KR20060110394A - Producing mothod of hydrogen gas using anaerobic microorganism complex - Google Patents

Abstract

A method for producing hydrogen gas by using the anaerobic microorganism complex is provided to improve production speed of hydrogen gas and perform the production of hydrogen gas at all hours. The method for producing hydrogen gas comprises the steps of: collecting the concentrated sewage sludge produced from the precipitation tank in a sewage disposal plant; treating the concentrated sewage sludge with heat to concentrate hydrogen-producing microorganisms; adding the concentrated hydrogen-producing microorganisms into a substrate; and producing hydrogen by using anaerobic fermentation processes, wherein the heat treatment temperature is 80-100 deg. C; the heat treatment is performed at 85-95 deg. C for 10-30 minutes just one time; and the substrate is sewage produced from tofu(bean-curd) production.

Description

Producing Mothod of Hydrogen Gas Using Anaerobic Microorganism Complex}

1 is a graph showing the growth and fermentation characteristics of microbial complexes isolated from sewage sludge according to the present invention.

Figure 2 is a graph showing the concentration and type of organic acid produced during anaerobic fermentation by anaerobic microbial complex according to the present invention

Figure 3 is a preferred embodiment according to the present invention the results of analysis of dominant species microorganisms present in the fermenter with and without hydrogen production excellent

Figure 4 is a graph of the pH change in hydrogen and incubator generated when the semi-continuous culture for 25 days using the anaerobic microorganism complex according to the present invention [●: gas, △: hydrogen, ■: pH]

5 is a block diagram of an apparatus for treating tofu manufacturing wastewater presented as a preferred embodiment according to the present invention;

6 is a graph showing the amount and pH change of starch when the anaerobic microbial complex according to the present invention produces hydrogen using starch

The present invention relates to a method for producing hydrogen using an anaerobic microbial complex for hydrogen production, and more particularly, to produce a microbial complex that can only generate hydrogen from the sludge generated from the sedimentation tank of the sewage treatment plant and heat treatment for a predetermined time. And a method for producing hydrogen by an anaerobic fermentation step using the microbial complex obtained therefrom.

Biological hydrogen production by anaerobic fermentation, which obtains energy and electrons necessary for microbial growth and hydrogen generation from organic matter, is faster than hydrogen production by photosynthetic bacteria or algae that use sunlight, and thus can be industrially produced in large quantities. The advantages are as follows.

First, related anaerobes have faster hydrogen production than photosynthetic microbes.

Second, unlike photosynthetic microorganisms using sunlight, fermentation occurs in dark conditions, so it is possible to produce hydrogen day and night with organic matter as a substrate.

Third, the cell growth rate is fast and convenient for continuous culture or maintenance of large facilities.

Fourth, it can be used as a treatment technology of organic waste, such as food factory wastewater, food waste, agricultural product collection waste.

Bacteria capable of anaerobic fermentation are complex microorganisms in natural ecosystems with anaerobic-deficient anaerobic environments, such as sewage sludge or riverbeds, and these microbial complexes undergo biological reactions to maintain the ecosystem in certain conditions. Doing. That is, such a microbial complex is composed of microorganisms that liquefy high polymer materials, microorganisms that generate hydrogen, microorganisms that consume hydrogen, and microorganisms that are not related to hydrogen metabolism.

Acid producing bacteria present in anaerobic conditions produce organic acids, carbon dioxide and hydrogen from organics, which are converted back to methane and carbon dioxide by bacteria that consume hydrogen. That is, while methane bacteria consuming the produced hydrogen gas and acid producing bacteria generating hydrogen gas coexist in the anaerobic state, they are maintained in a natural state in which hydrogen gas, methane gas and organic acid in the sewage sludge are balanced.

Many studies that have been conducted for the purpose of continuously generating hydrogen have various treatments such as adjusting the pH, maintaining organic materials in the fermenter, holding time in the fermenter, and filling of nitrogen gas in order to maintain various conditions in the fermenter producing only hydrogen. The operation was carried out but was not successful in producing hydrogen continuously.

In addition, organic waste resources such as food factory wastewater, sewage sludge, agricultural market waste, and food waste are domestically available resources, and at the same time, they are also wastes that are prohibited by landfilling from 2005.

Biological hydrogen production from organic waste resources is a technology capable of treating organic waste as well as alternative and clean energy production, which serves two purposes: energy production and environmental treatment.

