A method for promoting chitin degradation using sulfate-reducing bacteria

Abstract

This invention discloses a method for promoting chitin degradation using sulfate-reducing bacteria, belonging to the field of environmental microbiology. The method involves inoculating sulfate-reducing bacteria into an anaerobic medium containing sulfate for activation culture. After the bacteria have stabilized, crystalline chitin is added to the culture system and co-cultured under anaerobic conditions, allowing the sulfate-reducing bacteria to attach to the chitin surface and form a biofilm. During the culture process, fresh medium is periodically added to maintain bacterial activity and sulfate supply. The culture is continued for 10–60 days, utilizing the sulfur-containing active substances produced by the sulfate-reducing bacteria's metabolism to non-enzymatically corrode the crystalline chitin, thereby promoting its degradation. This invention eliminates the need for expensive enzyme preparations, is simple to operate, and has low cost. It is particularly suitable for the resource utilization of chitin-rich marine biomass waste such as shrimp and crab shells, providing a new technical approach for the efficient degradation and high-value utilization of chitin waste.

Description

Technical Field

[0001] This invention belongs to the field of environmental microbiology technology, and specifically relates to a method for promoting chitin degradation using sulfate-reducing bacteria. Background Technology

[0002] Chitin is the second most abundant natural biopolymer after cellulose, widely found in the exoskeletons of crustaceans such as shrimp and crabs, as well as in the cell walls of fungi. With the rapid development of aquaculture, seafood processing, and bio-fermentation industries, a large amount of chitin-rich waste, such as shrimp shells, crab shells, and fungal residues, is generated annually. Due to a lack of economical and efficient treatment technologies, a large amount of chitin waste remains unutilized, resulting not only in resource waste but also potential environmental pollution.

[0003] Currently, the biodegradation of chitin mainly relies on chitin hydrolases and oxidases produced by microorganisms. Chitin hydrolases hydrolyze the β-1,4 glycosidic bonds in the chitin molecule, while polysaccharide monooxygenase (LPMO) oxidatively destroys the chitin crystal structure; both work synergistically to promote chitin degradation. However, chitin in nature mostly exists in a highly crystalline form, characterized by its dense structure, insolubility in water, and high stability. This dense crystal structure limits the contact between enzyme molecules and the substrate, significantly restricting the efficiency of enzymatic degradation, especially in the early stages of degradation where it is difficult to effectively destroy the crystal structure. Furthermore, existing enzymatic degradation systems typically rely on microorganisms to synthesize and secrete large amounts of extracellular enzymes, resulting in long enzyme production cycles, high costs, and sensitivity to environmental conditions. In low-oxygen or anaerobic environments such as marine sediments and the interior of shrimp and crab shells, the activity of some oxidases is limited, further affecting the chitin degradation efficiency.

[0004] Sulfate-reducing bacteria (SRB) are a group of functional microorganisms widely found in marine sediments and other anaerobic environments. They use sulfate as an electron acceptor to oxidize organic matter and produce sulfur-containing metabolites such as hydrogen sulfide. For a long time, SRB have been considered a significant driver of biocorrosion of materials such as metals and concrete in marine environments. Studies have shown that SRB can form biofilms on material surfaces and cause continuous corrosion and structural damage by producing metabolites such as hydrogen sulfide and organic acids. Statistical data shows that microbial-induced material corrosion accounts for more than 20% of total corrosion, with approximately 70% of microbial corrosion caused by sulfate-reducing bacteria. Under anaerobic conditions, SRB can utilize organic matter attached to the material surface as a carbon source to reduce sulfate to hydrogen sulfide. Their metabolites interact with the material matrix, accelerating the corrosion process.

[0005] However, current technologies primarily focus on the role of sulfate-reducing bacteria in sulfur cycling and material corrosion, with no reports on utilizing sulfate-reducing bacteria to promote the degradation of crystalline chitin. While some studies have reported on the co-culture of chitin-degrading and sulfate-reducing bacteria, the role of sulfate-reducing bacteria in these systems is merely to provide growth factors to promote the enzymatic activity of chitin-degrading bacteria, rather than directly destroying the chitin crystal structure. Furthermore, no technical solutions have been found that utilize sulfur-containing reactive substances produced by sulfate-reducing bacteria to directly destroy the chitin crystal structure.

