant phylum; Figure 7 As shown in the genus level heatmap, Bacteroides, Syntrophobacter, Sporanaerobacter and other groups were further enriched during the acclimation process.

[0048] Table 2 Comparison of key performance indicators (first generation and fifth generation) Example 3: Application of acclimated bacterial flora in anaerobic acid production of algal sludge The effect of exogenous addition of acclimated bacterial flora on the dissolution, hydrolysis and acid production of algal sludge was evaluated by sequencing batch fermentation experiment.

[0049] (1) Substrate and inoculation: The TS of algal sludge is 14%; the mass ratio of substrate to inoculated sludge (in TS) is 2:1. The mixture is dispensed into serum bottles with a total volume of 120 mL and a working volume of 80 mL.

[0050] (2) Group setup: A total of 6 experimental groups were set up, namely the CK group (no exogenous bacteria were added) and 5 experimental groups with the first to fifth generation domesticated bacteria; BES was added to each group and the initial pH was adjusted to 10; 3 parallel samples were set up in each group.

[0051] (3) Treatment and addition of exogenous microbial community: Take the domesticated microbial community obtained in Example 1, collect it by centrifugation, and wash it twice with buffer solution to remove residual soluble carbon source in the culture medium; add exogenous microbial solution at 10% (v / v) of the working volume of the fermentation system.

[0052] (4) Acidification conditions: Add 2 g / L of BES to the fermentation system to inhibit the methanogenesis process; use NaOH solution to adjust the initial pH of the fermentation system to 10.

[0053] (5) Anaerobic replacement and fermentation: The headspace of the serum bottle was purged with high-purity nitrogen for 5 minutes and then sealed. It was then placed under anaerobic conditions of 37℃ and 120r / min for 10 days.

[0054] (6) Sampling and testing: Samples were taken at 0, 2 and 10 days of reaction to determine SCOD, total VFA and TS and other indicators.

[0055] (7) Results: such as Figure 8 As shown, compared with the CK group, the system with the addition of the 5th generation domesticated bacteria increased SCOD from 1645 mg / L to 2103 mg / L after 2 days of reaction; total VFA increased from 1.41 g / L to 1.90 g / L after 10 days of reaction; and the reaction endpoint TS decreased from 7.11% to 5.23%, indicating that the exogenous addition of domesticated bacteria can promote the dissolution and hydrolysis of algal mud and increase acid production accumulation.

[0056] also, Figure 8 (a) The addition of different generations of domesticated bacterial groups showed differences in the dissolution of algal mud: after 2 days of reaction, the SCOD of the first to fifth generation bacterial groups were 1806, 1825, 2005, 2036 and 2103 mg / L, respectively, all of which were higher than those of the CK group.

[0057] Table 3 Comparison of key indicators for anaerobic acid production in algal sludge (CK vs. 5th generation bacterial colony addition) Comparative Examples and Optional Implementation Methods To facilitate the attribution analysis of the key technical features of the present application in the process of anaerobic acid production of algal sludge, and to improve the interpretability and repeatability of comparison, the following control tests can be set based on Example 3. Unless otherwise specified, each control test is the same as Example 3 in terms of substrate and inoculation (including substrate TS and substrate / inoculated sludge mass ratio), reactor form and working volume, anaerobic replacement and sealing method, fermentation temperature, stirring / oscillation conditions, reaction time, exogenous bacteria liquid treatment method (if applicable), and sampling and detection method. Only the conditions described in the control item are different.

[0058] Comparative Example 1: Blank control / no addition of exogenous domesticated bacteria This comparative example does not add exogenous domesticated bacteria (the amount of exogenous bacteria liquid added is 0), and the rest of the conditions are the same as Example 3. By comparing SCOD, total VFA, and TS indicators with Example 3 (for example, the group that adds the 5th generation of domesticated bacteria), the strengthening contribution of exogenous domesticated bacteria to the dissolution, hydrolysis, and acid accumulation of algal sludge is evaluated. As an example, the relevant comparison results are shown in Figure 8 and Table 3: Compared with the group that adds the 5th generation of domesticated bacteria, the blank control group has lower SCOD at 2 days of reaction and lower total VFA at 10 days of reaction, and higher TS at the end point, indicating that the addition of domesticated bacteria can promote the release of soluble organic matter and the accumulation of VFA, and accelerate the conversion of solid organic matter to liquid phase.

