Straw decomposition scheduling method for multi-strain continuous gradient inoculation

Abstract

The invention relates to a straw decomposition scheduling method for multi-strain continuous gradient inoculation. The method comprises the following steps: identifying a metabolic signal induction marker through a microenvironment evolution trend in a straw decomposition process; triggering a subsequent strain to start environment-driven continuous inoculation by taking a signal that the concentration of the induction marker reaches a threshold value; by screening complementary metabolic pathways between strains, designing a relay enzyme activity pairing mechanism between front and back gradient bacteria aiming at an intermediate product; metabolic intermediates generated by the strains in each stage are optimal substrates or activating factors of the next gradient bacteria; the ecological regulation and control capability of the strains is integrated, and alkali-producing yeast and scavenging bacteria are introduced; in the straw decomposition stage, trace alkaline metabolites are released to neutralize lignin toxins, and the buffer stability of the system is maintained; a strain decline interference window is constructed by adjusting environmental parameters in each gradient inter-strain process, so that new and old flora naturally relay; based on lignin stress accumulation characteristics, the inoculated fungus strain is induced to generate stress type laccase transient expression, so that the lignin is deeply depolymerized.

Description

Technical Field

[0001] This invention relates to a method for scheduling straw decomposition, specifically a method for scheduling straw decomposition through continuous gradient inoculation of multiple microbial strains. Background Technology

[0002] Currently, in straw decomposition scheduling methods, such as the Chinese patent CN105918615A "A method for producing large-scale rice and wheat straw microbial feed", there are still several core deficiencies and technical bottlenecks. In particular, improvements are urgently needed in areas such as microbial expression rhythm control, intelligent system scheduling, metabolic chain synergy, and raw material adaptability. These limitations restrict the further expansion and industrialization of existing technologies in the process of refined straw bioconversion and high-value utilization.

[0003] First, the existing technology adopts a fixed-stage inoculation and batch fermentation mode. Although its microbial composition covers a variety of microorganisms such as white-rot fungi, lignocellulose-degrading bacteria, brewer's yeast, and lactic acid bacteria, its scheduling logic is essentially based on artificial staged inoculation and artificially set culture batches. Although this method achieves the synergistic effect of functional bacteria to a certain extent, its microbial expression switching depends on artificial time-series control and lacks adaptive expression scheduling ability. It cannot accurately identify the expression status of each microbial community in a complex metabolic environment, nor can it automatically determine when to take over the dominant expression based on the metabolite accumulation status or environmental signals. Therefore, it has obvious shortcomings in terms of the continuity of degradation rhythm and the intelligence of system operation. Secondly, regarding the synergistic mechanism among microbial expression, existing technologies employ a crude parallel synergistic design, which involves directly adding white-rot fungi and degrading bacteria in the first stage of fermentation, followed by the sequential addition of Bacillus, yeast, lactic acid bacteria, etc. in subsequent stages. This results in passive competition among microbial species within the culture system rather than expression crossover, which easily leads to problems such as metabolic pathway conflicts, resource contention, and even premature depletion of functional bacteria. Especially in the stages of deep lignin degradation and protein synthesis, the inability to match the expression rhythm often leads to a decrease in the utilization efficiency of enzyme products.

[0004] Third, existing technologies lack systematic and coordinated control over the metabolic competition among microbial communities. Taking the three-stage inoculation process described in this patent as an example, lactic acid bacteria, Bacillus, and Saccharomyces cerevisiae participate in different stages of expression. However, in actual systems, these strains have highly overlapping requirements for oxygen, pH, and nutrients, easily leading to metabolic conflicts. Furthermore, due to the lack of a dynamic environmental regulation mechanism in this patent, once the expression becomes unbalanced at a certain stage, subsequent microbial communities may not be able to successfully take over, resulting in broken degradation chains or metabolic accumulation. In addition, existing technologies are mainly designed for rice and wheat straw. Although they have some universality, their core microbial system and process parameters are not adjustable to accommodate differences in different straw components (e.g., corn straw has a higher proportion of lignin, while wheat straw has a higher proportion of hemicellulose), lacking raw material-oriented process adjustment capabilities. Summary of the Invention

[0005] The purpose of this invention is to provide a method for scheduling straw decomposition through continuous gradient inoculation of multiple microbial species, thereby addressing some of the drawbacks and shortcomings pointed out in the background art.

[0006] The present invention addresses the aforementioned technical problems by employing the following technical solution: a method for scheduling straw decomposition through continuous gradient inoculation of multiple microbial species, comprising: identifying metabolic signal induction markers by observing the microenvironmental evolution trends, including oxygen pressure, electrical conductivity, and organic acid accumulation, during straw decomposition; and triggering the initiation of subsequent microbial species by using a threshold concentration of the induction marker as a signal, thereby enabling environment-driven continuous inoculation. By screening complementary metabolic pathway pairs between strains, a relay enzyme activity pairing mechanism was designed between the preceding and following gradient bacteria for intermediate products including arabinose, acetic acid, and furfural; so that the metabolic intermediates produced by the strains at each stage become the preferred substrates or activators for the next gradient bacteria. When designing the inoculation sequence and density, the ecological regulation capabilities of the strains were considered, and alkali-producing yeasts and H2O2-scavenging bacteria were introduced. This allowed the release of trace amounts of alkaline metabolites during the straw decomposition stage, neutralizing lignin toxins and maintaining the system's buffer stability. During the inoculation process at each gradient, environmental parameters were adjusted to construct a strain reduction interference window, allowing new and old strains to naturally take over. Based on the characteristics of lignin stress accumulation, stress-induced transient expression of laccase was induced in the introduced fungal strains, resulting in deep lignin depolymerization.

[0007] Furthermore, the process of triggering subsequent bacterial initiation by inducing a marker concentration reaching a threshold includes: Straw is divided into multiple decomposition stages based on its degradation characteristics, with each stage dominated by microbial strains with different functions. In any stage, the preceding strains naturally release metabolites during the degradation of straw. The activation or expression of subsequent strains is passively induced when the metabolites reach a threshold concentration in the system. The subsequent bacterial strains are in a dormant, low-metabolic, or passive coexisting state before being activated.

