Water treatment apparatus and water treatment process comprising high-efficiency biohydrogen production using microbial strain activated by magnetite carrier

Abstract

According to an embodiment of the present invention, provided is a water treatment apparatus comprising a fermentation tank for receiving wastewater containing organic matter and decomposing the organic matter contained in the wastewater to produce hydrogen, wherein the fermentation tank comprises a microbial strain activated by a magnetite carrier. The water treatment process and apparatus according to the present invention can maintain high hydrogen production efficiency regardless of the environment in the fermentation tank, and thus are effective in the industrial production of biohydrogen.

Description

Water treatment device and water treatment process including high-efficiency biohydrogen production using microbial strains activated by magnetite carrier

[0001] The present invention relates to a water treatment device and process for producing hydrogen from biomass contained in wastewater using a microbial strain activated by a magnetite carrier.

[0002] Hydrogen exists primarily in the form of water on Earth, making it an abundant and environmentally friendly energy source. Typically, hydrogen is produced by reforming fossil fuels like natural gas or coal into water or through electrolysis, or as biohydrogen through fermentation with hydrogen-producing microorganisms.

[0003] Representative methods of biological hydrogen production through microbial fermentation can be divided into photolysis and photofermentation, which require light energy, and dark fermentation, which does not use light energy. In particular, dark fermentation is emerging as a promising process due to its non-light-intensive nature, low energy intensity, rapid decomposition of organic matter from various sources, and relatively accommodating undemanding cultivation conditions. Nevertheless, despite its rapid fermentation rate, dark fermentation exhibits low efficiency in converting the energy value contained in the substrate into hydrogen. Numerous studies have been conducted to overcome these limitations of anaerobic hydrogen production, but most have focused on optimizing culture and process parameters, such as inoculum strains, substrates, and reactor configurations.

[0004] Meanwhile, strains of the genus Clostridium are representative microorganisms that produce hydrogen using carbon sources under anaerobic conditions. These microorganisms can produce biohydrogen using biomass such as energy crops, lignocellulose, algae, food waste, wastewater, or household waste as substrates. Volatile fatty acids (VFAs), such as lactate, formate, acetate, and butyrate, produced by Clostridium strains, have been suggested to suppress sugar consumption by inhibiting metabolic pathways when accumulated under limited buffering capacity in batch conditions, thereby reducing hydrogen production.

[0005] Thus, it has been reported that lactate consumption by Clostridium strains does not directly mediate additional hydrogen production, but rather, when accumulated, may act as a factor that hinders hydrogen production, and thus, the role of lactate in improving hydrogen production has been required.

[0006] The purpose of the present invention is to provide a water treatment device and process capable of producing hydrogen from biomass contained in wastewater with high efficiency even when the environment within the reactor changes by utilizing a microbial strain activated by a magnetite carrier.

[0007] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions described below. In addition, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated into this specification in their entirety by reference to more clearly explain the level of the technical field to which the present invention belongs and the contents of the present invention.

[0008] Figure 1 illustrates a water treatment device according to one embodiment of the present invention.

[0009] A water treatment device according to the present invention comprises a fermentation tank that receives wastewater containing organic matter and decomposes the organic matter contained in the wastewater to produce hydrogen, wherein the fermentation tank comprises a microbial strain activated by a magnetite carrier. In the water treatment device according to the present invention, the microbial strain is activated by the magnetite carrier, and specifically, the magnetite carrier can stimulate the metabolic pathway of the microbial strain to increase the hydrogen production yield. Accordingly, even if the composition of the wastewater changes to a point that is unfavorable to the microbial metabolism, the microbial strain can perform metabolism for hydrogen production with high efficiency. Therefore, a water treatment process utilizing a microbial strain can be efficiently performed without a complex pretreatment process.

[0010] According to one aspect of the present invention, a hydrogen production step may be performed in which wastewater containing organic matter is supplied to a fermentation tank or a wastewater storage tank, and the organic matter contained in the wastewater is decomposed to produce hydrogen. In this case, the hydrogen production step may be performed by supplying lactate to a microbial strain activated by a magnetite carrier.

[0011] First, looking at the strains utilized in water treatment devices and processes, the term "strain (or microorganism)" in the present invention includes both wild-type microorganisms and microorganisms that have undergone natural or artificial genetic modification, and may be microorganisms that have had a specific mechanism weakened or strengthened due to causes such as the insertion of an external gene or the enhancement or inactivation of the activity of an endogenous gene, and may be microorganisms that include genetic modification for the production of a desired polypeptide, protein or product.

[0012] The strain of the present invention may be a strain having hydrogen production ability. In particular, the strain may be a microorganism that naturally has hydrogen production ability or a microorganism (e.g., a recombinant strain) that has been endowed with hydrogen production ability through recombination or the like. The hydrogen-producing strain described above may be a strain of the genus Clostridium, and as another example, may be Clostridium butyricum.

[0013] The strains described above can be cultured in a separate medium and then activated by a magnetite carrier.

[0014] In the present invention, the term "cultivation" refers to growing the strain of the present invention under appropriately controlled environmental conditions. The culturing process of the present invention can be performed using any suitable medium and culture conditions known in the art. This culturing process can be easily adjusted and used by those skilled in the art depending on the selected strain. Specifically, the culturing process may be batch, continuous, and / or fed-batch, but is not limited thereto.

[0015] In the present invention, the term "medium" refers to a material containing nutrients as main components necessary for culturing the strain of the present invention, and supplies nutrients and growth factors, including water essential for survival and growth. Specifically, the medium and other culture conditions used for culturing the strain of the present invention may be any medium used for culturing general microorganisms without particular limitation, but the strain of the present invention may be cultured under aerobic conditions while controlling temperature, pH, etc. in a general medium containing an appropriate carbon source, nitrogen source, phosphorus source, inorganic compound, amino acid, and / or vitamin.