[0059] Comparative Example 2: Un-domesticated / low-generation bacteria source control This comparative example adds un-domesticated bacteria or low-generation bacteria as a control. The un-domesticated bacteria can be selected as the original anaerobic sludge, or the initial generation of domesticated bacteria (for example, the 1st generation of bacteria) is used as a low-generation representative. The rest of the conditions are the same as Example 3. Preferably, to exclude the interference of introducing exogenous soluble carbon source on acid production evaluation, the added bacteria liquid can be centrifuged and washed with buffer solution before addition according to Example 3. By comparing the SCOD release, total VFA accumulation, and TS changes of different domesticated generations (un-domesticated / low-generation and high-generation) under the same algal sludge acidification conditions, the contribution of "directed domestication" to the strengthening effect and the trend of improvement with generation are evaluated. As an example, Figure 8 (a) shows that with the increase of domesticated generation, the SCOD at 2 days of reaction generally shows an increasing trend (for example, the 1st generation group is lower than the 5th generation group), indicating that the improvement of domesticated generation helps to enhance the promotion of algal sludge dissolution / hydrolysis.

[0060] Optional implementation Without departing from the overall concept of the present application, which is to obtain a cellulose-like polysaccharide hydrolysis-acid producing functional bacteria group by "restrictive cellulose-like polysaccharide selection pressure + auxiliary start-up carbon source transition exit" and to construct an acidification niche for VFA enrichment, the present application can also use the following optional implementation: (1) Alternative and equivalent selection of restrictive substrate: In addition to sodium carboxymethylcellulose (CMC-Na), the restrictive substrate can also be selected from microcrystalline cellulose, filter paper powder, cotton cellulose, regenerated cellulose, cellulose powder or cellulose derivatives such as hydroxyethyl cellulose and hydroxypropyl methyl cellulose, or a combination thereof, to continuously exert selective pressure on the hydrolysis of cellulose-like polysaccharides. Preferably, at the end of the domestication terminal generation (e.g. the last 1 generation or the last 2 generations), the restrictive substrate becomes the only additional fermentable carbon source in the system to avoid the dependence of the microbial community on easily degradable carbon sources and to facilitate the stable acquisition of the target functional phenotype. For insoluble or difficultly dispersed restrictive substrates, mass transfer can be improved by pre-wetting, particle size control, and increasing stirring / oscillation intensity; the dosage can be adjusted according to the equal COD or equal carbon content principle within the concentration range defined in claim 3.

[0061] The theoretical COD value (or measured COD value) of each cellulose material can be used as a conversion basis. The conversion formula is as follows: C X = C CMC-Na × COD CMC-Na / COD X In the formula, C X is the dosage mass concentration of the alternative substrate (g / L), C CMC-Na is the mass concentration of CMC-Na to be replaced (g / L), COD CMC-Na and COD X are the unit mass COD values of CMC-Na and the alternative substrate, respectively (g O2 / g).

[0062] According to literature and measured data, the theoretical COD value of sodium carboxymethylcellulose (CMC-Na) is about 1.07 g O2 / g, and the theoretical COD value of microcrystalline cellulose is about 1.18 g O2 / g. When the dosage concentration of CMC-Na in the examples is to be replaced by microcrystalline cellulose, the calculated dosage concentration is about: Cmicrocrystalline cellulose = (2.0 g / L x 1.07 g O2 / g) / (1.18 g O2 / g) ≈ 1.81 g / L Accordingly, when the mass concentration of sodium carboxymethylcellulose described in the present application is 0.5-5.0 g / L, the equivalent COD dosage of microcrystalline cellulose is about 0.45-4.53 g / L. Other alternative substrates can be converted according to their specific COD values.

[0063] Preferred particle size range: for insoluble or hardly soluble restrictive substrates such as microcrystalline cellulose, filter paper powder, cellulose powder, etc., to improve their dispersibility and microbial accessibility, their physical form is preferably controlled as powder, and the particle size range is preferably 75 μm to 850 μm (about 200 mesh to 20 mesh). Within this particle size range, the material has a suitable specific surface area, which is conducive to microbial adhesion and enzymatic hydrolysis, while avoiding agglomeration or operational difficulties caused by excessively fine particle size. For fibrous substrates such as cotton cellulose, appropriate shearing or grinding pretreatment can be performed to achieve similar size effects.