[0008] Furthermore, the triggering mechanism of the metabolites includes establishing a response threshold for microorganisms to small molecule products. The response is mediated by a cell membrane surface signal sensing system, triggering the expression of enzymes or promoter sequences encoded in downstream regulatory pathways, thereby enabling the self-initiation of bacterial expression. The dormant state of the subsequent bacterial strain before activation is physically isolated by low-temperature spore embedding or a degradable carrier, and the carrier depolymerizes in a metabolically acidic environment, forming an environmentally triggered release mechanism.

[0009] Furthermore, the preceding and following gradient bacteria share a class of intermediate metabolites, which are both decomposition products of the preceding strains and substrates for metabolic enzyme pathways absorbed and activated by the subsequent strains, thus creating a metabolic interlocking functional inheritance relationship between the strains.

[0010] Furthermore, the inoculation sequence allows some bacterial species to overlap and express for a short period under staged coexistence conditions, and during the overlap, the transitional balance of the bacterial community structure is maintained through relative metabolic inhibition and competitive substrate allocation mechanisms; by setting a complex response relationship of multiple inducing markers, multiple bacterial species are activated sequentially or simultaneously, enabling multi-site and multi-rhythm synergistic degradation of the mixed materials; This invention allows for short-term overlapping expression of bacterial species under phased coexistence conditions and drives a dynamic and coordinated scheduling mechanism of the bacterial community through a multi-inducible marker complex response. Combined with an original expression regulation function, it enables a multi-rhythmic, multi-site coordinated degradation process. The following expression regulation function is introduced to determine the expression dominance of a particular bacterial species at a specific time point: in: For the first Each bacterial species in time The transient expression potential index is used to determine whether it has entered the dominant expression period; strains The fitness function of the composite response to the inducer combination, numerical range This represents the overall sensitivity to induced markers in the environment (such as lignin, ferulic acid, and acetic acid); strains The potential metabolic release gradient under the current environment, i.e., the difference between the theoretical maximum activity capacity and the capacity under the current inhibited state; This is a resource accessibility function for the substrates currently available to this strain, reflecting its access advantages in terms of carbon source and substrate structure; strains Other coexisting bacterial species The degree of metabolic behavior disturbance reflects the interspecies competition / antagonism effect; The sensitivity coefficient is used to control the nonlinear amplification of the formula output by changes in variables. This can enhance the advantages of highly responsive bacteria; when When the expression threshold is set, determine the bacterial species. Entering the expression window; allowing multiple bacterial species to coexist and express for a short period under specific conditions, only when their expression potential values ​​are at the dynamic equilibrium boundary; interference function. As a negative feedback mechanism for system scheduling, it prevents a bacterial population from expanding indefinitely due to first-mover advantage; induced response function It is calculated from the inducing factor concentration and the induction curve of the bacterial species, and allows for multi-marker-based co-initiation.

[0011] Furthermore, the treatment method for enabling the natural succession of old and new microbial communities and deep depolymerization of lignin includes: Microbial communities with different metabolic preferences were configured to dominate the degradation of specific components at different stages of straw decomposition. During the alternation of microbial community dominance, system parameters were controlled to form a transition window from strong to weak metabolic activity. In the early stage, the microbial community spontaneously withdrew from dominant expression. In the later stage, the microbial community rapidly expressed enzymes after acquiring the ecological niche, maintaining the overall continuity of degradation. When aromatic metabolites accumulate to a set threshold in the system, the introduced fungal sense should be stimulated to produce short-term high-level laccase expression, thus completing the deep degradation of lignin.

[0012] Furthermore, the reduction in the metabolism of the early-stage microbial community is achieved through a variable oxygen supply mode, which includes setting the oxygen flux to be gradually reduced in hourly increments to induce delayed metabolic suppression in aerobic bacteria, thereby releasing living space for anaerobic or facultative bacteria. By monitoring the level of extracellular enzyme activity in real time, when the downward trend continuously exceeds a set threshold, it is determined to be a microbial community replacement window period and triggers an environmental fine-tuning strategy.

[0013] Furthermore, the lignin stress-induced mechanism is reversibly regulated. After the burst expression of laccase, the laccase level returns to the baseline by reducing the concentration of lignin accumulation or restoring neutral environmental conditions. The enhancement of laccase expression is assisted by external non-nutritive physical factors, including short-cycle weak light pulse irradiation or intermittent low-voltage micro-electric field application, to improve the sensitivity of fungal stress response.

[0014] Furthermore, the set threshold is defined as an extracellular enzyme activity decrease rate of 10% every 6 hours or 20 U / L / h, and when this condition is met in two consecutive monitoring cycles, it is determined to be an effective trend.

[0015] Furthermore, the preceding and following gradient bacteria are initially introduced in a single release, surviving in the system as spores, embedded bodies, or in an inducible state, respectively, with their expression sequence naturally triggered by their respective response thresholds to environmental factors; based on the structural composition differences of various straw raw materials, including rice straw, corn straw, wheat straw, and their mixtures, the adaptive decomposition scheduling across raw material systems is achieved by adjusting the metabolic window threshold and induction triggering parameters.

[0016] The beneficial effects of this invention are as follows: By setting response thresholds for specific metabolic signals (such as organic acids, aromatic compounds, pH, etc.) to each bacterial species, the automatic activation of the species from dormant or low-metabolic states to the expression state is achieved. The entire bacterial community expression process is naturally regulated by the internal metabolic dynamics of the system, eliminating the need for staged bacterial introduction or forced human intervention, thus greatly simplifying the process. A continuous gradient design is adopted, with interlocked metabolic pathways constructed between different gradient strains through intermediate metabolites. The intermediate products produced by the early-stage bacterial community serve as both induction signals for subsequent strains and their preferred substrates, achieving a smooth relay of metabolic functions and effectively avoiding degradation interruptions or functional breakdowns caused by imbalances in bacterial community turnover.

[0017] By adjusting the induction threshold parameter and metabolic window recognition conditions, this method can adapt to raw materials with different structural components, such as rice straw, corn straw, wheat straw, and their mixtures, achieving intelligent decomposition scheduling across substrates. It possesses good versatility and flexibility in field applications. It supports reversible regulation of the stress expression behavior of fungal laccases, including induction-outburst-regression mechanisms. This ensures high-intensity deep lignin cleavage while promptly reducing the induction intensity after enzyme activity reaches its peak, preventing enzyme resource waste and excessive cell load, and improving overall metabolic efficiency. Attached Figure Description

[0018] Figure 1 This is the main flowchart of the straw decomposition scheduling for continuous gradient inoculation of multiple microbial species according to the present invention.