[0016] The carbon source to be added to the above-described medium may include sugars (carbohydrates) such as glucose, saccharose, lactose, fructose, sucrose, maltose, etc.; sugar alcohols such as mannitol, sorbitol, etc.; organic acids such as pyruvic acid, lactic acid, citric acid, etc.; amino acids such as glutamic acid, methionine, lysine, etc. In addition, natural organic nutrients (i.e., biomass) such as algae hydrolysate, starch hydrolysate, molasses, blackstrap molasses, rice winter, cassava, sugarcane bagasse, and corn steep liquor may be used, and specifically, sugars contained in algae hydrolysate may be used, and other appropriate amounts of carbon sources may be used in a variety of ways without limitation. These carbon sources may be used alone or in combination of two or more, but are not limited thereto.

[0017] Nitrogen sources to be added to the above-described medium include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, ammonium nitrate, etc.; organic nitrogen sources such as amino acids such as glutamic acid, methionine, glutamine, etc.; peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product, etc. These nitrogen sources may be used alone or in combination of two or more, but are not limited thereto.

[0018] The components added to the above-described medium may include potassium phosphate monobasic, potassium phosphate dibasic, or their corresponding sodium-containing salts. Inorganic compounds may include sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. In addition, amino acids, vitamins, and / or suitable precursors may be included. These components or precursors may be added to the medium in a batch or continuous manner, but are not limited thereto.

[0019] In addition, during the cultivation of the strain of the present invention, compounds such as sodium bicarbonate, sodium hydroxide, ammonium hydroxide, potassium hydroxide, ammonia, hydrochloric acid, phosphoric acid, sulfuric acid, etc. can be added to the medium in an appropriate manner to adjust the pH of the medium. In addition, during the cultivation, foaming can be suppressed by using an antifoaming agent such as sterile silicone oil or fatty acid polyglycol ester. In one embodiment of the present invention, a commercially available antifoaming agent (antifoam) is added, but the present invention is not limited thereto. In addition, in order to maintain the aerobic state of the medium, oxygen or an oxygen-containing gas may be injected into the medium, or in order to maintain the anaerobic and microaerobic state, no gas may be injected, or nitrogen, hydrogen, or carbon dioxide gas may be injected, but the present invention is not limited thereto.

[0020] In the culture of the present invention, the culture temperature can be maintained at 20 to 40°C, specifically 25 to 37°C, and the culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0021] The strain is activated by a magnetite carrier. In some cases, the strain may be provided supported or immobilized on the magnetite carrier.

[0022] The magnetite carrier contains magnetite (Fe3O4). Magnetite is an oxide of iron and can serve as a source of Fe (or iron ions) when added to a medium. The magnetite may also be referred to as "magnetite." The magnetite carrier may be provided in the form of a carrier capable of supporting the above-described microorganisms. The carrier may be a solid material having internal and external voids.

[0023] The magnetite carrier containing the microbial strain can be provided within the fermentation tank in the form of pellets. The magnetite carrier can be provided within the fermentation tank in the form of a Packed Bed Reactor (PBR) or floating within the tank. In this case, stirring can be performed within the fermentation tank to increase contact between the magnetite carrier and the organic matter in the wastewater.

[0024] Rather than adding microorganisms and magnetite separately, the magnetite-induced microbial metabolism promotion effect can be enhanced by creating a magnetite carrier and immobilizing microorganisms within it. Specifically, microorganisms immobilized within the pores of the magnetite carrier absorb organic matter entering and exiting wastewater through the pores. Furthermore, because they are in contact with the magnetite, the magnetite-induced microbial metabolism promotion can occur immediately.

[0025] Furthermore, since the microorganisms are immobilized on the magnetite carrier, if the activity of the microorganisms or the magnetite carrier decreases, the magnetite carrier and the microbial strains immobilized thereon can be recovered, and new magnetite carriers and / or microbial strains can be easily introduced. In particular, since magnetite carriers are generally denser than wastewater, a sedimentation tank can be utilized to settle the magnetite carriers, if necessary, and they can be easily separated from the water treatment device.

[0026] Magnetite may be included in a concentration of about 0.05 g / L to about 11 g / L based on the total volume of the mixture of wastewater, magnetite, and microbial strains provided to the fermenter. When magnetite is included in the above-mentioned concentration range, the microbial activation effect by magnetite may be exhibited. In particular, when microorganisms activated by magnetite meet lactate, the hydrogen production efficiency is improved, and the water treatment process for hydrogen production may operate regardless of the concentration of substances contained in the wastewater, such as the concentration of lactate. In some cases, magnetite may be included in a concentration of about 6 g / L or more, about 7 g / L or more, about 8 g / L or more, about 9 g / L or more, about 10 g / L or more, and about 11 g / L or less, and specifically, magnetite may be included in a concentration of about 8 to about 11 g / L. By providing magnetite to be mixed in the above-mentioned range, the microbial metabolism promotion effect by magnetite may be maximized.

[0027] Magnetite activates microbial strains, particularly hydrogen-producing bacteria, by stimulating their metabolic pathways. Hydrogen-producing bacteria can promote the consumption of lactate and / or acetate in the metabolic process that produces butyrate and hydrogen from lactate and / or acetate.

