A method for cultivating diatoms using sediments in a resource-efficient manner

Abstract

This invention provides a method for cultivating diatoms using sediment in a resource-efficient manner. By adding an appropriate amount of agar to water containing sediment and subjecting it to hydrodynamic disturbance to promote an aerobic state, a large number of diatoms can be obtained by cultivating them for 15-30 days, attaching and growing on the inner wall of the container and the surface of the sediment. The cell density of the attached diatoms can reach as high as 9.8 × 10⁻⁶ cells / day. 5 cells / cm 2 This method utilizes nutrients in sediments to cultivate diatoms, achieving the resource utilization of these nutrients. The resulting diatoms can be used as live feed for aquaculture and can also be used to clean the environment, thus expanding the methods for diatom cultivation.

Description

Technical Field

[0001] This invention relates to the field of algae cultivation methods, and more particularly to a method for cultivating diatoms by utilizing sediments in a resource-efficient manner. Background Technology

[0002] In aquaculture, nitrogen input from feed accounts for 90% of the total nitrogen input in the entire aquaculture system. However, approximately 30% of the total nitrogen from feed enters the pond water or sediment (also known as bottom mud) in the form of uneaten feed and feces. Sediment is the concentration point of nitrogen and other elements in aquaculture ponds. Sediment in aquaculture ponds contains abundant nutrients, such as carbon, nitrogen, and phosphorus, which are important factors affecting water quality. Changes in the flux of nitrogen, phosphorus, and other nutrients at the sediment-water interface are a direct means of studying the storage, balance, and cycling of nutrients at the interface. This directly reflects the nutrient conditions and water quality status of the water body, and is of great significance for evaluating the primary productivity level of ponds, regulating water quality, and protecting aquaculture ecosystems. The formation of pond sediment is influenced by various factors such as the species, stocking density, and feeding. Sediment is also a significant cause of eutrophication and algal blooms in aquaculture waters. Therefore, proper sediment management is a crucial aspect of water quality management in eutrophic water bodies.

[0003] Currently, to prevent eutrophication and hypoxia caused by nutrients in sediments, methods such as bottom aeration, dredging, application of modified materials, and chemical passivation are often used to enhance the treatment of nutrients in sediments. Aeration of sediments can improve dissolved oxygen levels, enhance the activity conditions of microorganisms, and reduce the adverse effects of nutrients. Dredging directly removes sediments from the water system, reducing the amount of nutrients stored and released, thus controlling eutrophication. The application of modified clay and modified biochar can treat ionic compounds in water, control algal blooms, and treat organic pollutants. Chemical passivation of nutrients mainly works by passivating phosphorus in sediments, inhibiting the release of endogenous phosphorus; commonly used passivating agents include iron salts, aluminum salts, and modified clays. Under suitable conditions, these methods can achieve good results in controlling eutrophication, and their basic characteristic is reducing the utilization of nutrients in sediments by algae.

[0004] Besides managing and controlling nutrients in sediments, the resource utilization of these nutrients is also an important research direction. Since sediments are rich in nutrients, their primary function is as agricultural fertilizer. With eutrophication of water bodies, harmful algae, especially harmful cyanobacteria, easily proliferate under high summer temperatures. If diatoms preferred by aquaculture species can be cultivated using the nutrients in sediments, harmful cyanobacteria can be controlled, and the resources of sediments can be utilized. In particular, some aquaculture species have a high demand for diatoms, especially attached diatoms. One of the most economically valuable marine aquaculture species in northern China in recent years, the sea cucumber (Apostichopus japonicus), prefers to feed on attached diatoms. Sea cucumbers are benthic omnivorous organisms that obtain nutrients by feeding on detritus, organic matter in the sediment, algae, bacteria, and protozoa. Therefore, the resource utilization of nutrients in sediments for diatom cultivation has a promising application prospect.