[0019] Figure 2 This is a diagram showing the functional relationship and activation dependency of continuous gradient inoculation of multiple bacterial species according to the present invention.

[0020] Figure 3 This is a schematic diagram of the multi-species staged synergistic expression and dynamic regulation structure of the present invention.

[0021] Figure 4 This is a schematic diagram of the phased synergistic degradation process and regulation structure of multi-gradient microbial communities in southern rice straw according to an embodiment of the present invention.

[0022] Figure 5 This is a diagram showing the functional modules of multi-gradient microbial community in the staged degradation of rice straw in southern China, according to an embodiment of the present invention. Detailed Implementation

[0023] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0024] Combined with appendix Figure 1 As shown, this invention provides a method for scheduling straw decomposition through continuous gradient inoculation of multiple microbial species. This method constructs an environment-induced, driven continuous inoculation mechanism based on microenvironmental changes during straw decomposition. Specifically, in practical applications, key microenvironmental parameters in the straw decomposition system are first monitored in real time using sensing devices. These parameters include, but are not limited to, oxygen partial pressure (oxygen pressure), system conductivity, and the concentration of organic acids (such as acetic acid and lactic acid) representing intermediate stages of organic matter decomposition. These parameters dynamically reflect the intensity of microbial activity and decomposition rate at different stages. By analyzing and modeling the evolution trends of these parameters, specific metabolic signal induction markers can be identified. These induction markers are typically intermediate metabolites released by the dominant microbial species in the previous stage during their metabolic activities. When these products accumulate to a certain concentration in the system, reaching a preset threshold, they can serve as a signal trigger mechanism to initiate the next stage of functional microbial species.

[0025] The threshold is set based on the sensitivity curves of different strains to metabolites, ensuring that the activation process is selective and time-dependent. To achieve environment-driven continuous inoculation, this method pre-distributes multiple functionally complementary microbial strains in the system according to phased degradation targets, using spores, embedded bodies, or dormant states. Initially, these strains are not expressed or coexist at low levels. When the concentration of a specific metabolic induction marker reaches a set value, the cell membrane surface signal sensing system is activated, triggering the expression of downstream intracellular regulatory pathways and initiating the expression of specific degradation enzymes or genes, thus achieving the transition of the strain from a non-expressing state to an expressed state. This mechanism enables the self-organized relay renewal of the microbial community through the system's own metabolic evolution process without artificial addition or external stimulation, effectively improving the dynamic synergy and degradation efficiency of straw decomposition.

[0026] This method further enhances straw decomposition efficiency and strengthens the sequential degradation process by constructing metabolic complementarity between upstream and downstream strains. Specifically, it first analyzes and classifies the main intermediate metabolites in straw decomposition, focusing on representative components such as arabinose, acetic acid, and furfural. These components are typically generated by the initial functional microbial community during the degradation of hemicellulose, cellulose, and lignin primary structures, exhibiting high metabolic activity and the potential to serve as metabolic initiation substrates for downstream strains. Based on this, a systematic screening of candidate strains is conducted using microbial metabolomics analysis, biochemical pathway reconstruction, and enzyme expression profile comparison. This identifies strain pairs with complementary characteristics in metabolic pathways; that is, the initial strain can effectively generate a specific type of metabolic intermediate, while the later strain possesses a dedicated enzyme system or metabolic pathway for further conversion and utilization of this intermediate, thus achieving a metabolic relay-style substrate transfer.

[0027] To ensure the high efficiency and stability of metabolic synergy, this method further designed a screening strategy based on relay enzyme activity pairing. In vitro enzyme activity interoperability experiments were used to verify the absorption and conversion efficiency of specific intermediate products between successive gradient strains. Their enzymatic kinetic parameters were coupled and evaluated, including optimal pH, Km value, Vmax, and response characteristics to substrate concentration gradients. Straw combinations exhibiting high conversion efficiency and matched response times in natural fermentation environments were screened. The establishment of this pairing mechanism ensures that metabolic intermediates generated by strains at each stage during straw decomposition not only do not accumulate in the system to form inhibitory feedback, but also become preferred substrates or metabolic activators for the next stage strains, effectively reducing the risk of metabolic blockage or resource waste, thereby achieving seamless relay of multi-gradient microbial communities throughout the decomposition process.

[0028] Helper strains with specific metabolic regulatory functions, such as alkali-producing yeasts and H2O2 scavenging bacteria, are introduced to mitigate the accumulation of potentially harmful chemical factors during decomposition. Alkali-producing yeasts can release trace amounts of alkaline metabolites during the decomposition of specific substrates, neutralizing acidic or toxic intermediates such as lignins released during the mid-decomposition of straw. This maintains the pH of the system within a relatively stable buffer zone, preventing functional inhibition or enzyme inactivation caused by extreme environments. Meanwhile, H2O2 scavenging bacteria actively remove H2O2 accumulated by specific bacterial populations during decomposition by secreting antioxidant enzymes such as catalase, preventing its oxidative damage to lignin-degrading enzymes (such as laccase and peroxidase), thus achieving homeostatic regulation of the active enzyme environment within the system. To ensure the natural relay effect of bacterial communities at different stages during the transition of bacterial species, this method designed a bacterial species reduction interference window mechanism. That is, during the alternation period of adjacent gradient strains, environmental parameters (such as oxygen flux, temperature, and substrate concentration) are gradually adjusted to gradually reduce the metabolic activity of the previous stage strains, while creating suitable ecological niches for the expression of the next gradient strains. This transition process can be confirmed by real-time monitoring of extracellular enzyme activity trends using sensors, ensuring that the transition between bacterial communities is not drastic but rather a natural retreat.

[0029] Furthermore, in response to the need for deep degradation of complex components such as lignin, this method further introduces a fungal stress-induced mechanism based on the stress accumulation characteristics of lignin. That is, when the aromatic intermediates (such as lignin and ferulic acid) in the system reach a set concentration threshold, the introduced fungal strain senses the stress signal and rapidly initiates the transient high expression of laccase-like degrading enzyme system, which accelerates the breaking of phenolic ether bonds and aromatic rings in the lignin structure, thereby achieving deep depolymerization of lignin and subsequent carbon source release.