[0006] In summary, existing chitin degradation technologies have the following main shortcomings: (1) they rely on enzymatic hydrolysis, which is limited by the dense structure of crystalline chitin, resulting in low degradation efficiency; (2) enzyme production has a long cycle and high cost, and poor adaptability to actual environmental conditions such as anaerobic environments; (3) there is no technical solution that utilizes the chemical corrosion properties of sulfate-reducing bacteria metabolites to directly act on the chitin crystal structure. Therefore, it is urgent to develop a new degradation technology that can overcome the structural barrier of crystalline chitin, is easy to operate, and is suitable for anaerobic environments. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method for promoting chitin degradation using sulfate-reducing bacteria. This method achieves non-enzymatic chemical corrosion of crystalline chitin using sulfur-containing active substances produced by sulfate-reducing bacteria metabolism, without relying on chitin hydrolases and oxidases. This effectively destroys the dense structure of crystalline chitin, significantly improves chitin degradation efficiency, reduces processing costs, simplifies the operation process, and is applicable to anaerobic environments and sulfate-rich marine biomass waste treatment scenarios.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: A method for promoting chitin degradation using sulfate-reducing bacteria includes the following steps: (1) Sulfate-reducing bacteria were inoculated into an anaerobic medium containing sulfate for activation culture; (2) After the bacterial cells have grown stably, crystalline chitin is added to the culture system and co-cultured under anaerobic conditions so that sulfate-reducing bacteria attach to the surface of chitin to form a biofilm. Fresh culture medium is added regularly during the culture process to maintain bacterial activity and sulfate supply. The culture is continued for 10 to 60 days. The sulfur-containing active substances produced by the metabolism of sulfate-reducing bacteria are used to non-enzymatically corrode the crystalline chitin, thereby promoting the degradation of crystalline chitin.

[0009] In the above scheme, the sulfate-reducing bacteria are selected from one or more of the genera Desulfovibrio, Desulfotomaculum, Desulfobulbus, Desulfobacter, Desulfococcus, Desulfobacterium, Thermodesulfovibrio, and Thermodesulfobacterium.

[0010] Furthermore, the sulfate-reducing bacterium is *Desulfovibriobizertensis*.

[0011] In the above scheme, the sulfate concentration in the culture system is 0.5–10 g / L, with SO42- as the main component. 2- count.

[0012] In the above scheme, the culture temperature is 20-40℃ and the pH of the culture system is 6.5-8.5.

[0013] In the above scheme, the culture temperature is 37±1℃.

[0014] In the above-mentioned scheme, each liter of the culture medium comprises: 25.00g sodium chloride, 0.50g dipotassium hydrogen phosphate, 1.00g ammonium chloride, 1.00g sodium sulfate, 0.10g calcium chloride dihydrate, 2.00g magnesium sulfate, 2.00g sodium DL-lactic acid, 1.00g yeast extract, 0.50mL sodium resazurin solution, 0.10g sodium mercaptoacetate and 0.10g ascorbic acid.

[0015] In the above scheme, the crystalline chitin exists in the form of powder, flakes, granules or blocks, and 1 to 20 g of crystalline chitin is added per liter of culture medium.

[0016] In the above protocol, a portion of fresh culture medium is added every 24 to 72 hours.

[0017] In the above scheme, the sulfur-containing active substances include hydrogen sulfide, polysulfides, hydrogen sulfide ions, and combinations thereof.