[0005] Therefore, the resource utilization of nutrients in sediments to cultivate beneficial algae has become a method of algal regulation technology, requiring research into how algae utilize nutrients. Algal regulation is related to various factors such as temperature, light, nutrient concentration, nutrient type, and hydrodynamic disturbance. Existing algal regulation research has focused most on nitrogen and phosphorus, especially inorganic nitrogen and phosphorus, while less attention has been paid to organic substances. To date, the formation mechanisms of cyanobacterial blooms, diatom blooms, and euglena blooms in natural water bodies are not fully understood. The most clearly defined mechanism is that eutrophication leads to algal blooms, but eutrophic water bodies do not necessarily produce algal blooms. In addition to nitrogen and phosphorus, the mechanisms by which the form of substances affects the formation of dominant algal species are still insufficiently studied.

[0006] Large-scale or mass cultivation of algae is a key technology in the field of microalgae biotechnology and a bottleneck in the development and utilization of microalgae resources and the production of their bioproducts. Common methods for large-scale microalgae cultivation include photobioreactors (closed-system) and racetrack-type circulating bioreactors (open-system). Photobioreactors mainly include tubular, flat-plate, and fiber optic photobioreactors, characterized by the largest surface area to volume ratio, the most efficient light source system, the shortest light path for light energy transfer to microalgae, and the most efficient mixing and circulation, thus achieving high-density, high-yield, and high-efficiency cultivation. Racetrack-type circulating bioreactors are the most traditional microalgae cultivation mode, with simple structure, low cost, ease of construction and operation, and mature cultivation technology. They have been successfully applied to the large-scale outdoor cultivation of Spirulina, Chlorella, and Dunaliella. The selection of the culture medium is crucial in these cultivation techniques; a suitable medium can yield higher concentrations of algal cells. Some of these media have publicly available formulations, while others are proprietary formulations developed in-house. Common algal culture medium formulations mainly consist of various inorganic substances with low organic content. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for cultivating diatoms by utilizing sediments in a resource-efficient manner, so as to utilize the nutrients stored in the sediments as a source of material for diatom growth. The cultivated diatoms can be used as biological feed for aquaculture, and also have many other benefits such as cleaning the environment and developing new energy sources.

[0008] The technical problem it aims to solve can be addressed through the following technical solutions.

[0009] A method for cultivating diatoms using sediments in a resource-efficient manner, the method comprising the following steps:

[0010] S1. Obtain the sediment and add it to the container;

[0011] S2. Add water to the sediment;

[0012] S3. Add agar to the mixture of sediment and water;

[0013] S4. Perform hydrodynamic disturbance on the system;

[0014] S5. After 15 to 30 days of cultivation, a large number of diatoms will grow on the inner wall of the container and on the surface of the sediment.

[0015] The diatoms that grow there are mainly *Neptunus* and *Neptunus*, but these attached diatoms are generally small, with cell lengths rarely exceeding 25 μm. The diatom cell density at dense attachment sites can reach as high as 9.8 × 10⁻⁶. 5 cells / cm 2 This means that diatom dominance emerged, realizing the conversion of matter and energy in the sediment into diatoms.

[0016] in,

[0017] In step S1, the sediment used can come from various water bodies, such as lakes, rivers, aquaculture ponds, paddy fields, etc. The sediment used can be organic matter-rich gray-brown or black silt, or ordinary silt. The nitrogen content in the dry matter of the sediment should be in the range of 0.5 to 20 g / kg, and the thickness of the added sediment should be 0.2 to 10 cm.

[0018] In step S2, the algae content in the added water should not be high, and the added water needs to come from a body of water with a transparency of 70cm or more. It can be a variety of water sources or a mixture of water sources, such as water from lakes, rivers, aquaculture ponds, tap water, or even pure water. The concentration of nutrients in the added water should not be too high, and the concentrations of total nitrogen and total phosphorus should be below 30mg / L and 3mg / L, respectively.

[0019] In step S3, the added agar can be common strip or powdered agar, and the concentration of added agar must be less than 30 mg / L.