[0030] Combined with appendix Figure 2As shown, to achieve temporal regulation and autonomous expression switching during the continuous gradient inoculation process of multiple microbial species, this method adopts a microbial activation mechanism based on the concentration threshold of metabolically induced markers. The initiation process is designed as follows: First, the target decomposing raw material straw is divided into several degradation stages with phased decomposition characteristics according to the complexity of its structural components and the difficulty of degradation. Each stage is dominated by a type of functionally specific microbial species, forming a clear gradient microbial community structure. For example, in the early stage, saccharophilic bacteria that mainly degrade soluble oligosaccharides and oligosaccharides dominate; in the middle stage, filamentous fungi or facultative bacteria that depolymerize cellulose and hemicellulose are the core; and in the later stage, fungal species that can process lignin and aromatic compounds take over the dominant role. In actual operation, each preceding bacterial species in each gradient naturally releases specific metabolites during its growth and metabolic activities, such as acetic acid, lactic acid, arabinose, furfural, or lignans. These metabolites gradually accumulate in the system. When their concentration reaches a preset sensing threshold for the next gradient of bacterial strains, they can be recognized and responded to by the subsequent bacterial species through their membrane surface signal sensing system. This activates their intracellular gene regulatory network, triggering the expression of related enzyme systems or initiating signaling pathways, causing the strain to transition from a dormant or low-expression state to a dominant expression state, completing the relay switch of bacterial community dominance. To ensure that the expression rhythms of preceding and following bacterial species do not conflict, the subsequent bacterial strains remain in a dormant, low-metabolic, or passive coexisting state until they are activated. Their existence can take the form of spores, low-temperature embedded bodies, or a low-metabolic self-inhibitory state, exhibiting good environmental tolerance and metabolic inertia, and not actively affecting resource allocation and metabolic activities in the system, until the sensed metabolites reach the activation threshold before initiating expression.

[0031] For typical small molecule products generated by the metabolism of preceding microbial strains during straw decomposition, such as arabinose, acetic acid, and furfural, a response threshold system for subsequent microbial strains to these metabolites is established. This system is configured with a membrane surface signal recognition system via genetic or natural signal sensing devices. When the concentration of a specific metabolite reaches a preset value, this system activates downstream regulatory pathways within the cell, thereby initiating the expression process of enzymes encoding specific degradation products or promoter sequences. This allows subsequent strains to achieve self-initiated metabolic activation without external induction. To prevent premature expression by microbial strains before activation conditions are met, interfering with system stability, this method employs low-temperature spore encapsulation or carrier-based encapsulation techniques to pre-isolate the strains within the system. The degradable carrier is a pH-responsive material whose physical stability can be maintained in neutral or alkaline environments. During metabolism, it gradually depolymerizes in acidic environments caused by the accumulation of organic acids, thus achieving a passive release mechanism based on environmental state changes. This ensures synchronous coupling between the physical release of the strains and metabolic signal sensing, guaranteeing precise temporal and spatial separation of the activation process. Furthermore, this method designs a metabolic interlocking relationship between the preceding and following gradient strains to ensure the continuity and directionality of the system's metabolic flow. That is, the intermediate metabolites not only serve as metabolic releases from the degradation of straw structure by the preceding strains, but also as metabolic substrates or even key activating factors required by the subsequent strains. This makes the metabolites themselves both signals and material carriers for energy and carbon source transfer in the system. The preceding and following gradient strains exhibit a typical functional inheritance and relay pattern in the metabolic pathway, effectively avoiding resource waste and metabolic discontinuity.

[0032] Combined with appendix Figure 3 As shown, this method allows some functional strains to exhibit short-term overlapping expression under phased coexistence conditions, thereby adapting to the asynchronous differences in spatial distribution and decomposition rate of different structural components in straw. During this overlapping expression window, to avoid fierce competition among bacterial communities due to substrate overlap, this method introduces a relative metabolic inhibition mechanism and a competitive substrate allocation strategy. Specifically, by adjusting the metabolic activity response intensity of bacterial strains, substrate affinity weights, and the accessibility of environmental inducers, a dynamic transitional equilibrium is constructed, causing different strains to exhibit temporal misalignment, substrate selectivity shift, or differences in expression amplitude during the active expression period, effectively maintaining the stability and dynamic controllability of the system's bacterial community structure. Meanwhile, to ensure that multiple structural sites (such as hemicellulose layer, cellulose layer, and lignin blocks) in mixed straw raw materials are effectively identified and decomposed in different spatiotemporal dimensions, this method sets up multiple metabolic induction markers (such as lignin, ferulic acid, acetic acid, furfural, etc.) as scheduling signal sources and establishes a composite response model among inducers. This allows multiple microbial species to be activated sequentially or simultaneously based on the sensitivity of multiple induction signals, thereby achieving parallel and synergistic processing of different substrate sites and decomposition rhythms and enhancing the coupling and efficiency of the overall decomposition process.

[0033] To scientifically determine the dominant expression level of a strain at a specific time point, this method further introduces an original expression regulation function. Defined as: ,in For the first Each bacterial species in time The transient expression potential index is used to determine whether it has entered the dominant expression period; This indicates the adaptive response of the bacterial strain to the compound inducer, with a numerical range of [value missing]. This represents its overall sensing ability to environmentally induced markers; The metabolic release potential gradient under the current environment is the difference between the theoretical maximum activity capacity and the actual capacity under the limited state. This represents the resource accessibility of the substrates currently available to this strain, reflecting its competitive advantage in terms of carbon source or structural substrate; strains Other coexisting bacterial species The degree of metabolic behavior disturbance is a competitive or antagonistic disturbance parameter used to reflect the ecological tension within the system; To express the sensitivity coefficient and control for the nonlinear amplification effect of the formula on changes in the variable, where This can enhance the expression advantage of highly responsive strains, making their expression dominance more prominent. When the expression threshold is reached, the bacterial species is considered to have entered the expression window, allowing it to initiate the expression of relevant enzyme systems and form a short-term co-expression state with other strains within a specific time period, provided that their respective expression potentials are within the dynamic equilibrium boundary, thereby achieving a controllable coexistence structure; simultaneously, the interference function... As a negative feedback regulatory mechanism of the system, it can effectively inhibit the excessive expression expansion caused by the first-mover advantage of a certain bacterial species, and maintain the dynamic stability of the entire bacterial community structure; induced response function By combining the concentration of inducing factors with the response curve of the strain, the system can be ensured to drive the switching of microbial expression based on multiple marker signals, and finally realize the distributed and efficient biodegradation process of straw with complex multi-components at multiple sites and rhythms. This reflects the technical innovation of the present invention in expression scheduling modeling and ecological logic nested control.