[0018] Through the above technical solution, the method for promoting chitin degradation using sulfate-reducing bacteria provided by the present invention has the following beneficial effects: (1) This invention is the first to apply the metabolic characteristics of sulfate-reducing bacteria that cause material corrosion to the field of crystalline chitin degradation, providing a novel non-enzymatic degradation technology route that is different from traditional enzymatic hydrolysis. Existing chitin degradation technologies all rely on the catalytic action of chitin hydrolases or oxidases, while this invention utilizes sulfur-containing active substances produced by sulfate-reducing bacteria to chemically corrode chitin without relying on any exogenous or endogenous enzyme preparations, thus breaking through the technical bottleneck that enzyme molecules cannot effectively penetrate the dense structure of crystalline chitin; (2) The present invention can effectively destroy the dense structure of crystalline chitin. After sulfate-reducing bacteria colonize the surface of chitin and form a biofilm, sulfur-containing active substances such as hydrogen sulfide and polysulfides produced by their metabolism are continuously enriched on the surface of chitin. Through chemical corrosion, the crystal structure of chitin is loosened, a large number of cracks and pores are formed on the surface, the specific surface area is significantly increased, the accessibility of the substrate is significantly improved, and favorable conditions are created for further degradation. (3) The present invention has high degradation efficiency and better treatment effect than the traditional enzyme-producing bacteria route. After treatment with sulfate-reducing bacteria, the quality of crystalline chitin is significantly reduced, and the degradation effect is better than that of chitin-enzyme hydrolyzing bacteria alone, which fully demonstrates that the non-enzymatic corrosion route has a significant advantage in degradation efficiency; (4) This invention has low operating costs and good economic efficiency. It does not require the addition of expensive enzymes, strong oxidants, or chemical corrosives; the structural destruction of chitin can be achieved solely through the metabolic activities of sulfate-reducing bacteria. The culture medium is simple and readily available, and the culture conditions are mild, without involving harsh conditions such as high temperature, high pressure, or strong acids and alkalis. Furthermore, the marine environment and waste materials such as shrimp and crab shells are naturally rich in sulfates, which can directly meet the needs of sulfate reduction metabolism without the need for additional electron acceptors, further reducing operating costs. (5) This invention is simple to operate and has a clear process route. It uses a single functional strain to directly treat chitin, which avoids the uncertainty of degradation effect caused by the fluctuation of complex mixed bacterial community composition. It has good repeatability and controllability and is easy to achieve industrial scale-up. (6) This invention is highly compatible with anaerobic environments and has a wide range of applications. Sulfate-reducing bacteria are anaerobic microorganisms, and their metabolic activity is stronger under low oxygen or anaerobic conditions. They are particularly suitable for actual anaerobic treatment scenarios rich in chitin, such as marine sediments and shrimp and crab shell deposits. (7) This invention can be widely applied to the resource utilization of various biomass wastes rich in chitin, such as shrimp shells, crab shells, krill shells, insect exoskeletons and fungal mycelia. It can also be used as a pretreatment method for crystalline chitin, combined with subsequent enzymatic hydrolysis or microbial degradation processes, to further improve the high-value utilization efficiency of chitin and provide a new technical approach for the recycling of marine biomass resources. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0020] Figure 1 This is the chitin mass change curve after treatment in Example 1 of the present invention; Figure 2The images are SEM images of chitin during the reaction process in Example 1, where (a) is day 0, (b) is day 15, and (c) is day 30. Figure 3 The change of sulfur (S) in chitin during the reaction process in Example 1; Figure 4 This refers to the changes in sulfur-containing functional groups of chitin during the reaction process in Example 1; Figure 5 The remaining chitin mass in different treatment groups. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0022] This invention provides a method for promoting chitin degradation using sulfate-reducing bacteria, and specific embodiments are as follows: Example 1 This embodiment provides a method for promoting chitin degradation using sulfate-reducing bacteria, including the following steps: (1) Sulfate-reducing bacteria Desulfovibrio bizertensis The culture medium was inoculated into anaerobic medium and cultured in 2 L headspace vials. The medium composition was as follows: per liter, 25.00 g sodium chloride (NaCl), 0.50 g dipotassium hydrogen phosphate (K₂HPO₄), 1.00 g ammonium chloride (NH₄Cl), 1.00 g sodium sulfate (Na₂SO₄), 0.10 g calcium chloride dihydrate (CaCl₂·2H₂O), 2.00 g magnesium sulfate (MgSO₄), 2.00 g sodium DL-lactate (Na-DL-lactate), 1.00 g yeast extract, 0.50 mL sodium resazurin solution (0.1% w / v), 0.10 g sodium thioglycolate (Na-thioglycolate), and 0.10 g ascorbic acid. The culture temperature was controlled at 37 ± 1 °C.