In advanced countries, including Japan, a large project called 'Development of Hydrogenation Technology for Environmental Harmony' has been actively researched for about 29 billion won over the past eight years to produce hydrogen from food processing wastewater and sludge biologically.

Among the wastewater from food factories, most of the tofu manufacturing wastewater is produced in the market or apartment complexes.The wastewater generated during manufacturing is significantly high at 27,440 mg / l and 9,800 mg / l in COD and BOD, respectively. It is becoming.

Even in the wastewater treatment facilities within the manufacturing plant itself, the wastewater itself has high starch and sugar content and low nitrogen source concentration, making it difficult to treat efficiently. Particularly, the conventional conditions, including pH, are difficult to coexist with acid-producing bacteria and methane-producing bacteria, resulting in delayed or limited decomposition of organic substances in wastewater, resulting in inconsistent treatment efficiency.

Hydrogen-producing bacteria use carbohydrates under anaerobic fermentation to accumulate various organic acids and organic solvents in the culture, and simultaneously generate hydrogen and carbon dioxide. Typical as Clostridium butyric rikeom (Clostridium butyricum), Clostridium Pas Teng Ria over (Cl. Pasteurianum), Clostridium Oh Shetty Com (Cl. Aceticum), Clostridium Cluj berry (Cl. Kluyveri) and Enterobacter Enterobacter aerogenes is the best known anaerobic hydrogen-producing bacterium, and research on hydrogen production using these is being actively conducted.

However, long-term production of hydrogen from organic wastewater or waste using these single bacteria is practically difficult. Because many microorganisms already exist in the tofu wastewater itself, hydrogen production efficiency may be lowered or other gases may be generated. In addition, the high concentration of organic wastewater in the food industry, agriculture, water and livestock is not only complicated in its sex box, but more research is needed on the characteristics of wastewater by considering the difference and size according to the process or season of discharge.

The present invention is presented to solve the problems of the prior art,

It is an object of the present invention to obtain a microbial complex that can only generate hydrogen from the sludge generated from the sedimentation tank of the sewage treatment plant and heat treatment for a predetermined time, by using this anaerobic fermentation process stable and mass without night and day To provide a method for producing hydrogen.

The present invention as a means for achieving the above object comprises the steps of collecting the concentrated sewage sludge generated in the sedimentation tank of the sewage treatment plant; Heat-treating the concentrated sewage sludge to concentrate hydrogen-producing microorganisms; And it provides a method for producing hydrogen using the anaerobic microbial complex comprising the step of adding the concentrated hydrogen-producing microorganism to the substrate to produce hydrogen using an anaerobic fermentation process.

According to the present invention, the heat treatment temperature provides a method for producing hydrogen using an anaerobic microorganism complex, characterized in that carried out at 80 ℃ to 100 ℃.

According to the present invention, the heat treatment provides a method for producing hydrogen using an anaerobic microorganism complex, characterized in that it is carried out once for 10 to 30 minutes at 85 ℃ to 95 ℃.

According to the present invention, there is provided a method of producing hydrogen using an anaerobic microbial complex further comprising the step of continuously stirring the reactant during the generation of hydrogen gas, and / or removing the generated gas.

Hereinafter, the content of the present invention in more detail as follows.

The present invention collects the concentrated sewage sludge generated in the sedimentation tank of the sewage treatment plant; Heat-treating the concentrated sewage sludge to concentrate hydrogen-producing microorganisms; And a method for producing hydrogen using the anaerobic microorganism complex comprising adding the concentrated hydrogen-producing microorganism to a substrate to generate hydrogen using an anaerobic fermentation process.

At this time, the sedimentation tank of the sewage treatment plant is preferably a secondary sedimentation tank, and the concentrated sewage sludge generated includes both methane-producing microorganisms and hydrogen-producing microorganisms. In the present invention, hydrogen production efficiency is enhanced by a technique for maintaining the activity of hydrogen-producing microorganisms while killing or inactivating microorganisms involved in anaerobic fermentation without hydrogen production such as methane-producing microorganisms that consume hydrogen or lactic acid fermentation. You can.

To this end, the present invention is intended to achieve the above effects by simply heat treatment the concentrated sewage sludge obtained from the secondary sedimentation tank of the sewage treatment plant. In this case, the heat treatment is carried out in a water bath at 80 ℃ to 100 ℃, preferably at 85 ℃ to 95 ℃ once for 10 to 30 minutes. At this time, an excessive level of heat treatment may rather kill or inactivate the hydrogen-producing microorganisms, so be careful.