[0034] To achieve efficient and continuous metabolic relay and deep lignin degradation in a multi-strain system during straw decomposition, the method employs a stage-dominant functional microbial community configuration strategy. This involves configuring microbial communities with dominant expression capabilities at different decomposition stages based on the metabolic preferences of different microorganisms for carbon source structures. These communities then dominate the degradation of components such as hemicellulose, cellulose, and lignin at different stages of straw decomposition. To ensure natural relay rather than abrupt alternation within the microbial community, this method establishes a transition window during the alternation of microbial dominance, where metabolic activity gradually weakens. This window is adjusted progressively by controlling system parameters such as oxygen supply rate, temperature, or substrate concentration. This allows the activity of the dominant strains in the early stages to gradually decrease and spontaneously relinquish their dominant expression positions. In the later stages, the microbial community, sensing the release of metabolic space, can rapidly occupy the ecological niche and initiate the expression of relevant degradative enzyme systems, ensuring the continuity and stability of the system's degradation process. Specifically, to achieve deep processing of recalcitrant components such as lignin, when aromatic metabolites such as lignans and ferulic acid accumulate to a set threshold in the system, pre-prepared fungal strains in the system will sense this stimulus signal, initiate a stress response, trigger short-term high-level laccase expression, and rapidly destroy the phenolic ether bonds and aromatic skeleton structure of lignin, completing the deep depolymerization of lignin. To achieve the natural exit of the weakened bacterial community metabolism in the early stage, this method designs a variable oxygen supply scheme. That is, in the later stage of the dominant bacterial community expression, the oxygen flux is gradually reduced in hourly increments, so that aerobic bacteria are subjected to delayed metabolic suppression, thereby releasing new ecological niches that can be occupied by facultative anaerobic bacteria or fungal communities. By real-time monitoring of extracellular enzyme activity levels, such as changes in cellulase and hemicellulase activity, When the rate of decrease in activity is detected to exceed 10% every 6 hours or 20 U / L / h for two consecutive cycles (e.g., 12 hours), the system is determined to have entered the microbial community replacement window. Subsequently, an environmental fine-tuning strategy is automatically triggered to assist in microbial community replacement. In addition, the stress-induced mechanism of laccase is reversibly regulated. That is, after the short burst expression of laccase is completed, the system parameters are adjusted, such as reducing the concentration of lignins or restoring the system to a neutral pH value, so that the expression of laccase can return to the baseline state and prevent the oxidative imbalance of the system. In order to improve the sensitivity of fungal stress response and expression efficiency, this method introduces external non-nutritive physical stimuli, such as short-cycle weak light pulse irradiation or intermittent low-voltage micro-electric field intervention, so that the fungal sensing mechanism can respond in advance under low concentration of inducers, thereby improving the timeliness of lignin degradation initiation. Regarding strain inoculation, all preceding and following gradient strains were initially introduced in a single-use strategy, existing in the system as spores, degradable embedded bodies, or uninduced cells, respectively. Their expression and activity initiation depended entirely on their respective response thresholds to environmental inducing factors, forming an adaptive expression sequence that could be achieved without artificial addition. Meanwhile, to adapt to various straw raw materials with different structural component ratios, including but not limited to rice straw, corn straw, wheat straw, and their mixtures, this method presets different metabolic window thresholds and induction triggering parameters during the system initialization stage, allowing the strain expression logic to be automatically adjusted according to the raw material structure.

[0035] Example 1: Combined with appendix Figure 4 As shown, taking a typical rice-producing area in southern China as an example, the local annual rice yield is approximately 700 kg / mu. Based on a straw-to-rice ratio of 1.1, 770 kg / mu of rice straw can be obtained annually. This straw has a high content of siliceous husk, hemicellulose, and highly crystalline cellulose structure, while the lignin content is approximately 14.2%. Ferulic acid and p-hydroxybenzoic acid account for over 60% of the lignins, serving as important signaling factors regulating the expression of the decomposition micro-ecosystem. To adapt to the characteristics of this raw material, the straw decomposition process is first divided into three main stages: The first stage is the initial stage of sugar and oligomer degradation, with Bacillus subtilis ZH-1 and Saccharomyces cerevisiae HX-3 as the dominant bacteria. The main metabolites are lactic acid, arabinose, and trace amounts of acetic acid. The activation thresholds for subsequent bacteria are set, such as arabinose ≥1.2g / L and acetic acid ≥0.8g / L. The second stage is the co-degradation stage of cellulose and hemicellulose, dominated by Clostridium cellulolyticum FJ-6 and white-rot fungus Trametesversicolor SD-9. Their expression is triggered by the accumulation of metabolites in the previous fungal species. When the concentration of arabinose reaches 1.5 g / L, the transcription factor SigX is triggered through the sensor response mechanism, which regulates the expression of extracellular β-1,4-glucanase by 3.4 times. The third stage is the lignin structure degradation stage, dominated by the actinomycete Streptomycessp.LS-2 and the schizophyllum commune YN-8. Their expression depends on the aromatic metabolites reaching specific concentration thresholds, such as ferulic acid ≥0.6 g / L and p-hydroxybenzoic acid ≥0.4 g / L. Experimental results showed that when the ferulic acid concentration in the system reached 0.67 g / L on the 7th day of fermentation, the expression level of the endogenous laccase gene ScLac1 in Schizophyllum commune instantaneously increased to 5.2 times the baseline level, marking the start of the lignin cleavage stage.