[0020] In step S4, hydrodynamic disturbance can take various forms, such as agitation, aeration, and circulation. The purpose of hydrodynamic disturbance is to increase dissolved oxygen in the water, creating an aerobic or oxygen-rich environment. Simultaneously, the hydrodynamic action provides support for diatom growth. The intensity of hydrodynamic disturbance should not be too high to prevent excessive suspension of sediments, which could inhibit light. The water transparency should be controlled between 50 and 150 cm to facilitate suitable lighting conditions.

[0021] In step S5, water temperature and light conditions are the most important influencing factors. The water temperature for cultivation should be between 5 and 30°C. The light conditions for cultivation are related to the season. Light is more suitable in spring, autumn and winter. In the sunny weather of summer (June to August), the light is strong and the temperature is high. It is advisable to reduce the light by increasing the water depth to 1.5 to 2 m or by shading 50% to 70% and obtaining a suitable water temperature.

[0022] The basic principle of this invention is to utilize nutrient-rich sediments to provide essential inorganic nutrients such as nitrogen and phosphorus for diatom growth; simultaneously, agar provides an adhesive medium and nutrients for diatoms to attach to the sediment surface and the inner wall of the container, promoting their attachment and growth. The diatoms in the system originate from the sediments and added water. The nitrogen, phosphorus, and other nutrients in the sediments and water, along with the agar and suitable light conditions, provide the material basis and light conditions for diatom attachment and growth.

[0023] Agar, scientifically known as agar jelly, is a long-chain polysaccharide composed of galactose and its derivatives. It is a natural high-molecular-weight polysaccharide refined and purified using scientific methods from high-quality natural seaweed such as Gracilaria, Porphyra, and others. It is a component of cell walls and contains complex carbohydrates, calcium, and sulfates. Agar is the best solidifying agent for preparing solid culture media for bacteria. Solid culture media prepared with agar can be used for high-temperature cultivation without melting, and will not kill the culture during inoculation before solidification. Agar easily adheres to the surface of sediments and the inner walls of containers under hydrodynamic disturbance.

[0024] In the technical solution of this invention, the addition of agar, the supply of nutrients from the sediment, and hydrodynamic disturbance are the three key factors for achieving diatom attachment and growth. Changes in any of these factors will affect the time and density required for diatom cultivation. Because sediment is added to this method, it contains abundant nutrients, eliminating the need for additional nutrient supplementation. The nutrients stored in the sediment can be directly utilized, thus achieving resource utilization of the sediment. Furthermore, the sediment can be reused during the cultivation process because the interface between the sediment and the overlying water is often very thin. Agitation of the sediment below the sediment-water interface can promote the release of more nutrients. In particular, the agar concentration should not be too high. Excessive agar concentration can easily lead to the attachment and growth of various algae on the fine agar particles, such as green algae, cyanobacteria, and diatoms, making it difficult to establish a dominant diatom attachment.

[0025] This method did not show a large number of diatoms floating and growing in the water. The underlying mechanism is still unclear and further research is needed on the interaction of various factors such as nutrients, light, temperature, and hydrodynamics.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] 1. The nitrogen, phosphorus and other nutrients required for diatom attachment and growth all come from sediments, realizing the conversion of substances stored in sediments into substances for diatom growth, and providing a method for the resource utilization of sediments.

[0028] 2. Agar has a certain viscosity in water, which can adhere to the surface of sediments and the inner wall of containers, providing a good medium for the attachment and growth of diatoms; in addition, agar itself contains complex nutrients such as carbohydrates, calcium and sulfates, which can promote the rapid attachment and growth of diatoms.

[0029] 3. The greatest benefit of this invention is that it is the first time that agar and sediment have been discovered to be used together for diatom cultivation. Existing literature often describes agar as a bacterial culture medium, but there are no reports of its use for diatom cultivation. Attached Figure Description

[0030] Other features, objects, and advantages of the invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: all algae photographs in the embodiments were taken under an optical microscope at 400x magnification.

[0031] Figures 1-1 to 1-9 The brown substance adhering to the inner wall of treatment II in Example 1 is mostly diatoms.