[0064] (2) Alternative and decreasing exit of auxiliary starting carbon source: In addition to glucose, the auxiliary starting carbon source can also be selected from easily fermentable monosaccharides or disaccharides such as fructose, sucrose, maltose, lactose, xylose, etc., or combinations thereof; the auxiliary starting carbon source is preferably provided only at the initial stage of acclimation to provide starting drive, and is gradually decreased to zero during continuous passage, so as to maintain a sustained selection pressure on the restrictive substrate while taking into account the starting efficiency. The decreasing method can be linear decreasing, segmented decreasing, or self-adaptive decreasing according to the pH drop and VFA accumulation rate of the culture solution.

[0065] Example of equimolar carbon amount conversion: Glucose (C6H 12 O6, molar mass 180 g / mol) contains 40% carbon. If the first generation of auxiliary carbon source is replaced by sucrose (C 12 H 22 O 11 , molar mass 342 g / mol, carbon content 42.1%), to provide approximately equimolar carbon amount, the addition amount of sucrose can be calculated as follows: m 蔗糖 = m 葡萄糖 × (40.0% / 42.1%) ≈ 2.0 g / L×0.40 / 0.421 ≈ 1.90 g / L Accordingly, a sucrose decreasing gradient example for a 5-generation acclimation can be: 1.9, 1.4, 1.0, 0.5, 0 g / L.

[0066] (3) Adjustable transition gradient setting and switching criteria: Under the basic principle of "restrictive substrate gradually increasing from generation to generation, auxiliary starting carbon source gradually decreasing to zero from generation to generation", the concentration gradient of each generation can be routinely optimized according to the pH change curve of the acclimation system, the reducing sugar change, the restrictive substrate degradation rate and the cellulase activity improvement trend; preferably, the total COD of the external carbon source of each generation is kept basically constant or changes within a controllable range, so that the selection pressure mainly comes from the difference in carbon source form rather than the total carbon amount. As an example, the carbon source gradient setting can be referred to Figure 1See Table 1; the endpoint criteria for each generation can be found in the pH stability criteria described in this instruction manual. For example, total COD can be controlled by keeping the total dosage of CMC-Na and auxiliary carbon source constant across generations; taking Table 1 as an example, the total dosage of CMC-Na and glucose is 3.0 g / L for each generation. When taking COD... CMC-Na When COD_glucose≈ 1.07 g O2 / g, the total COD of each generation of added carbon sources is approximately 3.21 g O2 / L, which remains basically constant.

[0067] (4) Scalability and adaptability of substrates: In addition to algal sludge (including cyanobacterial sludge), the microbial community can also be used for the anaerobic acidification of VFA production from other wet biomass substrates containing cellulose-like polysaccharides or EPS / structural polysaccharide networks with limited hydrolysis, such as microalgal sludge, algal residue, floating aquatic plant residue, paper / pulp fiber sludge, or food processing sludge containing cellulose-like polysaccharides. During application, within the parameter range defined in this invention, the substrate-to-inoculation sludge mass ratio, the amount of exogenous bacterial solution added, the amount of methanogenic inhibitor added, and the initial pH setting can be adjusted according to the substrate TS, buffering capacity, and salinity conditions to obtain a VFA-enriched fermentation broth.

[0068] (5) Preservation and recovery of microbial community: Optionally, in order to meet the needs of engineering addition, the microbial community can be preserved by continuous subculturing, short-term preservation at 4°C or cryopreservation at -80°C in a protectant containing 10% to 30% (v / v) glycerol; before use, it can be recovered for 1 to 2 generations in an anaerobic culture medium with limiting substrate as the main carbon source to restore the functional phenotype.

[0069] The above embodiments and comparative examples are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. Those skilled in the art can make reasonable substitutions, equivalent transformations, or combinations of the step sequence, process parameters, material selection, etc., in the various embodiments without departing from the concept of the present invention; all technical solutions obtained thereby, if not exceeding the scope of protection defined by the claims, should fall within the scope of protection of the present invention.

[0070] Unless otherwise expressly stated, the numerical ranges mentioned in this specification should be understood to include their endpoints; the technical features disclosed in the various embodiments can be combined with each other without creating contradictions. The scope of protection of this invention is determined by the claims, and the specification and drawings are used to interpret the claims.