[0028] Referring to the diagram, hydrogen production occurs through the reduction of hydrogen ions by the reducing power of NADH and Fd. In both cases, hydrogen ions catalyze the reaction. First, in the case of NADH-mediated hydrogen production, the NADH produced in the process is oxidized, and 2 moles of hydrogen molecules are produced through the reduction of hydrogen ions. Second, in the case of Fd-mediated hydrogen production, pyruvate is oxidized to acetyl-CoA, and Fd is reduced. As a result, the reduced Fd is oxidized again, producing 2 moles of hydrogen molecules. As shown in Figure 2, when the final product is acetate, a total of 4 moles of hydrogen molecules are produced. However, in the case of the final product, butyrate, because it involves a reaction that consumes NADH, 2 moles of hydrogen molecules are ultimately produced.

[0029] The environment within the fermenter of the present invention may increase the expression of at least one selected from the group consisting of hydrogenase, electron-transfer flavoprotein, and pyruvate ferredoxin oxidoreductase in a hydrogen-producing strain compared to an environment in which neither the magnetite carrier nor lactate is added.

[0030] In the present invention, the term "hydrogenase (Hyd)" is an enzyme that catalyzes the reversible oxidation of hydrogen molecules (H2) as follows: (1) H2+ A 산화된 → 2H + + A 환원된 or (2) 2H + + D 환원된 → H2+ D 산화된 . The above hydrogenase may be used interchangeably with “hydrogenase”, “Hyd”, etc.

[0031] The amino acid sequence of the above Hyd protein can be obtained from a known database such as NCBI's Genbank.

[0032] For example, the Hyd protein of the present application may be derived from a microorganism. As another example, the microorganism may be, specifically, a strain of the genus Clostridium sp., or as another example, a strain of Clostridium butyricum.

[0033] In the present invention, the term "electron-transferring flavoprotein (EtfAB)" refers to an enzyme that participates in electron bifurcation by forming a complex with proteins such as butyryl-CoA dehydrogenase, caffeyl-CoA reductase, and lactate dehydrogenase (LDH) in anaerobic microorganisms. The electron-transferring flavoprotein may be used interchangeably with "electron-transferring flavoprotein", "EtfAB", etc.

[0034] The amino acid sequence of the above EtfAB protein can be obtained from a known database such as NCBI's Genbank.

[0035] For example, the EtfAB protein of the present application may be derived from a microorganism. As another example, the microorganism may be, specifically, a strain of the genus Clostridium sp., or as another example, a strain of Clostridium butyricum.

[0036] The EtfAB protein of the present application may be encoded by the etfAB gene. For example, the etfAB gene may be a polynucleotide encoding the EtfAB protein derived from Clostridium butyricum, and as another example, may include a base sequence encoding the amino acid sequence of SEQ ID NO: 3.

[0037] In the present invention, the term "pyruvate ferredoxin oxidoreductase (Pfor)" is an enzyme that catalyzes the interconversion of pyruvate and acetyl-CoA. The pyruvate ferredoxin oxidoreductase may be used interchangeably with "pyruvate:ferredoxin oxidoreductase", "Pfor", etc.

[0038] The amino acid sequence of the above Pfor protein can be obtained from a known database such as NCBI's Genbank. For example, the Pfor protein of the present application may be derived from a microorganism. As another example, the microorganism may be, specifically, a strain of the genus Clostridium (Clostridium sp.), or as another example, a strain of Clostridium butyricum.

[0039] The Pfor protein of the present application may be encoded by a pfor gene. For example, the pfor gene may be a polynucleotide encoding a Pfor protein derived from Clostridium butyricum.

[0040] Thus, the main factor in the increased hydrogen productivity confirmed by magnetite supplementation in the present invention was the activation of the lactate utilization pathway. The decrease in lactate concentration was analyzed to be the result of lactate oxidation to pyruvate catalyzed by LDH. This regeneration of pyruvate through lactate utilization can produce additional hydrogen through redox with Fd. The increase in butyrate production by magnetite supplementation can also be interpreted through this metabolic flux. That is, the oxidation of lactate to pyruvate is the effect of flavin-based electron bifurcation by the etfAB-LDH complex, and the conductivity of magnetite was analyzed to increase the electron transfer rate in the enzymatic reaction. Therefore, magnetite supplementation contributed to the regeneration of pyruvate by activating lactate oxidation, which in turn increased hydrogen production through its downstream pathway.

[0041] In particular, in the past, sugars such as glucose provided in organic wastewater were mainly used, and the usability for substances other than sugars was low, but according to the present invention, organic acids such as acetate in wastewater can be used, so there is an advantage in that even acidified organic waste can be treated.

[0042] As described above, lactate may be added to the fermentation tank to increase hydrogen production and improve organic matter treatment efficiency by adding magnetite. In the present invention, the term "lactate" refers to the conjugate base of lactic acid (CH3CH(OH)COOH, lactic acid), and the lactate may also be referred to as "lactate salt" or "lactate anion."

[0043] In the present invention, lactate may be included in a concentration of about 0.5 g / L or more, about 0.6 g / L or more, about 0.7 g / L or more, about 0.8 g / L or more, about 0.9 g / L or more, about 1 g / L or more, and about 2.7 g / L or less based on the total volume of the mixture of wastewater, magnetite, microbial strains, etc., and specifically, may be included in a concentration of about 0.1 to about 2.7 g / L. That is, the lactate concentration in the present application may mean the total amount of lactate in the entire mixture regardless of the inflow path of the lactate (existing in the wastewater from the beginning, added in a subsequent process, etc.). If lactate is added below or above the above concentration range, the increase in hydrogen production is insignificant compared to the control group, and thus addition of lactate outside the above concentration range may be unnecessary. The efficiency of the water treatment process can be significantly increased when the lactate in the mixture is included in the above-described range, but the water treatment process according to the present invention can also be operated outside the above-described lactate concentration range.