In the microbial complex according to the present invention obtained through the above process, it was determined that strain of Clostridium sp. The anaerobic strain is known to accumulate hydrogen and various organic acids in glycolysis metabolism. That is, 4 mol of hydrogen is generated in the acetic acid fermentation process per 1 mol of glucose in the process of fermenting the organic acid with glucose as a substrate, and 2 mol of hydrogen is obtained in the butyric acid fermentation process.

However, when the concentrated sewage sludge obtained from the secondary sedimentation tank is not subjected to an operation such as heat treatment, methane-producing bacteria or Lactobacillus spp. Occupy a large number, and thus hydrogen consumption or lactic acid fermentation process is performed. When lactic acid fermentation is performed on one mole of glucose, no hydrogen is produced.

Therefore, the heat treatment process for the concentrated sewage sludge obtained from the secondary sedimentation tank according to the present invention includes a process of killing or inactivating microorganisms performing methane production, lactic acid fermentation, etc. irrelevant to hydrogen production. In addition, this heat treatment process should not be involved in the killing or inactivation of anaerobic bacteria associated with hydrogen production. This may be performed by adjusting the temperature and time, such as heat treatment conditions, as mentioned above.

The anaerobic microbial complex according to the present invention can be used for the purpose of treating organic wastewater. When the anaerobic microorganism complex according to the present invention is used for hydrogen production, it provides an advantage faster than the hydrogen production rate by conventional photosynthetic microorganisms.

In addition, unlike photosynthetic microorganisms using sunlight, the fermentation process can be performed in dark conditions, so that hydrogen can be produced using organic matter as a substrate.

In addition, the growth rate of the cells is fast and convenient for continuous culture and maintenance of large facilities, and is very useful for the treatment of organic wastes such as food factory wastewater, food waste, farm picking waste.

In order to increase the final hydrogen production during the fermentation process for hydrogen production, it is desirable to continuously remove the hydrogen gas produced in the stirring or incubator. Continuous agitation and gas removal resulted in an increase of about 1.2 times more hydrogen in stationary cultures. The effect of continuous agitation is to remove hydrogen gas in the incubator to lower the partial pressure of hydrogen, and to prevent the mass transfer phenomenon from being inhibited by the cells sitting on the incubator through agitation. This is a very important factor in the size of the incubator.

Hereinafter, the hydrogen production process using the tofu manufacturing wastewater which is one of the organic waste as a substrate as follows.

Fig. 5 shows an apparatus configuration for generating hydrogen by treating tofu manufacturing wastewater. Hydrogen growth value for the treatment of tofu production wastewater is fermenter (4), substrate (2), discharge tank (8), pump (3), pH regulator (7), alkali (9), stirrer (6), gas collection Device 10 is included.

First, 2 to 5 g / l of yeast extract and 5 to 10 g / l of ammonium chloride were added to the tofu manufacturing wastewater as a nutrient for growth of the cells, and then the anaerobic microorganism complex separated according to the present invention was about 10 to 20% by volume ratio. Add to At this time, the pH is maintained at a level of about 6.8, and the argon gas is substituted for about 5 to 20 minutes for the anaerobic fermentation process to remove air in the fermentor (4).

The fermentation process is carried out at about 30 ~ 40 ℃, the gas generated during the fermentation (including hydrogen) and the liquid after the fermentation is moved to the tank (8), the gas is measured in the gas collecting device (10). Tofu production waste water is produced by the fermentation proceeds hydrogen and organic acid, so the pH is continuously lowered to prevent the pH probe is installed in the fermenter (4), it is adjusted to 5.5 to 6.5 through the pH controller (7).

When the organic matter in the tofu manufacturing wastewater is consumed, no more hydrogen is produced, so new feedstock wastewater is continuously supplied. At this time, since a large amount of hydrogen-producing microorganisms are present in the tank 4, it is not necessary to supply new microorganisms.

The tofu manufacturing wastewater has a high starch content, so that the decomposition rate is slow, but as shown in FIG. 6, the pH can be decomposed after about 22 hours by adjusting the pH to 6.5.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the content of the present invention, and the scope of the present invention should not be construed as being limited to these embodiments.