[0036] During the transition phases, subsequent strains, such as Clostridium FJ-6, were encapsulated in chitosan-sodium alginate bilayer microcapsules using freeze-dried spore embedding. Their degradation initiation point was related to the system's pH; the pH decrease caused by arabinose accumulation from 6.8 to 5.6 increased the microcapsule release rate by 80%, thus triggering the expression of subsequent strains under the dual induction of metabolite accumulation and environmental acidification. During a simulated 12-day continuous fermentation process, the total straw decomposition rate under the segmented activation mechanism increased from 48.3% in the control group to 71.4%, with the lignin degradation rate increasing from 12.5% ​​to 28.6%. Through inducer concentration-expression time-series modeling, it can be calculated that when the arabinose concentration threshold is set at 1.5 g / L and the actual production rate is 0.21 g / L / d, the expression index of Clostridium in the system can be expected on day 7. Approaching the set threshold =0.8, calculated based on the aforementioned expression regulation function, its ≈0.85, =0.62, =0.25, =0.12, χ²=1.3, then the expression potential index is: This indicates that the bacterial strain has entered its dominant expression phase. This example fully illustrates the adaptability, operability, and responsiveness of the microbial community relay expression mechanism constructed based on metabolite concentration thresholds in the southern rice straw decomposition system, achieving coordinated scheduling and efficient division of labor among the microbial community through environmental induction and dynamic control of the expression window.

[0037] This embodiment uses a double-cropping rice field in Guigang, southern China, as a model. The average annual accumulated temperature is relatively high, and the rice straw return rate exceeds 70%. Analysis of the original straw samples shows that the carbon-to-water ratio is 68:1, the hemicellulose content is about 23.6%, the proportion of xylan and arabinogalactan is relatively high, and the lignin content is 13.8%, which is suitable for multi-gradient microbial relay degradation. In this project, the first-stage inoculated Bacillus subtilis ZH-1 released a large amount of arabinose during the degradation of rice straw hemicellulose. The daily release rate reached 0.22 g / L / d between the 3rd and 5th days of fermentation, and the cumulative concentration measured by the 5th day was 1.1 g / L, which was close to the activation threshold of subsequent strains. The second-gradient inoculated strain was the anaerobic cellulose-decomposing bacterium Clostridium thermocellum YX-3, which has a concentration threshold of 1.25 g / L for sensing arabinose. It has a specific RbsB binding protein sensor on its cell membrane surface, which activates the intracellular XylR transcription factor after binding, regulating the promoter regions encoded by β-xylanase and endoglucanase. Experiments showed that its expression level was 4.8 times higher than the basal state when arabinose ≥1.3 g / L, and the transcription delay was no more than 6 hours. To achieve precise time-sequential expression of Y-3 bacteria, freeze-dried spores were initially embedded in a composite microcarrier of polylactic acid (PLA) and sodium alginate, achieving an embedding rate of 89%. The carrier remained stable under neutral conditions, but the acidolysis rate significantly increased at pH ≤ 5.5. In the locally acidic environment caused by arabinose accumulation, 90% of the spores were released within 48 hours. On day 6, the pH of the system decreased from the initial 6.9 to 5.4, meeting the conditions for carrier depolymerization and achieving environment-triggered bacterial release. The inoculated Clostridium bacteria rapidly activated expression after obtaining arabinose and a locally hypoxic environment. Simultaneously, the extracellular glucanase activity in the system increased from 22 U / mL on day 6 to 45 U / mL on day 8, representing a 67% increase in substrate degradation rate.

[0038] Further analysis after day 8 revealed that the concentration of acetic acid in the newly released intermediates increased to 1.6 g / L, and the concentration of ferulic acid, a lignin degradation product, was 0.48 g / L. This provided conditions for the initiation of the third-gradient fungal relay strain, namely *Schizophyllum commune* SC-1. Its cell membrane is equipped with a PheR-type aromatic sensor protein, which activates the laccase promoter ScLac1 upon sensing aromatic metabolites. The expression level reaches a high state when the ferulic acid concentration is ≥0.5 g / L. Laccase expression in this process was initiated using a stress-induced initiation method, with enhanced sensitivity through exogenous micro-electric field stimulation. Under an intermittent mode with an application of 0.5 V / cm and a cycle of 6 hours, laccase expression increased by approximately 2.3 times compared to the unstimulated control group, reaching 95 U / L per day, and the lignin degradation rate increased from 13% to 29.1%.

[0039] The above examples clearly demonstrate that in the system of this invention, the preceding and following gradient strains construct a metabolic interlocking mechanism by sharing intermediate metabolites. The arabinose produced by the preceding strains serves as both an induction signal and a preferred carbon source for the following strains. After acquiring their niche, the following strains can immediately metabolize and further generate substrates and signaling substances for third-gradient expression, forming a metabolic relay chain among the bacterial communities. This avoids substrate breakage and competition conflicts among the bacterial communities, while simultaneously achieving controllability of the self-organized expression rhythm. Throughout the scheduling process, all strains are deployed once in the initial stage via spore encapsulation without intermediate intervention. Driven by metabolite concentration, self-regulating promoters, pH-responsive release systems, and external non-nutritive factors, the orderly succession and expression control of the strains in each stage are completed.

[0040] Example 2: Combined with appendix Figure 5As shown, in a rice straw return project in a southern paddy field, rice straw with a moisture content of 60%, a dry weight lignin content of 13.4%, a hemicellulose content of 25.6%, and a soluble sugar content of 3.1% was selected. Its decomposition requires multi-site synergistic degradation by microorganisms. We divided the inoculated microbial communities into three gradients: ① early stage soluble sugar degrading bacterium S. cerevisiae HX-3 (referred to as strain 1); ② mid-stage hemicellulose / cellulose degrading bacterium Clostridium thermocellum YX-3 (strain 2); ③ late stage lignin degrading bacterium Schizophyllum commune SC-1 (strain 3). Among them, the decomposition structure of rice straw is not strictly linear, with a multi-site structure of cavity → core → cuticle, resulting in a non-uniform distribution of inducer release rhythm. Therefore, a multi-inducible marker composite response and stage overlap strategy was adopted to optimize expression scheduling.