[0032] Figures 1-10 to 1-14The brownish substance adhering to the inner wall of treatment III in Example 1 is mainly a group of bacteria.

[0033] Figures 2-1 to 2-17 The transparent agar particles in Example 2 and the bacteria and attached algae on them.

[0034] Figures 2-18 to 2-22 This refers to the aggregates formed after a large number of bacteria and some tiny algae grow on the fine agar particles in Example 2. Detailed Implementation

[0035] Based on the current state of the technology, the inventors of this application have discovered methods for cultivating diatoms using Microcystis blooms and methods for cultivating diatoms using aquatic plants. The main characteristic of these methods is that, under hydrodynamic disturbance conditions, diatom growth is achieved by utilizing the substances and energy stored in cyanobacterial blooms and aquatic plants. The technology for cultivating attached diatoms mainly utilizes the characteristic of diatoms easily attaching and growing, achieving diatom attachment growth by introducing diatom seed sources. Currently, methods for cultivating diatoms in planktonic growth mainly involve certain marine species. There is a need for more large-scale diatom cultivation methods in production, including for aquaculture and the extraction of medical nutrients.

[0036] Therefore, based on the existing foundation, the inventors further studied more diatom cultivation methods, focusing on the characteristics of freshwater diatom growth and the needs of production practice, and further expanded the cultivation methods and technologies for freshwater diatoms.

[0037] The present invention will be further described and illustrated in detail below with reference to specific embodiments and accompanying drawings, with more specific implementation methods.

[0038] Example 1:

[0039] The culture was conducted using 10L wide-mouth glass bottles, divided into 8 treatments, as shown in Table 1. In the treatment with added sediment, 200g of air-dried, crushed soil (i.e., sediment) that had passed through a 100-mesh sieve was added to each bottle. This soil originated from black sediment deposited in urban rivers, and its total nitrogen and total phosphorus contents were 7.85g / kg and 3.26g / kg, respectively. Tap water was added to each bottle to a volume of 10L, and then powdered agar was added according to Table 1. The selected powdered agar was of biopharmaceutical grade, with excellent quality and purity. Continuous aeration was performed in each glass bottle to promote an aerobic environment and ensure that the dissolved oxygen concentration was above 4mg / L.

[0040] All the glass bottles were placed in a glass greenhouse for cultivation.

[0041] Table 1: Design and Measures for Each Treatment

[0042] I yes 0 0 II yes 0.03 3.0 III yes 0.10 10.0 IV yes 0.20 20.0 V no 0 0 VI no 0.03 3.0 VII no 0.10 10.0 VIII no 0.20 20.0

[0043] Initially, sediment was added, which became largely suspended due to hydrodynamic disturbance, resulting in turbid water. After 3 days of cultivation, the water in all containers became clear. By day 10, some phytoplankton had begun to grow in some treatment containers. By day 12, significant changes were observed in each treatment, as described in Table 2. On day 20, the phenomena were even more pronounced, as described in Table 2. Treatment II, which had 3 mg / L agar and added sediment, developed a large amount of brown material on its inner wall. Microscopic examination revealed that the brown material on the inner wall of Treatment II was diatoms. Figures 1-1 to 1-9 Furthermore, the density of attached diatoms in densely packed areas reached 9.8 × 10⁻⁶. 5 cells / cm 2 On day 20, brown deposits on the lower inner wall of treatment I container were examined under a microscope and found to be the same diatoms as those in treatment II. Surface sediment from treatment II on day 20 also showed some diatom growth. Microscopic examination of the brown deposits on the inner wall of treatment III on day 20 revealed primarily bacterial communities (including some larger cyanobacteria, also known as blue-green algae), with no significant diatom growth. Figures 1-10 to 1-14 ).

[0044] After another 10 days of cultivation, the area of ​​brown diatoms attached to the inner wall of treatment II was larger, and the amount of diatoms attached to the inner wall of treatment I was also increased.