Example 1 Preparation of Anaerobic Microbial Complex

Experiment process

(1) sewage sludge and heat treatment

Concentrated sewage sludge from secondary sedimentation tank of Daejeon Metropolitan City sewage treatment plant was collected and 20ml of sewage sludge was filled in a 53ml serum bottle, and the gas was removed from a 90 ℃ water bath and heated for 20 minutes.

(2) Culture conditions

2 ml (10%) sewage sludge in a 53 ml serum bottle was prepared from PYG synthetic media containing 1% glucose [0.9 g of K ₂ HPO ₄ ; 0.9 g KH ₂ PO ₄ ; 0.9 g NaCl; 0.9 g (NH ₄ ) SO ₄ ; 0.09 g MgSO ₄ ; 0.09 g of CaCl ₂ ; 10.0 g peptone; 5.0 g of yeast extract; Cysteine-HCl 0.5 g; 4.0 g Na ₂ CO ₃ 10 H ₂ O; 100 µg ρ-aminobenoic acid; 10 μg biotin; 10 g of glucose] was added to 18 ml, sealed with a stopper, and replaced with argon gas for 15 minutes to make anaerobic conditions, and passaged at 37 ° C. for 7 hours or 16 hours to observe hydrogen production of sewage sludge strains. The pH was initially 7.0 and then not adjusted.

(3) analysis

Hydrogen content produced in the culture was analyzed by gas chromatography (Shimazu 14-B) by taking 100 μl of a head space of a 33 ml serum bottle with a gas-tight microsyringe. The column used was 300mm × 2mm (length × diameter) glass, and Molecular Sieve 5A (Supelco Inc.) was used as a filler and analyzed by a thermal conductivity detector (TCD). The analytical conditions for quantifying hydrogen gas in the generated gas were column temperature of 80 ° C, injector temperature of 100 ° C, and detector temperature of 120 ° C. The carrier gas was maintained at a flow rate of 35 ml / min as argon. Cell concentration in the culture was measured by the ultraviolet-visible spectrophotometer (Shimadzu UV-1601) at a wavelength of 660nm. Glucose concentration used as a carbon source in the culture was quantified by the dinitrosulfonic acid (DNS) method. When culturing with glucose as a substrate, a sample obtained by separating the cells and the supernatant by centrifuging the culture solution collected at regular intervals at 10,000 g for 5 minutes was used for reducing sugar analysis. After adding 3 ml of DNS to 1 ml of the sample, the mixture was boiled and cooled at 100 ° C. for 5 minutes, and then absorbance was measured at 540 nm. A standard curve equation was prepared using glucose standard reagents of known concentration and the decomposition of reducing sugars was calculated accordingly. The pH of the culture was measured at room temperature with a pH meter (TOA, Model 30G).

(4) denaturation gradient gel electrophoresis (DGGE)

As a molecular biological method for the analysis of the microbial community for the analysis of DNA of hydrogen-producing strains in sewage sludge, the PCR-DGGE method, which has recently been attracting a lot of attention as an evaluation of wastewater treatment or decomposition of organic matter, was used.

DNA amplified by PCR was isolated for 6 hours at 100V, 60 ℃ using Dcode Universal Mutation Detection System (Bio-Rad Laboatories, Hercules, CA). The gel of DGGE consisted of 40% to 60% of 6% polyamide gel (acrylamide; N, N'-methylene-bisacrylamide ratio, 37.5: 1 [Bio-Rad]) concentration gradient with a mixture of urea and formamide. Used. After electrophoresis, the gel was stained with ethidium bromide (100 μl / L) and photographed (PHC-34, Vilber Lourmat, France).

(5) PCR amplification

DNA of hydrogen-producing strain in sewage sludge is MO. It was analyzed using the Ultraclean DNA kit prepared by Bio. Forward primer 968f (5'-AACGCGAAGAACCTTAC-3 ') with GC clamp (5'-CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCACGGGGGG-3') and reverse primer 1492R (5'-TACGGYTACCTTGTTACGACTT-3 ') with Taq polymerase, dNTP After mixing, 16S rDNA amplification was attempted at a total amount of 20 µl. The PCR reaction was warmed at 95 ° C. for 5 minutes before the first cycle started, and then 30 cycles were denatured at 95 ° C. for 30 seconds, annealed at 50 ° C. for 30 seconds, and extended at 72 ° C. for 90 seconds. And 10 min at 72 ° C. for complete extension. Before DGGE analysis, electrophoresis was performed on 0.8% agarose gel to confirm DNA amplification.