[0044] The present invention can also be applied to wastewater containing a high concentration of lactate. Specifically, the water treatment process according to the present invention can be performed even in a high-lactate environment, where the entire mixture of wastewater and microbial strains activated by a magnetite carrier contains lactate at a concentration of 16 g / L or less. Conventional techniques have been problematic in that water treatment processes using microorganisms do not operate or have significantly reduced efficiency when the composition of wastewater, including the lactate concentration, is not constant. However, since the water treatment process according to one embodiment of the present invention can function even in an environment containing a high concentration of lactate, it can be performed without a process for controlling the lactate concentration within a specific range.

[0045] In order to add lactate to the fermentation tank, the water treatment device may further be provided with a lactate supply unit. The lactate supply unit is a component for supplying stored lactate to the fermentation tank provided with the microbial strain activated by the wastewater and the magnetite support. However, if the wastewater to be treated already contains lactate, a separate lactate supply unit may not be required. The lactate supply unit may supply lactate stored in a liquid or solid form into the fermentation tank. Therefore, the form of the lactate supply unit may vary depending on the form of the stored lactate. For example, if the lactate is in a liquid form, the lactate supply unit may be provided in the form of a pump for supplying an appropriate amount of lactate from the storage tank storing the liquid into the fermentation tank.

[0046] The lactate supply unit may further be provided with a control device so as to supply an appropriate amount of lactate depending on factors such as the amount of wastewater supplied into the fermentation tank, the amount of microbial strains activated by the magnetite carrier, etc. Alternatively, the lactate supply unit may be controlled so that a preset amount of lactate is supplied into the fermentation tank when wastewater flows into the fermentation tank.

[0047] Fermentation tanks can be provided in batch or continuous reactor configurations. The shape and capacity of the fermentation tank can be determined by considering the time required for the microorganisms to perform metabolism and the water treatment capacity.

[0048] After the fermentation tank, a sedimentation tank, an anaerobic digestion tank, etc. may be provided.

[0049] In particular, a sedimentation tank may be provided to sediment the microbial strain activated by the magnetite carrier used in the fermentation tank. The microbial strain activated by the magnetite carrier is provided within the fermentation tank as described above, and depending on the method of provision, may be discharged out of the fermentation tank together with the treated wastewater after wastewater treatment in the fermentation tank. In particular, when the size of the magnetite carrier is not large or the magnetite carrier is not fixed within the fermentation tank, the magnetite carrier and the microbial strain fixed thereto can be recovered and recycled by utilizing the sedimentation tank.

[0050] Anaerobic digesters can be used to produce hydrogen and convert remaining byproducts, such as butyrate, into energy.

[0051] In some cases, a wastewater collection tank may be further provided in front of the fermentation tank according to one embodiment of the present invention. The wastewater collection tank is a device that stores wastewater to be treated in the fermentation tank. The wastewater is not directly introduced into the fermentation tank, but rather a certain amount may be collected in the wastewater collection tank before being transferred to the fermentation tank. The wastewater collection tank may be provided with a filter membrane or a filtering device to remove suspended solids, etc.

[0052] Water treatment equipment can be applied to food waste treatment facilities, etc. Furthermore, hydrogen gas produced in water treatment equipment can be converted into biogas and used as energy through a biogas purification facility. Biogas purification facilities may include, for example, wet desulfurizers, biogas dehumidifiers, and biogas storage facilities.

[0053] The water treatment device or method may additionally include a device and / or step for recovering hydrogen gas from the fermenter. The recovery may be accomplished by collecting the desired hydrogen gas using a suitable method known in the art, depending on the operating method of the fermenter using the microorganism of the present invention, such as batch operation, continuous operation, etc. For example, the hydrogen gas may be collected from the headspace of the fermenter using a gas sampling syringe, and the desired hydrogen gas may be recovered using a suitable method known in the art.

[0054] Additionally, the water treatment process of the present invention may additionally include a purification step. This purification may be performed using any suitable method known in the art. In one example, if the hydrogen gas production method of the present invention includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously, simultaneously, or integrated into a single step, but this is not limited thereto.

[0055] A water treatment device and water treatment process according to one embodiment of the present invention can secure high hydrogen productivity by simultaneously utilizing microbial strains activated by a magnetite carrier and lactate. This solves the following problems associated with conventional hydrogen production technologies, such as hydrogen-producing bacteria improvement technology, culture process optimization technology, and microbial immobilization technology.

[0056] First, hydrogen-producing bacteria improvement technology regulates the strain's metabolism by suppressing competing pathways within the metabolic pathway and enhancing the hydrogen production pathway. However, this technology faces the challenges of maintaining the reproducibility of improved strains and the need to maintain a constant concentration of antibiotics, making it uneconomical.

[0057] Furthermore, cultivation process optimization technology aims to derive optimal cultivation and operating factors for the growth of hydrogen-producing bacteria and hydrogen production. However, this technology faces the challenge of maintaining stability due to the high inflow / outflow rates of the reactor, which often results in numerous internal environmental changes.

[0058] Next, microbial immobilization technology aims to achieve physicochemical immobilization of microorganisms to maintain high concentrations of hydrogen-producing bacteria within the reactor. This technology suffers from the drawback of requiring an additional process for initial microbial immobilization.

[0059] Thus, existing technologies have the problem that microorganisms can only produce hydrogen with high efficiency under limited conditions, and that hydrogen production efficiency drops sharply when conditions change. However, given the nature of water treatment processes, where wastewater composition, temperature, and pH conditions constantly change, existing technologies that can only produce hydrogen under limited conditions have limited utility.