[0041] Based on continuous dynamic data, the concentrations of the inducers detected on day 6 were as follows: arabinose 1.3 g / L, ferulic acid 0.42 g / L, and acetic acid 1.1 g / L. The main structure of the substrate consisted of undepolymerized hemicellulose and some lignin blocks. Although strain 1 had not yet completely terminated expression, strains 2 and 3 showed partial responsiveness to the above combination and may have entered a short-term expression window. We used the expression regulation function defined in this invention to determine the expression dominance of each strain: Define the following environment parameters: Induced fitness function (normalized by induction curve): (The sugar response is still in the high expression tail phase) (Strong compatibility with arabinose and acetic acid) (Ferulic acid did not fully reach the maximum sensitive area) Metabolic release potential gradient (difference between theoretical maximum activity and current environmental activity): Resource accessibility function (related to carbon source structure / affinity): Interspecific interference (degree of competition / antagonism): The expression sensitivity coefficient is set to The expression threshold is set to The expression potential was calculated as follows: Strain 1: Strain 2: Strain 3: Based on the above calculations, strain 2 is currently approaching its expression threshold. However, while the expression potential of strain 3 had entered the candidate window, the system's triggering strategy employed a multi-inducible marker complex response + short-term overlapping expression mechanism, allowing strains 2 and 3 to be initiated in parallel during the tail end of strain 1's expression phase. During this period, structural stability was maintained through competitive substrate regulation. For example, in hemicellulose substrate allocation, soluble components were preferentially allocated to strain 1 (substrate affinity index 1.4 times), while strains 2 and 3, which have high affinity for acetic acid and lignin substrates, achieved expression advantage shift by adjusting the spatial distribution of inducible factors. Experimental results showed that from day 7 to day 9, strain 1 expression decreased by 43%, while the enzyme activities of strains 2 and 3 increased by 67% and 35%, respectively. No abrupt changes in degradation efficiency occurred during co-expression, and the system structure remained stable, validating the effectiveness of the short-term overlapping expression and multi-marker synergistic triggering mechanism.

[0042] This embodiment uses a double-cropping rice paddy in southern China as the experimental setting. The raw material is rice straw with a moisture content of 65%. The dry matter contains 38.5% cellulose, 25.4% hemicellulose, and 13.6% lignin, as well as trace amounts of lignin, ferulic acid, and a siliceous shell. A multi-stage relay of microbial strains is required to achieve efficient decomposition. Initially, three types of gradient microbial communities are deployed in the inoculation system: ① aerobic carbohydrate and hemicellulose degrading bacteria, mainly Bacillus subtilis ZH-1 and Saccharomyces cerevisiae HX-3; ② facultative anaerobic cellulose degrading bacteria, such as Clostridium thermocellum YX-3; ③ fungal lignin degrading bacteria, represented by Schizophyllum commune SC-1 and Trametesversicolor SD-9. These three types of bacteria are added to the system all at once via spores or embedded bodies, and their expression is triggered based on different metabolites and environmental thresholds.

[0043] In the initial stage of system operation, sufficient oxygen is provided (flux rate of 2.5 L / min / 5 L reaction system) to activate the first gradient bacterial community and its extracellular enzyme activity. -xylosidase and -arabinofuranosidase peaked at 32.1 U / L and 28.5 U / L on day 2, and began to gradually decline on day 4. To induce a natural transition between the old and new bacterial communities, a variable oxygen supply control was implemented from day 5, decreasing the flux by 0.3 L / min per hour, down to 0.1 L / min by hour 8, thus inhibiting the metabolic capacity of aerobic bacteria. Real-time monitoring of extracellular enzyme activity revealed that, starting from day 5, the rate of decrease in enzyme activity exceeded 10% / 6h for two consecutive cycles (days 5–6). The -xylosidase decrease rate was 12.4% (11.2% on day 6–7), indicating the start of the microbial replacement window. The system automatically triggered a fine-tuning strategy, adjusting the temperature to 36℃ and adding 0.5% trace manganese salt to promote Clostridium expression. Simultaneously, under hypoxic conditions, its extracellular cellulase activity increased to 58.6 U / L on day 7, and the microbial community subsequently occupied the dominant ecological niche.

[0044] Meanwhile, metabolites from cellulose degradation, such as acetic acid and lignins, accumulated continuously in the mid-stage fungal community from days 8 to 9. Ferulic acid reached 0.52 g / L and p-hydroxybenzoic acid reached 0.44 g / L, exceeding the set induction threshold (ferulic acid ≥ 0.5 g / L), thus activating the Lac1 gene expression system in fungal SC-1 and initiating laccase synthesis. To enhance its stress response, a low-voltage micro-electric field stimulation (0.5 V / cm, 6 hours per cycle) and weak light flash irradiation (450 nm, 20 µmol / m² / s) were applied externally. On day 9, laccase activity rapidly increased to 102.3 U / L, 2.6 times that of the unstimulated control group, achieving efficient lignin cleavage. Subsequently, on day 11, residual lignin decreased to 5.3%, with a degradation rate of 61.0%, while the control system only achieved 31.7%. The stress mechanism of laccase is reversible. After day 12, the pH was adjusted back to neutral (6.8) and the inducer was reduced to below 0.2 g / L. Laccase activity naturally decreased to the baseline level, and the system returned to stability.

[0045] Ultimately, over the entire 12-day reaction cycle, the overall degradation rate of straw reached 75.4%, with cellulose conversion rates of 88.1%, hemicellulose of 82.3%, and lignin of 61.0%, representing increases of 23%, 31%, and 44%, respectively, compared to the traditional non-regulated inoculation method. Furthermore, this mechanism demonstrates cross-material adaptability. By adjusting the induction marker concentration threshold and oxygen downregulation curve, in comparison fermentation experiments with corn straw (17.8% lignin) and wheat straw (higher siliceous shell proportion), only the Clostridium activation pH threshold (corn: 5.8; wheat: 5.4) and the lignin concentration response curve in the fungal induction response function needed to be modified to achieve an efficient relay mechanism adaptation, with degradation rates reaching 68.5% and 63.2%, respectively. This verifies the universality and precise control capability of this invention in various straw decomposition systems. This implementation case demonstrates that by designing a microbial community gradient with metabolic preference differences, a variable oxygen regulation, metabolite-induced expression, a stress laccase triggering mechanism, and a dynamic niche switching strategy, this invention effectively achieves the phased degradation of multiple components of straw and the natural relay of microbial strains. It effectively solves the problems of microbial expression mismatch, metabolic interruption, and high lignin residue in traditional inoculation methods, and has significant engineering practical value and broad application prospects.