Experiment result

As a result of collecting concentrated sewage sludge from secondary sedimentation tank of Daejeon Metropolitan City sewage treatment plant and using it as a spawn, heat treatment using water tank resulted in killing or inactivating methane-producing microorganisms in sewage sludge and concentrating only hydrogen producing microorganism. Indicated. However, two to three consecutive heat treatments also killed all of the hydrogen-producing bacteria (Table 1).

<Table 1> Cell and Hydrogen Production by Sewage Sludge Heat Treatment and Sludge Complex

Heat treatment (90 ℃, 20 minutes) - + ++ +++ Cell growth and hydrogen production 1 day ○ ○ △ × 2 days ○ ○ × × 3 days × ○ × × 4 days × ○ × × ~ 2 months × ○ × ×

○: Excellent cell growth and hydrogen production

△: slightly cell growth and hydrogen production

× :: No cell growth and no hydrogen production

-: Does not heat treat sewage sludge.

+: 14 hours of incubation in PYG medium using sewage sludge as a spawn

++: 14 hours incubation in PYG medium after heat treatment of +

+++: Incubated for 14 hours in PYG medium after re-treating the treated culture solution

In addition, when the sewage sludge was used as a seed without heat treatment, the hydrogen production rate was high at the beginning of the cultivation, but after 2 days, the cells did not grow under anaerobic culture conditions and no hydrogen was generated.

After the heat treatment, the composite bacteria for producing hydrogen were named "sewage C-1", and passaged for about two months to observe hydrogen production and cell growth. . That is, referring to FIG. 1, hydrogen productivity is 1.2 ml H ₂ / mg-dry cell weight after 10 to 15 hours of culture in PYG synthetic medium containing 1% glucose, which corresponds to 2.94 ml H ₂ / ml-culture. . The dry weight of the cells was 2.2 mg / ml, and the initial pH 7.00 began to decrease after about 4 hours of incubation and dropped to pH 4.48.

The organic acid generated at this time is 17, 35 and 35 mM of acetate, lactate and butyrate, respectively, according to the results of FIG. And over time the butyrate concentration increased.

Assuming a maximum conversion rate of acid producing anaerobes producing about 4M H ₂ and 2M CO ₂ and acetate from 1M glucose, a maximum of about 4.93 LH ₂ can be generated from l-culture from 1% glucose.

Sewage C-1 generated 2.94 ml of hydrogen and had an efficiency of about 59% in converting glucose to hydrogen. The generated organic acid is mixed with lactate and butyrate as well as acetate. That is, when glucose is converted into lactate by the strain of Lactobacillus in the complex, no hydrogen is generated at all, and when converted to butyrate, only about 2M of hydrogen is generated, which is only about 50% of the conversion to acetate ( See formula below).

(1) C ₆ H ₁₂ O ₆ + 2H ₂ O → 4H ₂ + 2CO ₂ + 2CH ₃ COOH

(2) C ₆ H ₁₂ O ₆ + H ₂ O → 2CH ₃ CHOHCOOH

(3) C ₆ H ₁₂ O ₆ + H ₂ O → 2H ₂ + 2CO ₂ + CH ₃ (CH ₂ ) ₂ COOH

The sewage C-1 complex produced almost all of the 1% glucose in about 5-6 hours to generate organic acids. Acids generated were mainly acetate, lactate, butyrate, and ethanol, an organic solvent, was also produced. This lowered the pH of the culture so that glucose was broken down into about 33% organic acid at 3 hours of culture, and the pH was lowered to 5.29. At this time, the cells grew to 1.823 g-dry cell weight / L-culture medium. The combined bacteria lasted about 5 hours in logarithmic growth, with generation time of 18 minutes. Hydrogen evolution began at the same time that glucose was broken down to produce organic acids, and hydrogen production was stopped when glucose was stopped. Glucose was not degraded by more than 87.5%, because the pH of the culture had already been lowered to 4.5 and the complex bacteria sewage C-1 reached the lowest pH at which it could grow anymore. In further experiments, all the glucose was degraded when the pH was maintained between 6.8 and 7.0 during incubation. The distribution of microbial populations was examined by culturing sewage sludge C-1 isolated from sewage sludge after using heat treated sewage sludge itself as a spawn for hydrogen production.