[0060] In contrast, according to one embodiment of the present invention, by introducing a microbial strain activated by a magnetite carrier into a fermentation tank and combining it with lactate as needed, excellent hydrogen production is possible regardless of changes in the fermentation tank environment.

[0061] The water treatment process and device according to the present invention are useful for the industrial production of biohydrogen because they can maintain high hydrogen production efficiency regardless of the environment within the fermentation tank.

[0062] Figure 1 illustrates a water treatment device according to one embodiment of the present invention.

[0063] Figure 2 is a diagram showing the proposed metabolic pathway for enhancing hydrogen production through magnetite supplementation. ldh, lactate dehydrogenase; pfl, pyruvate formate lyase; pyc, pyruvate carboxylase; mdh, malate dehydrogenase; fum, fumarate hydratase; pct, propionate-CoA transferase; pfor, pyruvate ferredoxin oxidoreductase; pta, phosphate acetyltransferase; ack, acetate kinase; thl, thiolase; hbd, 3-hydroxybutyryl-CoA dehydrogenase; crt, crotonase; bcd, butyryl-CoA dehydrogenase; ptb, phosphate butyryl transferase; buk, butyrate kinase; adhE1, aldehyde dehydrogenase; adhE2, alcohol dehydrogenase; hyd, hydrogenase; Fd, ferredoxin; etfAB, electron-transferring flavoprotein.

[0064] Figure 3 is a diagram showing gas production through lactate and / or magnetite supplementation; (A) Hydrogen production profile (B) Comparison of hydrogen production and yield.

[0065] Figure 4 compares the final volatile fatty acids (VFAs) production and consumption under lactate and / or magnetite supplementation conditions. Negative values ​​indicate that VFAs are ultimately consumed.

[0066] Figure 5 shows the results of analyzing the changes in VFAs concentration and pH according to operating time under lactate and / or magnetite supplementation conditions; (A) negative control, (B) magnetite supplementation, (C) lactate supplementation, (D) lactate and magnetite supplementation.

[0067] Figure 6 shows the results of comparing the expression levels of genes involved in hydrogen production in the lactate and / or magnetite supplementation conditions compared to the negative control group; (A) pfor, pyruvate ferredoxin oxidoreductase, (B) hyd, hydrogenase, (C) Fd, ferredoxin, (D) etfAB, electron-transfer flavoprotein.

[0068] Figure 7 is a comparative analysis graph of hydrogen production and yield according to the concentration of added lactate and the presence or absence of added magnetite carrier.

[0069] Figure 8 is a graph showing the analysis of metabolites of strains according to the concentration of added lactate.

[0070] Figure 9 is a comparative analysis graph of lactate consumption rate according to lactate addition concentration.

[0071] Figure 10 is a comparative analysis graph of biogas and hydrogen gas production according to the dilution ratio of wastewater.

[0072] Figure 11 is a comparative analysis graph of biogas and hydrogen gas production depending on whether magnetite is added to wastewater containing 5 g / L of glucose.

[0073] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0074]

[0075] Experimental Example 1. Analysis of hydrogen production under conditions of simultaneous supplementation of lactate and magnetite.

[0076] To analyze the optimal culture conditions for hydrogen production, lactate and magnetite were added singly or simultaneously to the Clostridium butyricum strain.

[0077] Specifically, to preculture the hydrogen-producing strain Clostridium butyricum (C. butyricum) DSM10702 (Korean Culture Center of Microorganisms), 800 μL of a 25% (w / w) glycerol stock of the C. butyricum strain was inoculated into 100 mL of Reinforced Clostridia Media (RCM) medium (10 g / L peptone, 10 g / L beef extract, 3 g / L yeast extract, 5 g / L sodium chloride, 3 g / L sodium acetate, 0.5 g / L L-cysteine, 5 g / L sodium bicarbonate, 100 μL / L antifoam 203, initial pH 7.0) in a 160 mL serum bottle under anaerobic conditions. The serum bottle was sealed and incubated overnight at 37°C for activation. Optical density (OD) was measured at 600 nm using a spectrophotometer, and OD 0.1 of C. butyricum strain was inoculated into 100 mL of RCM containing 5 g / L glucose in a 160 mL serum bottle and cultured at 37°C and 60 rpm. All cultivations were performed in an anaerobic chamber (Coy Laboratory Products, MI, USA) under 99.9% nitrogen gas conditions. For anaerobic conditions, the headspace of the serum bottle was replaced with nitrogen gas containing 2.5% hydrogen gas using the anaerobic chamber. Since the hydrogen gas in the initial headspace was below the calibration limit, it was not detected when measuring the hydrogen production produced from the strain. Subsequently, 5 g / L sodium bicarbonate was added as a buffer to the medium to alleviate the decrease in pH due to the production of volatile fatty acids (VFAs) from the C. butyricum strain. Additionally, 100 μL / L of antifoam (Sigma-Aldrich, USA) was added to prevent errors in headspace analysis due to bubbles in the serum bottle caused by gas production.

[0078] Next, to prepare a magnetite-only supplemented medium, 10 g / L of sterilized magnetite (Junsei Chemical Co., Japan) was added to the medium. The size and specific gravity of the magnetite used were 325 mesh and 5.18, respectively. For the lactate-only supplemented medium, 1 g / L of lactate was added to the medium. For the lactate and magnetite simultaneous supplemented medium, 10 g / L of magnetite and 1 g / L of lactate were added. The RCM medium contains lactate at a concentration of approximately 0.6 g / L, and the lactate concentration under each condition is the sum of the initial lactate concentration and the added lactate concentration.