Claims

1. A method for scheduling straw decomposition through continuous gradient inoculation of multiple microbial strains, characterized in that... Includes the following steps: By observing the microenvironmental evolution trends, including oxygen pressure, electrical conductivity, and organic acid accumulation, during straw decomposition, metabolic signal induction markers are identified; the activation of subsequent strains is triggered by the concentration of the induction marker reaching a threshold, enabling continuous inoculation driven by the environment. By screening complementary metabolic pathway pairs between strains, a relay enzyme activity pairing mechanism was designed between the preceding and following gradient bacteria for intermediate products including arabinose, acetic acid, and furfural; so that the metabolic intermediates produced by the strains at each stage become the preferred substrates or activators for the next gradient bacteria. When designing the inoculation sequence and density, the ecological regulation capabilities of the strains are considered, and alkali-producing yeasts and H2O2 scavenging bacteria are introduced to release trace amounts of alkaline metabolites during the straw decomposition stage, neutralize lignin toxins, and maintain the system's buffer stability. During the inoculation process at each gradient, a decreasing interference window for fungal species was constructed by adjusting environmental parameters, allowing the old and new fungal communities to take over naturally. Based on the characteristics of lignin stress accumulation, stress-induced transient expression of laccase was induced in the inoculated fungal strains, resulting in deep depolymerization of lignin.

2. The method for scheduling straw decomposition through continuous gradient inoculation of multiple microbial strains according to claim 1, characterized in that... The process of triggering subsequent bacterial initiation when the induced marker concentration reaches a threshold includes: Straw is divided into multiple decomposition stages based on its degradation characteristics, with each stage dominated by microbial strains with different functions. In any stage, the preceding strains naturally release metabolites during the degradation of straw. The activation or expression of subsequent strains is passively induced when the metabolites reach a threshold concentration in the system. The subsequent bacterial strains are in a dormant, low-metabolic, or passive coexisting state before being activated.

3. The method for scheduling straw decomposition through continuous gradient inoculation of multiple microbial strains according to claim 2, characterized in that... The triggering mechanism of the metabolites includes establishing a response threshold for microorganisms to small molecule products. The response is mediated by a cell membrane surface signal sensing system, triggering the expression of enzymes or promoter sequences encoded in downstream regulatory pathways, thus enabling the self-initiation of bacterial expression. The dormant state of the subsequent bacterial strain before activation is physically isolated by low-temperature spore embedding or a degradable carrier, and the carrier depolymerizes in a metabolically acidic environment, forming an environmentally triggered release mechanism.

4. The method for scheduling straw decomposition through continuous gradient inoculation of multiple microbial strains according to claim 3, characterized in that... The preceding and following gradient bacteria share a class of intermediate metabolites, which are both decomposition products of the preceding strains and substrates for metabolic enzyme pathways absorbed and activated by the following strains, thus creating a metabolic interlocking functional inheritance relationship between the strains.

5. The method for scheduling straw decomposition through continuous gradient inoculation of multiple microbial strains according to claim 4, characterized in that... The inoculation sequence allows some bacterial species to overlap for a short period of time under phased coexistence conditions, and during the overlap, the transitional balance of the bacterial community structure is maintained through relative metabolic inhibition and competitive substrate allocation mechanisms. By setting a complex response relationship of multiple inducing markers, multiple bacterial species are activated sequentially or simultaneously, enabling multi-site and multi-rhythm synergistic degradation of the mixed materials.

6. The method for scheduling straw decomposition through continuous gradient inoculation of multiple microbial strains according to claim 1, characterized in that... The treatment method that enables the natural succession of old and new microbial communities and deep depolymerization of lignin includes: Microbial communities with different metabolic preferences were configured to dominate the degradation of specific components at different stages of straw decomposition. During the alternation of microbial community dominance, system parameters were controlled to form a transition window from strong to weak metabolic activity. In the early stage, the microbial community spontaneously withdrew from dominant expression. In the later stage, the microbial community rapidly expressed enzymes after acquiring the ecological niche, maintaining the overall continuity of degradation. When aromatic metabolites accumulate to a set threshold in the system, the introduced fungal sense should be stimulated to produce short-term high-level laccase expression, thus completing the deep degradation of lignin.

7. The method for scheduling straw decomposition through continuous gradient inoculation of multiple microbial strains according to claim 6, characterized in that... The reduction in the metabolism of the early-stage microbial community is achieved through a progressive oxygen supply mode, which includes setting the oxygen flux to be gradually reduced in hourly increments to induce delayed metabolic suppression in aerobic bacteria, thereby releasing living space for anaerobic or facultative bacteria. By monitoring the level of extracellular enzyme activity in real time, when the downward trend continuously exceeds a set threshold, it is determined to be a microbial community replacement window period and triggers an environmental fine-tuning strategy.

8. The method for scheduling straw decomposition through continuous gradient inoculation of multiple microbial strains according to claim 7, characterized in that... The lignin stress-induced mechanism is reversible. After the burst expression of laccase, the laccase level returns to the baseline by reducing the concentration of lignin accumulation or restoring neutral environmental conditions. The enhancement of laccase expression is assisted by external non-nutritive physical factors, including short-cycle weak light pulse irradiation or intermittent low-voltage micro-electric field application, to improve the sensitivity of fungal stress response.

9. The method for scheduling straw decomposition through continuous gradient inoculation of multiple microbial strains according to claim 7, characterized in that... The set threshold is defined as an effective trend when the rate of decrease in extracellular enzyme activity reaches 10% every 6 hours or 20 U / L / h, and this condition is met in two consecutive monitoring cycles.

10. The method for scheduling straw decomposition through continuous gradient inoculation of multiple microbial strains according to claim 9, characterized in that... The preceding and following gradient bacteria are initially introduced in a single application, surviving in the system as spores, embedded bodies, or in an inducible state, respectively. Their expression sequence is naturally triggered by their respective response thresholds to environmental factors. Based on the structural composition differences of various straw raw materials, including rice straw, corn straw, wheat straw, and their mixtures, the adaptive decomposition scheduling across raw material systems is achieved by adjusting the metabolic window threshold and induction triggering parameters.

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