As shown in FIG. 3, the microorganisms present in the hydrogen-producing anaerobic medium showed a constant band pattern as in lanes 5, 6, and 7, and extracted the bands to amplify DNA by PCR to match the 16S rRNA gene. Gene Bank BLAST was run to find the most likely microorganisms. From the anaerobic broth with excellent hydrogen production, many anaerobic strains were matched with the strains of Clostridium (Figs. 3-E to G), while a large amount of microorganisms of Lactobacillus species were detected from the non-hydrogen broth (Fig. 3). -CD). These results indicate that strains of Lactobacillus species generally have lactose and CO ₂ production from glucose and thus do not produce hydrogen gas, whereas strains of Clostridium species have hydrogen and various organic acids. The result is consistent with what is known to accumulate. Some of Bifidobacterium species was detected from the culture medium that did not produce hydrogen (FIGS. 3-A and B).

Stirring and degassing during gas production and incubation affected total gas and hydrogen production. After substituting argon for air in the incubator, the hydrogen content in the generated gas was measured to be lower than the hydrogen concentration produced by the complex bacteria in the incubator. Continuous stirring during incubation and degassing in the incubator increased hydrogen production by about 1.2 times over stationary culture. Continuous stirring showed a better effect than removing the gas in the incubator. Such a phenomenon is that if the stirring is not performed, the cells sink to the bottom of the incubator, thereby inhibiting the mass transfer phenomenon. Such continuous agitation and degassing effect is considered to be an important factor in the enlargement of the incubator.

<Table 2> Effect of agitation and degassing of fermenter on hydrogen production when anaerobic fermentation of complex bacteria isolated from sewage sludge

Total gas production (ml) H ₂ content(%) Ratio (ml H ₂ / ml Medium) Stirring × Degassing × 32.7 (± 4.22) 36.7 (± 1.11) 1.77 (± 0.08) Degas only 36.7 (± 2.89) 43.3 (± 3.11) 1.85 (± 0.06) Only stirring 48.3 44.5 2.1 Perform stirring and degassing 52.7 (± 7.56) 45.7 (± 1.11) 2.17 (± 0.15)

The complex strain isolated from the heat treated sludge produced hydrogen stably for about 1 month in a semi-continuous culture (HRT = 24hr) under the condition that pH was not controlled (FIG. 4). Hydrogen accounted for 60-70% of the total gas produced, with CO ₂ remaining. Reducing sugar was consumed in 18 to 20 hours, but for convenience, the medium was changed every 24 hours. During incubation, the pH initially reached 6.7-6.8 and then decreased to 4.5 after 24 hours, and the cell concentration was maintained at absorbances of 3.9-4.2. 10 g / l of reducing sugar was decomposed about 70% for 15 to 17 hours, and the pH was reduced to about 4.5 due to the generation of organic acid. After 24 hours, the carbon source remained about 3.2 to 3.5 g / l. It was not decomposed due to the decrease in pH, and when the pH was raised to 6.7 to 6.8 again by medium exchange, 36% reducing sugar was decomposed for about 5 hours, and 63% was decomposed for 15 hours.

Example 2 Treatment of Tofu Manufacturing Wastewater

Experiment process

Yeast extract 3g / L and NH ₄ Cl 8g / L were added to the tofu manufacturing wastewater (Table 3) not more than 12 hours after tofu production, and the complex bacteria (sewage C-1) separated from the sewage sludge were About 20% was added, and after adjusting the pH to 6.8, argon gas was substituted for about 10 minutes to remove air in the fermentor (symbol 4 in FIG. 5). A fermentation apparatus was installed as shown in FIG. 5 and cultured at 37 ° C. The generated gas (including hydrogen) and the fermented liquor are transferred to the tank (8), where the gas is measured in the cylinder (10). The tofu manufacturing wastewater is hydrogen and organic acid is produced as the fermentation proceeds, so the pH continues to drop. To prevent this, the pH sensor was installed in the fermenter (4) and adjusted to 5.5 through the pH controller (7).

When the organic matter in the tofu manufacturing wastewater was consumed, no more hydrogen was produced, so a tank (2) was installed to continue supplying new tofu wastewater. At this time, since a large amount of hydrogen-producing microorganisms exist in the tank 4, it is not necessary to supply new microorganisms.