Claims

1. A method for the targeted domestication of cellulose-degrading acid-producing bacteria, characterized in that, Includes the following steps: S1) Prepare an anaerobic enrichment and acclimatization medium, wherein the medium contains inorganic salts, buffer salts, reducing agents and carbon sources, wherein the carbon source consists of a limiting substrate and an auxiliary starting carbon source, wherein the limiting substrate is cellulose and / or its derivatives, and the auxiliary starting carbon source is a fermentable sugar. S2) Using anaerobic sludge as the inoculum source, the anaerobic sludge is inoculated into the culture medium at an inoculation amount of 5% to 20% (v / v) of the culture medium volume, and cultured under anaerobic conditions at 30 to 40°C for 7 to 14 days to obtain the current generation culture medium; S3) The previous generation culture medium is transferred to the fresh culture medium at a transfer volume of 5% to 20% (v / v) for the next generation acclimatization. In each generation acclimatization, the mass concentration of the limiting substrate is increased and the mass concentration of the auxiliary starting carbon source is decreased until the mass concentration of the auxiliary starting carbon source is 0. S4) Repeat step S3) for a total of 4 to 8 generations to obtain cellulose-degrading acid-producing bacteria.

2. The method according to claim 1, characterized in that: The anaerobic enrichment and acclimatization culture medium contains 0.1–1.0 g / L NH4Cl, 0.05–0.5 g / L KH2PO4, 0.02–0.5 g / L MgSO4·7H2O, 0.005–0.1 g / L CaCl2, 0.5–10 g / L NaHCO3, and 0.01–0.5 g / L L-cysteine, and also contains 0.1–5 mL / L each of trace element solution and vitamin solution.

3. The method according to claim 1, characterized in that: The limiting substrate is sodium carboxymethyl cellulose, and the auxiliary starting carbon source is glucose; the mass concentration of sodium carboxymethyl cellulose is 0.5–5.0 g / L; the mass concentration of glucose in the initial generation of domestication is 0.5–3.0 g / L, and decreases gradually to 0 in subsequent generations of domestication.

4. The method according to claim 1, characterized in that: The culture temperature for steps S2) and S3) is 35±1℃, and the culture is carried out under stirring or shaking conditions, with a stirring or shaking speed of 80~160 r / min; and the criterion for the end of each generation of culture and the transfer is that the pH of the culture medium tends to stabilize after decreasing, preferably the pH change does not exceed 0.1 within 12~24 hours.

5. The method according to claim 3, characterized in that: The domestication was carried out for 5 generations, and the carbon source concentrations for each generation were 1.0, 1.5, 2.0, 2.5, and 3.0 g / L for sodium carboxymethyl cellulose and 2.0, 1.5, 1.0, 0.5, and 0 g / L for glucose, respectively.

6. The method according to claim 1, characterized in that: During successive generations of domestication, the sum of the mass concentration of the limiting substrate and the mass concentration of the auxiliary starting carbon source remains constant in each generation.

7. A group of cellulose-degrading acid-producing bacteria, characterized in that: Prepared by the method described in any one of claims 1 to 6.

8. A method for enhancing anaerobic acid production from algal sludge, characterized in that, The process includes the following steps: A1) Using algal sludge as a substrate, mix it with inoculated anaerobic sludge at a mass ratio (based on total solids TS) of 1:1 to 4:1 to obtain a fermentation system; A2) Centrifuge and collect the bacterial community described in claim 7, wash it 1 to 3 times with a buffer solution to prepare an exogenous bacterial solution, and add it to the fermentation system at a volume of 1% to 20% (v / v) of the working volume of the fermentation system; A3) Add 0.5 to 5 g / L of sodium 2-bromoethanesulfonate to the fermentation system and adjust the initial pH of the fermentation system to 9 to 11; A4) Replace the headspace of the fermentation system with an inert gas and seal it, and anaerobically stir or shake it at 30 to 40°C for 5 to 15 days to obtain a fermentation broth enriched with volatile fatty acids.

9. The method according to claim 8, characterized in that: The total solids (TS) of the algal sludge are 5% to 20%, and the anaerobic stirring or oscillation speed in step A4) is 80 to 160 r / min.

10. The method according to claim 8 or 9, characterized in that: The mass ratio (in TS) of the substrate to the inoculated anaerobic sludge is 1.5:1 to 2.5:1; the amount of exogenous bacterial solution added is 8% to 12% (v / v) of the working volume of the fermentation system; the amount of sodium 2-bromoethanesulfonate added is 1.5 to 2.5 g / L; the initial pH is 9.5 to 10.5; the anaerobic reaction temperature is 36 to 38°C and the reaction time is 9 to 11 days.

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