[0023] (2) After the bacterial cells have grown stably, add 10 g of flaky chitin (derived from krill shells) to the culture system and carry out anaerobic culture at 37±1℃. Replace 50% of the culture medium with fresh medium every 48 h during the culture process to maintain the concentration of nutrients and sulfate in the culture system, and culture continuously for 30 days.

[0024] Samples of flaky chitin crystals were collected at different time points during the cultivation process. After sonication, the samples were treated with 2.5N NaOH solution at 75℃ for 30 min to remove microbial cells and extracellular polymers adhering to the chitin surface. The treated samples were adjusted to neutral with hydrochloric acid, dried at 105℃ to constant weight, and weighed to calculate the mass change of chitin. Scanning electron microscopy (SEM) was used to observe the surface morphology changes of chitin; SEM-EDS and inductively coupled plasma atomic emission spectrometry (ICP) were used to determine the changes in sulfur content in the samples; and X-ray photoelectron spectroscopy (XPS) was used to analyze the changes in the functional group composition of the sample surface.

[0025] Experimental results like Figure 1 As shown, in Example 1, the mass of the plate-like chitin decreased from the initial 10g to 2.9g after 30 days of cultivation.

[0026] like Figure 2 As shown in the figure, SEM observation results indicate that the surface structure of uncultured crystalline chitin is dense and smooth, while that of cultured chitin is... Desulfovibrio bizertensis After treatment, depressions, cracks and pore structures gradually appeared on the surface of chitin, and the number and size of cracks increased with the extension of culture time.

[0027] like Figure 3 As shown in the figure, the ICP analysis results indicate that the sulfur content in the chitin sample continued to increase during the cultivation process.

[0028] like Figure 4 As shown, XPS analysis results further indicate that new sulfur-containing functional group characteristic peaks such as C=S, CS, and -SO3H appear on the surface of the treated chitin.

[0029] The above results indicate that sulfate-reducing bacteria Desulfovibrio bizertensis Under the influence of the action, the surface of the crystalline chitin undergoes significant structural changes, accompanied by the continuous accumulation of sulfur on the surface of the chitin.

[0030] Comparative Example 1 The blank control group had the same culture system composition and operating conditions as in Example 1, but was not inoculated with any bacteria.

[0031] Comparative Example 2 Chitin-enzyme hydrolyzing bacteria group: inoculated with chitin-enzyme hydrolyzing bacteria Flavobacterium beibuense And add 10g of flaky chitin crystals for culturing.

[0032] All experimental groups used the same culture system and conditions, and were cultured at 30±1℃ for 30 days. During the culture process, 50% of the volume of fresh culture medium was added every 48 hours to maintain stable operation. After culture, the flaky chitin crystals were removed, sonicated, and then treated with 2.5N NaOH solution at 75℃ for 30 minutes to remove microbial cells and extracellular polymers attached to the chitin surface. The samples were then adjusted to neutral pH and dried at 105℃ to constant weight before weighing.

[0033] Experimental results like Figure 5 As shown, after 30 days of cultivation, the remaining mass of crystalline chitin in each treatment group was as follows: blank control group (comparative example 1) 9.7g; sulfate-reducing bacteria group (Example 1) 2.9g; chitin-enzymatic bacteria group (comparative example 2) 4.8g.

[0034] The results showed that both sulfate-reducing bacteria and enzyme-producing bacteria could promote the degradation of crystalline chitin, but the degradation effect of sulfate-reducing bacteria was better than that of enzyme-producing bacteria. Combined with the results of Example 1, it can be seen that sulfate-reducing bacteria can destroy the dense structure of crystalline chitin, forming cracks and pores on the chitin surface, thereby increasing the accessibility of the substrate.

[0035] Example 2 The difference between this embodiment and Embodiment 1 is that sulfate-reducing bacteria are used. Desulfovibrio bizertensis Replace with Desulfobacter The strain is a sulfate-reducing bacterium. The remaining steps and conditions were the same as in Example 1. The results showed that this strain could also effectively promote the degradation of crystalline chitin.

[0036] Example 3 The difference between this embodiment and Example 1 is that the crystalline chitin exists in powder form, and the dosage is 5g. The remaining steps and conditions are the same as in Example 1. The results show that powdered chitin can also be effectively degraded.