[0079] The concentrations of VFAs in the medium and hydrogen in the biogas produced from the strain in the serum bottle were analyzed according to previous methods (Kim et al., Fuel 285, 119059., 2021; Park et al., Bioresour. Technol. 281, 318-325., 2019).

[0080] The volume and yield of hydrogen produced under each culture condition using the above-mentioned magnetite-only supplemented medium, lactate-only supplemented medium, or lactate and magnetite-only supplemented medium are shown in Figure 3 and Table 1.

[0081] Magnetite, lactate not addedMagnetite alone addedLactate alone addedMagnetite and lactate addedBiogas (mL)132.0±0.2155.7±3.3145.4±0.4198.5±1.9Hydrogen (mL),P72.6±1.191.2±2.485.2±1.2115.4±1.2Maximum hydrogen production rate (mL / h),r11.0±1.220.8±4.516.1±0.917.0±2.9Maximum hydrogen content in biogas (%)47.7±0.150.6±1.252.0±1.056.9±0.2Induction period (h),x04.0±0.43.8±0.54.8±0.13.6±0.6Hydrogen production rate (mol) H2 / mol.Glucose consumption)1.43±0.031.75±0.031.67±0.062.22±0.02R2 0.9910.9740.9980.978

[0082] As shown in FIG. 3 and Table 1, the volume and yield of hydrogen produced from the hydrogen-producing strain cultured in the medium supplemented with 10 g / L magnetite (+MGNT) were 91.2 mL and 1.75 mol-H2 / mol consumed glucose, respectively, which were 25.6% and 23.0% higher than those in the negative control group. In addition, the hydrogen production rate increased by 89.2% compared to the negative control group, and the maximum hydrogen content in the biogas increased by 2.9%. In addition, magnetite supplementation affected the increase in microbial growth rate and the decrease in the lag time for hydrogen gas production. The volume and yield of hydrogen produced from the hydrogen-producing strain cultured in the medium supplemented with 1 g / L lactate (+HLa) were 85.2 mL and 1.67 mol-H2 / mol consumed glucose, which were 17.4% and 17.1% higher than those in the negative control group. Lactate supplementation increased hydrogen production but prolonged the lag time. This was analyzed as being due to the inhibition of bacterial growth due to the increase in ionic strength resulting from the dissociation of lactate by lactate supplementation at neutral pH.

[0083] Meanwhile, the volume and yield of hydrogen produced from the hydrogen-producing strain cultured in the medium (+HLa+MGNT) simultaneously supplemented with 1 g / L lactate and 10 g / L magnetite were 115.4 mL and 2.22 mol-H2 / mol consumed glucose, respectively, which were 58.9% and 55.5% higher than the negative control group, confirming a significant increase in hydrogen production compared to the medium supplemented with only magnetite or lactate.

[0084]

[0085] Experimental Example 2. Analysis of Activation of the Lactate Utilization Pathway

[0086] In order to analyze the cause of the increase in hydrogen production due to the simultaneous supplementation of lactate and magnetite confirmed in the results of Experimental Example 1 above, changes in the production and consumption of final volatile fatty acids (VFAs), such as lactate, formate, acetate, and butyrate, according to changes in culture time and pH under each anaerobic culture condition of Experimental Example 1 above were analyzed.

[0087] As a result, in the medium supplemented with 1 g / L lactate (+HLa), hydrogen production increased by 17.4% compared to the negative control (Fig. 3 and Table 1), but total VFAs production decreased by 16.0% to 3.5 g / L compared to the negative control (4.2 g / L) (Fig. 4).

[0088] In the medium supplemented with 10 g / L of magnetite (+MGNT), hydrogen production increased by 25.6% compared to the negative control (Fig. 3 and Table 1), and total VFAs production increased by 9.8% to 4.6 g / L compared to the negative control (4.2 g / L) (Fig. 4).

[0089] Meanwhile, in the medium supplemented with lactate and magnetite simultaneously (+HLa+MGNT), hydrogen production increased by 58.9% compared to the negative control group (Fig. 3 and Table 1), and total VFAs production increased by 12.9% to 4.7 g / L compared to the negative control group (4.2 g / L) (Fig. 4).

[0090] Furthermore, in the medium supplemented with both lactate and magnetite, unlike in the medium supplemented with magnetite alone, the production of formate and acetate decreased by 16.5% and 18.3%, respectively, compared to the negative control group (Fig. 4). This suggests that lactate supplementation resulted in a decrease in the production of VFAs, including formate and acetate, suggesting that lactate supplementation can increase hydrogen production by reducing formate production, which is not accompanied by hydrogen production.

[0091]

[0092] Next, no significant changes in lactate utilization were observed in the medium supplemented with 1 g / L lactate (+HLa) (Fig. 5A and Fig. 3C). This suggests that lactate does not act as an initiator of the lactate utilization pathway, and the relatively low pH conditions caused by lactate supplementation were analyzed to increase enzyme activities involved in hydrogen production but suppress acid production.

[0093] In the medium supplemented with 10 g / L magnetite (+MGNT), unlike in the medium supplemented with lactate alone, formate and acetate production were not significantly affected, but lactate utilization and butyrate production were significantly affected. After a delay period, the lactate concentration decreased rapidly, indicating that the lactate utilization pathway was activated by magnetite (Fig. 5B and Fig. 3D).

[0094] From this, it can be seen that the lactate utilization pathway is activated when magnetite is supplemented, and by additionally supplementing lactate, the utilization of lactate by magnetite is maximized, thereby increasing hydrogen production.

[0095] Next, the quantitative relationship between VFAs related to lactate utilization and increased hydrogen compared to the negative control in the magnetite-only supplemented medium (+MGNT) and the medium supplemented with lactate and magnetite simultaneously (+HLa+MGNT) is shown in Table 2 through changes in the concentration of each component.