[0045] The above algal growth results show that adding sediment is beneficial to the growth of attached diatoms, and adding agar is even more beneficial. This indicates that the nutrients in the sediment provide a material basis for the growth of attached diatoms, while without sediment, adding agar alone does not easily promote the attached growth of diatoms.

[0046] Table 2: Phenomena observed in each treatment bottle on day 12 and day 20.

[0047]

[0048] Example 2:

[0049] The culture was carried out using plexiglass columns, which were cylindrical at the top and hemispherical at the bottom. The upper cylindrical section had a diameter of 40 cm and a height of 100 cm. There were three treatments, as shown in Table 3. Sediment was added to each column. 300 g of air-dried, crushed soil (i.e., sediment) that had passed through a 100-mesh sieve was added to each column. This soil originated from aquaculture ponds, and its total nitrogen and total phosphorus contents were 1.57 g / kg and 0.33 g / kg, respectively. Water was added to each column. The added water was from a small water body in an urban park, diluted with tap water. The water was light yellowish-green and contained various algae. The total volume after adding the water was 120 L.

[0050] Powdered agar was added according to Table 3. The selected powdered agar was of biopharmaceutical grade, with excellent quality and purity. Air stones were used for aeration in each column to promote aerobic conditions and ensure dissolved oxygen concentration above 4 mg / L. Initially, the added sediment was suspended in the water, causing turbidity.

[0051] Table 3: Settings for each process

[0052] I 3.6 30.0 II 6.0 50.0 III 8.4 70.0

[0053] All the acrylic columns were placed in a glass greenhouse for cultivation.

[0054] For the first two days of cultivation, there was little change, and the water was relatively turbid. Starting on the fourth day, the water color in all treatments lightened, becoming a pale yellowish-green. The agar powder, after absorbing water, swelled and appeared as light-colored flocculent matter in the water, suspended under hydrodynamic disturbance, with a small amount settling on the surface of the sediment. As time continued, the water in all three treatments gradually turned green, becoming noticeably green by the tenth day, with the appearance of fine green particles; the inner walls of the containers were very clean, with no attached growth. Cultivation continued until the twentieth day, the water color became even greener, and the colors of the three treatments were similar, with a significant amount of fine green particles present. Under a microscope, a small amount of algae and some bacterial communities could be seen attached and growing on the transparent agar particles. Figures 2-1 to 2-17 Furthermore, various types of algae appeared on the fine agar particles, mainly from the phyla Chlorophyta, Cyanophyta, and Diatomaceousta. However, the inner walls of the containers in each treatment remained clean, with no obvious attached material or growth. After 20 days of continued cultivation, the inner walls of the containers remained clean, with no obvious attached material. Meanwhile, the suspended algae in the water clearly aggregated on the fine agar particles, becoming fine particles suspended in the water along with a large number of bacteria. Figures 2-18 to 2-22 ).

[0055] This experiment shows that when the added agar concentration is as high as 30-70 mg / L, the nutrients released from the sediment, together with the agar, promote the growth of algae attached to the agar particles, and diatoms will grow on the agar particles. However, there is no obvious growth of algae attached to the inner wall of the container. This should be related to the fact that the concentration of algae in the water is relatively high, which inhibits the growth of algae attached to the inner wall of the container.

[0056] Example 3:

[0057] The experiment was conducted using 10L wide-mouth glass bottles. There were six treatments, as shown in Table 4. Treatments I, II, and III did not add sediment, while treatments IV, V, and VI added 100g of air-dried, crushed soil (sediment) obtained through a 100-mesh sieve. This soil originated from aquaculture ponds, and its total nitrogen and total phosphorus contents were 1.35g / kg and 0.22g / kg, respectively. No hydrodynamic disturbance was applied to any of the bottles. Water from a small urban park pond was added to each bottle, and then diluted with tap water. The resulting water was a light yellowish-green color and contained various algae. The total volume after adding the water was 10L.