The tofu manufacturing wastewater had a high starch content, so that the decomposition rate was slow, but when the pH was adjusted to 6.5 as shown in FIG. Using the above strain from the tofu wastewater (containing 1.4% starch, adding nitrogen source) and optimizing the conditions, 1.7lH ₂ / l-wastewater was generated. The acid generated was mainly acetate, lactate, butyrate, and 64, 120, respectively. , And 44 mM.

<Table 3> Composition of Wastewater for Tofu Manufacturing

Reducing Sugar (g / ℓ) Starch (g / ℓ) Ammonia nitrogen (ppm) TS (g / ℓ) SS (g / ℓ) VS (g / ℓ) BOD (mg / ℓ) COD (mg / ℓ) 0.736 8 to 15 5.66 17.3 1.4 12 9,060 22,000

TS: total solids, SS: suspended solids, VS: volatile solids

Hydrogen and Organic Acid Analysis

The amount of gas generated during the cultivation was measured by reading the scale of the measuring cylinder through a 0.1N NaOH solution when operating in a fermenter. The content of hydrogen was analyzed by gas chromatography by taking 100 μl of a head space gas of the fermentor with a syringe. The column used was filled with a molecular sieve 5A (Supelco. Inc) in a 300mm × 200mm (length × inner diameter) stainless steel, and a thermal conductivity detector (TCD) was used as a detector. A column temperature of 80 ° C., an injector temperature of 100 ° C., and a detector temperature of 120 ° C. were used as analysis conditions, and the argon flow rate was maintained at 35 ml / min.

The fermentation products by the biomass and anaerobic strain used in the experiment were collected at regular intervals to separate the impurities and supernatant from the sample, and the culture solution was separated from the cells and the supernatant, and 20 μl of the supernatant was equipped with a 0.2 micro filter. After filtration by syringe, it was analyzed. The organic acid was analyzed at room temperature using HPLC equipped with an organic acid analysis column, Aminex HPX-87H, 300 mm × 7.8 mm (length × inner diameter), and various organic acid peaks were measured at a wavelength of 210 nm using a UV detector. Elution was carried out at a flow rate of 0.6 ml / min using NH ₂ SO ₄ as the mobile phase.

TABLE 4

Time (min) formula acetate 14.95 y = 0.0000148x-0.9693 Lactate 11.03, 11.63, 12.41 y = 0.00000619x + 0.1961 Butyrate 22.0 y = 0.00001106x-0.1529 Formate 13.7 y = 0.00000788x-0.0131

Reducing Sugar and Starch

Reducing sugar quantification of the culture medium was carried out using the dinitrosalicyclic acid (DNS) coloration method, starch concentration was 0.2mL 5.5N HCl and 2mL sample was heated in 100 ℃ water for 30 minutes and 2.2mL Na ₂ CO ₃ Neutralized by addition and quantified by DNS method. The standard curve was quantified by the same method using soluble starch. The standard equation is as follows.

Glucose Standard Line Formula: y = 4.34x-0.46

(y = absorbance at glucose (g / l), x = 540 nm)

Starch Normal Line Formula: y = 53.8x-0.59

(y = absorbance at starch (g / l), x = 540 nm)

According to the present invention, by collecting the sludge generated from the sedimentation tank of the sewage treatment plant and heat treatment for a predetermined time to obtain a microbial complex that can only generate hydrogen, and through the anaerobic fermentation process using the same, stable and large quantities without night or day Hydrogen can be produced.

As described above, the present invention has been described with reference to the preferred embodiments, but those skilled in the art to which the present invention pertains vary the present invention without departing from the spirit and scope of the present invention as set forth in the claims below. It will be understood that modifications and changes can be made.

Claims (5)

Collecting concentrated sewage sludge generated in the sedimentation tank of the sewage treatment plant; Heat-treating the concentrated sewage sludge to concentrate hydrogen-producing microorganisms; And producing hydrogen using an anaerobic fermentation process by adding the concentrated hydrogen-producing microorganism to a substrate. The method according to claim 1, wherein the heat treatment temperature is performed at 80 ° C to 100 ° C. The method according to claim 1, wherein the heat treatment is performed at 85 ° C to 95 ° C for one time for 10 to 30 minutes. The method of claim 1, further comprising continuously stirring the reactants and / or removing the generated gas during generation of hydrogen gas. The method of claim 1, wherein the substrate is tofu manufacturing waste water.

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