[0096] Magnetite addition Magnetite and lactate addition change amount (mmol) Hydrogen * Change in percentage (mmol) for hydrogen * Ratio for Lactate consumption 0.55 0.66 1.22 0.64 Acetate consumption 0.18 0.22 0.26 0.14 Butyrate production 0.54 0.65 1.12 0.59 Hydrogen (H2) production 0.83 1.00 1.9 11.00 * Increased hydrogen production compared to the negative control group

[0097] As shown in Table 2 above, in the medium supplemented with lactate and magnetite simultaneously, the utilization of lactate increased compared to the medium supplemented with magnetite alone, resulting in greater production of butyrate and hydrogen. The quantitative ratios of lactate to hydrogen produced in the medium supplemented with lactate and magnetite simultaneously and the medium supplemented with magnetite alone were 0.66 and 0.64, respectively, and the quantitative ratios of butyrate to hydrogen produced were 0.65 and 0.59, respectively. This suggested that there was a quantitative correlation in the lactate utilization pathway among lactate, butyrate, and hydrogen. However, in the case of acetate, the quantitative ratios of hydrogen produced were 0.22 and 0.14 in the medium supplemented with lactate and magnetite simultaneously and the medium supplemented with magnetite alone, respectively, showing a relatively low correlation compared to lactate and butyrate.

[0098]

[0099] Experimental Example 3. Analysis of gene-level changes involved in hydrogen production.

[0100] The effects of lactate and / or magnetite-supplemented media on the expression levels of genes involved in hydrogen production in C. butyricum strains were investigated. Genes encoding proteins directly involved in hydrogen production and genes encoding proteins that promote electron transport, which are indirectly involved in hydrogen production, are shown in Figure 2.

[0101] Specifically, RNA sequencing was performed to investigate differences in gene expression according to each anaerobic culture condition of Experimental Example 1. Cell samples for RNA sequencing were obtained at 10 hours of operational time, which is the exponential phase of C. butyrium growth (Fig. 3A). To maintain RNA molecule stability, the cell pellet was resuspended in RNAlater (Sigma-Aldrich, USA) and stored at -20°C. Total RNA was extracted from the cell pellet using a miRNA isolation kit (Invitrogen, USA). An mRNA sequencing library was constructed using the TruSeq Storged mRNA Library Prep Kit (Illumina, USA). Then, a final strand-specific cDNA library was constructed through reverse transcription of mRNA and enrichment of cDNA. Finally, the prepared samples were sequenced using a NovaSeq 6000 sequencing system (Illumina, USA) with a read length of 2 x 100 bp. In each condition, the change in gene expression compared to the negative control was converted to log2 (absolute change).

[0102] As a result, the C. butyricum strains cultured in the medium supplemented with only magnetite (+MGNT) and the medium supplemented with both lactate and magnetite (+HLa+MGNT) showed a 0.71-fold and 2.14-fold increase in the expression of the hyd gene encoding hydrogenase, respectively, compared to the negative control group (Fig. 6).

[0103] In the case of the Fd gene encoding ferredoxin, gene expression in the strain cultured in a medium supplemented with magnetite alone increased 3.50-fold compared to the negative control group, and in the strain cultured in a medium supplemented with both lactate and magnetite, gene expression increased 1.94-fold compared to the negative control group, indicating that gene expression was higher in the strain cultured in a medium supplemented with magnetite alone (Fig. 6). Since both the Hyd gene and the Fd gene contain iron ions in the protein structures encoded by them, it was analyzed that their expression was increased by the influence of iron ions derived from magnetite. In particular, in the case of hydrates, iron ions are essential elements because they not only serve as the active site of enzymes but also are a component of the Fe-S cluster. Therefore, it can be seen that iron ions can directly or indirectly affect the activity of proteins involved in hydrogen production, thereby increasing hydrogen production.

[0104] In contrast, the expression levels of genes encoding ferrous iron transport protein B, siderophore ABC transporter substrate-binding protein, ZIP family metal transporter, and NEAT domain-containing protein, which are generally involved in iron ion transport, in C. butyricum strains cultured in a medium supplemented with magnetite alone and a medium supplemented with both lactate and magnetite were reduced by more than twofold compared to the negative control.

[0105] In addition, C. butyricum strains cultured in a medium supplemented with only magnetite and a medium supplemented with both lactate and magnetite showed an increase in the expression of the etfAB gene encoding electron-transferring flavoprotein by 1.55 and 2.37 times, respectively, compared to the negative control group, and an increase in the expression of the pfor gene encoding pyruvate ferredoxin oxidoreductase by 0.26 and 1.06 times, respectively, compared to the negative control group (Fig. 6).

[0106] Accordingly, it was confirmed that supplementation of magnetite alone activates the lactate utilization pathway and upregulates the expression of pfor, hyd, and etfAB genes involved in hydrogen production, and that when lactate and magnetite are supplemented simultaneously, the activation of the lactate utilization pathway and upregulation of pfor, hyd, and etfAB genes are significantly increased compared to supplementation of magnetite alone, thereby improving hydrogen productivity.

[0107] From the results of the above experimental example, it was confirmed that when lactate and magnetite are simultaneously supplemented to the medium during the cultivation of a hydrogen-producing strain, the activation of the lactate utilization pathway and the upregulation of the pfor, hyd, and etfAB genes are significantly increased, thereby improving hydrogen productivity, and thus it can be applied to the industrial production of biohydrogen.