[0037] Example 4 The difference between this example and Example 1 is that the culture temperature was 25℃, and the pH of the culture system was controlled between 7.0 and 7.5. The remaining steps and conditions were the same as in Example 1. The results showed that sulfate-reducing bacteria could still effectively promote chitin degradation even at lower culture temperatures.

[0038] Example 5 The difference between this embodiment and Embodiment 1 is that the crystalline chitin is derived from shrimp shells and is pretreated to obtain flaky crystalline chitin. The remaining steps and conditions are the same as in Embodiment 1. The results show that chitin derived from shrimp shells can also be effectively degraded.

[0039] Example 6 The difference between this embodiment and Embodiment 1 is that the crystalline chitin is derived from crab shells and pretreated to obtain granular crystalline chitin, with an addition amount of 15g. The remaining steps and conditions are the same as in Embodiment 1. The results show that granular chitin derived from crab shells can also be effectively degraded.

[0040] Example 7 The difference between this embodiment and Embodiment 1 is that the cultivation is carried out in an upflow anaerobic sludge bed reactor, with the crystalline chitin placed in a perforated carrier and suspended in the liquid phase region of the reactor. The remaining steps and conditions are the same as in Embodiment 1. The results show that this reactor configuration is also suitable for the method of this invention.

[0041] Example 8 The difference between this embodiment and Example 1 is that the culture time was 60 days, and 50% of the volume of fresh culture medium was added every 24 hours. The remaining steps and conditions were the same as in Example 1. The results showed that extending the culture time further improved the degree of chitin degradation.

[0042] Example 9 The difference between this example and Example 1 is that the sodium sulfate concentration in the culture system is 5.00 g / L, the culture temperature is 35℃, and the pH is 7.5. The remaining steps and conditions are the same as in Example 1. The results show that these culture conditions can also achieve effective degradation of chitin.

[0043] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for promoting chitin degradation using sulfate-reducing bacteria, characterized in that, The steps include the following: (1) Sulfate-reducing bacteria were inoculated into an anaerobic medium containing sulfate for activation culture; (2) After the bacterial cells have grown stably, crystalline chitin is added to the culture system and co-cultured under anaerobic conditions so that sulfate-reducing bacteria attach to the surface of chitin to form a biofilm. Fresh culture medium is added regularly during the culture process to maintain bacterial activity and sulfate supply. The culture is continued for 10 to 60 days. The sulfur-containing active substances produced by the metabolism of sulfate-reducing bacteria are used to non-enzymatically corrode the crystalline chitin, thereby promoting the degradation of crystalline chitin.

2. The method according to claim 1, characterized in that, The sulfate-reducing bacteria are selected from one or more of the genera Desulfovibrio, Desulfotomaculum, Desulfobulbus, Desulfobacter, Desulfococcus, Desulfobacterium, Thermodesulfovibrio, and Thermodesulfobacterium.

3. The method according to claim 2, characterized in that, The sulfate-reducing bacteria is Desulfovibriobizertensis.

4. The method according to claim 1, characterized in that, The sulfate concentration in the culture system is 0.5–10 g / L, with SO42- as the main component. 2- count.

5. The method according to claim 1, characterized in that, The culture temperature is 20–40℃, and the pH of the culture system is 6.5–8.

5.

6. The method according to claim 1, characterized in that, The incubation temperature was 37±1℃.

7. The method according to claim 1, characterized in that, Each liter of the culture medium comprises: 25.00 g sodium chloride, 0.50 g dipotassium hydrogen phosphate, 1.00 g ammonium chloride, 1.00 g sodium sulfate, 0.10 g calcium chloride dihydrate, 2.00 g magnesium sulfate, 2.00 g sodium DL-lactic acid, 1.00 g yeast extract, 0.50 mL resazurin sodium solution, 0.10 g sodium mercaptoacetate, and 0.10 g ascorbic acid.

8. The method according to claim 1, characterized in that, The crystalline chitin exists in the form of powder, flakes, granules or blocks, and 1 to 20 g of crystalline chitin is added per liter of culture medium.

9. The method according to claim 1, characterized in that, Replenish with fresh culture medium every 24–72 hours.

10. The method according to claim 1, characterized in that, The sulfur-containing active substances include hydrogen sulfide, polysulfides, sulfide ions, and combinations thereof.

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