[0058] Table 4: Specific measures for each treatment

[0059]

[0060]

[0061] Each container was incubated in a glass greenhouse. Due to the lack of hydrodynamic disturbance, the added sediment settled almost completely to the bottom within two days, the water became clear, and the added agar also settled. No significant changes were observed in any of the containers during the first 10 days of incubation. As the incubation period lengthened, by day 20, the water in treatments I, II, and III (without added sediment) became very clear, with no obvious phytoplankton growth, and the inner walls were also very clean, with no attached algae growth. In treatments IV, V, and VI (with added sediment), the water was light yellowish-green, with some phytoplankton growth, but the inner walls were also very clean, with no attached algae growth.

[0062] By day 30, the water in treatments I, II, and III, where no sediment was added, remained very clear with no obvious growth of phytoplankton and the inner walls were also very clean with no attached algae. In treatments IV, V, and VI, where sediment was added, the light yellow-green color became slightly more intense, and there was phytoplankton growth, but the inner walls were still clean with no obvious attached algae growth.

[0063] This experiment shows that when the added agar concentration is 5 mg / L or 10 mg / L and there is no hydrodynamic disturbance, algae growth is unlikely to occur regardless of whether there is sediment.

[0064] The present invention provides a method for cultivating diatoms using sediments. By adding an appropriate amount of agar to water containing sediments and subjecting it to hydrodynamic disturbance to promote an aerobic state, a large number of diatoms can be obtained by cultivating them for 15-30 days, attaching and growing on the inner wall of the container and the surface of the sediments. The cell density of the attached diatoms can reach as high as 9.8 × 10⁻⁶ cells / day. 5 cells / cm 2 This method utilizes nutrients in sediments to cultivate diatoms, achieving the resource utilization of these nutrients. The resulting diatoms can be used as live feed for aquaculture and can also be used to clean the environment, thus expanding the methods for diatom cultivation.

Claims

1. A method for cultivating diatoms using sediments in a resource-efficient manner, characterized in that, The following processing steps are included: S1. Obtain sediment and add it to a container. The nitrogen content in the dry matter of the sediment ranges from 0.5 to 20 g / kg, and the thickness of the added sediment is from 0.2 to 10 cm. S2. Add water to the sediment. The water should be sourced from water with a transparency of 70 cm or more. The total nitrogen and total phosphorus concentrations in the added water should be less than 30 mg / L and 3 mg / L, respectively. S3. Add agar to the mixture of sediment and water at a concentration of 3-20 mg / L; utilize the nutrient-rich sediment to provide inorganic nutrients for diatom growth; at the same time, agar provides an adhesive medium and nutrients for diatoms to attach to the sediment surface and the inner wall of the container, promoting the attachment and growth of diatoms. S4. The system is subjected to hydrodynamic disturbance; the sediments below the sediment-water interface are stirred to promote the release of more nutrients. After the hydrodynamic disturbance, the water transparency is controlled at 50-150cm. S5. After 15 to 30 days of cultivation, a large number of diatoms will grow on the inner wall of the container and the surface of the sediment. In sunny weather with strong light and high temperature, you can choose to increase the water depth to 1.5 to 2m or reduce the light by shading 50% to 70% and obtain a suitable water temperature.

2. The method for cultivating diatoms using sediments as described in claim 1, characterized in that: In step S1, the source water of the sediment is a lake, river, aquaculture pond or paddy field; the sediment is organic-rich gray-brown or black silt, or ordinary silt.

3. The method for cultivating diatoms using sediments as described in claim 1, characterized in that: In step S2, the added water comes from lakes, rivers, aquaculture ponds, tap water, pure water, or mixtures thereof.

4. The method for cultivating diatoms using sediments as described in claim 1, characterized in that: In step S3, the added agar is the common strip or powder form of agar.

5. The method for cultivating diatoms using sediments as described in claim 1, characterized in that: In step S4, the hydrodynamic disturbance is treated by agitation, aeration, or circulation.

6. The method for cultivating diatoms using sediments as described in claim 1, characterized in that: In step S5, the water temperature for cultivation is controlled between 5 and 30°C.

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