[0108]

[0109] Experimental Example 4. Hydrogen Production Experiment at Different Lactate Concentrations

[0110] In order to confirm the change in hydrogen production according to the concentration of added lactate and the presence or absence of added magnetite carrier, the experiment was repeated under the same conditions as in Experimental Example 1, changing the presence or absence of added magnetite carrier (NC, MGNT) and the amount of added lactate (HLa).

[0111] The experimental results are shown in Table 3 below and Fig. 7.

[0112] Lactate addition concentration 0.0 g / L 0.5 g / L 1.0 g / L 1.6 g / L 2.1 g / L Magnetite addition NCMGNT NCMGNT NCMGNT NCMGNT NCMGNT Biogas (mL) 135.5 174.3 133.4 206.9 138.0 216.2 133.2 26.1 147.5 208.4 Hydrogen (mL), P 48.3 72.1 47.0 97.6 50.9 95.0 52.8 104.1 57.1 103.3 Maximum hydrogen production rate (mL / h), r 7.0 19.17.5 36.8 12.2 24.8 10.5 22.18.1 12.3 Maximum hydrogen in biogas Content (%) 38.5 36.2 33.9 46.6 35.3 43.9 38.1 52.5 42.0 5 5.1 Hydrogen production yield (mol H2 / mol.glucose consumption)0.831.250.811.680.881.640.921.811.001.81R 2 1.000.991.000.991.000.991.001.001.000.99

[0113] The experimental results confirmed that when the magnetite support was added, the hydrogen production amount, hydrogen production yield, hydrogen production rate, and hydrogen concentration in the biogas tended to increase. In addition, the biogas production amount was high when the lactate addition concentration was 0.5 g / L to 1.6 g / L, and the biogas production amount actually decreased when the lactate addition concentration was 2.1 g / L. Meanwhile, the amount of hydrogen in the biogas was high when the lactate addition concentration was 0.5 g / L to 2.1 g / L. Next, a component analysis was performed on the metabolites prepared according to Experimental Example 4 above (Fig. 8).

[0114] Referring to Figure 8, when magnetite was added, lactate consumption tended to increase as the amount of added lactate increased. However, as the amount of added lactate increased, the lactate consumption tended to decrease relative to the amount added. Under experimental conditions, the strain was analyzed to be capable of consuming up to approximately 2 g / L of lactate.

[0115] Next, Fig. 9 compares the lactate consumption rate according to lactate concentration, and all experiments were performed under conditions in which a magnetite carrier was added.

[0116] The lactate consumption rate was calculated by comparing the initial concentration with the final concentration after metabolism according to the lactate addition. It was confirmed that the lactate consumption rate decreased compared to the initial lactate concentration starting from the condition of 1.6 g / L. Accordingly, it is analyzed that the lactate consumption rate is high when the lactate addition concentration is in the range of 0.5 g / L to 1.6 g / L, and thus hydrogen production is high.

[0117]

[0118] Experimental Example 5. Analysis of Wastewater Treatment Results

[0119] For food wastewater having a composition of pH 3.57, salinity 0.8%, glucose content 0.9 g / L, lactate content 27.8 g / L, acetate content 5.9 g / L, propionate content 1.5 g / L, and butyrate content 5.0 g / L, the strain used in Experimental Example 1 was cultured after centrifugation, filtration using glass fiber, and sterilization.

[0120] Referring to Fig. 10, it was confirmed that when the above-mentioned wastewater was diluted twice, the addition of a magnetite carrier increased hydrogen production by approximately 12.5%. The initial lactate concentration under the condition of diluting the wastewater twice was confirmed to be 16.0 g / L, and it was confirmed that the water treatment according to the present application can be applied to wastewater containing high concentrations of lactate.

[0121] In addition, referring to Fig. 11, it was confirmed that when glucose was added under the condition of diluting the wastewater by two times, the increase in hydrogen production due to the addition of magnetite carrier was improved.

[0122]

[0123] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. Includes a fermentation tank that receives wastewater containing organic matter and decomposes the organic matter contained in the wastewater to produce hydrogen. A water treatment device, wherein the above fermentation tank comprises a microbial strain activated by a magnetite carrier.

2. In paragraph 1, The above microbial strain is a water treatment device belonging to the genus Clostridium.

3. In paragraph 1, A water treatment device, wherein the wastewater provided to the above fermentation tank contains lactate.

4. In paragraph 1, A water treatment device further comprising a sedimentation tank for sedimenting the microbial strain activated by the magnetite carrier used in the fermentation tank.

5. In paragraph 1, A water treatment device further comprising an anaerobic digestion tank into which the wastewater that has passed through the fermentation tank is introduced.

6. Includes a hydrogen production step of supplying wastewater containing organic matter to a fermentation tank or wastewater storage tank and decomposing the organic matter contained in the wastewater to produce hydrogen. A water treatment process wherein the above hydrogen production step is performed by supplying lactate to a microbial strain activated by a magnetite carrier.

7. In paragraph 6, A water treatment process in which the magnetite carrier is supplied so that magnetite is provided at a concentration of 0.05 to 11 g / L to the entire mixture of the wastewater and the microbial strain activated by the magnetite carrier in the hydrogen production step.

8. In paragraph 6, A water treatment process in which lactate is supplied to the entire mixture of the wastewater and the microbial strain activated by the magnetite carrier in the hydrogen production step so that lactate is provided at a concentration of 0.1 to 2.1 g / L.

9. In paragraph 6, A water treatment process capable of producing hydrogen by decomposing organic matter even when lactate at a concentration of 16 g / L or less is provided to the entire mixture of the wastewater and the microbial strain activated by the magnetite carrier in the hydrogen production step.

10. In paragraph 6, A water treatment process further comprising a step of recovering the magnetite carrier and the fixed microbial strain after performing the hydrogen production step.

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