WO2011079827A1

WO2011079827A1 - Extreme halophilic bacteria, composite halophilic bacterium microbial agent and uses thereof

Info

Publication number: WO2011079827A1
Application number: PCT/CN2011/000145
Authority: WO
Inventors: 李捍东
Original assignee: 中国环境科学研究院
Priority date: 2009-12-31
Filing date: 2011-01-28
Publication date: 2011-07-07
Also published as: CN101712945A; CN101712945B

Abstract

Three extreme halophilic bacteria strains and composite halophilic bacterium microbial agent containing the three extreme halophilic bacteria strains and uses thereof are provided. The extreme halophilic bacteria strains obtained in the present invention are halomonas sp. CGMCC No.3081, pseudomonas sp. CGMCC No.3082 and bacillus sp. CGMCC No.3083.The composite halophilic bacterium microbial agent containing the extreme halophilic bacteria strains above are also provided. Three extreme halophilic bacteria strains which have tolerance of salinity of 15-25% are isolated. The composite halophilic bacterium microbial agent obtained by combining the three extreme halophilic bacteria strains can treat the wastewater produced in the production process of high-salt cyanuric chloride effectively.

Description

Extreme halophilic bacteria, and complex halophilic bacteria agents and applications

Technical field

The present invention relates to the field of microorganisms, and in particular to extreme halophilic bacteria, complex halophilic bacteria agents and applications.

Background technique

With the acceleration of China's industrialization process, many production areas will produce high-salt wastewater, such as printing and dyeing, papermaking, chemicals, pesticides, oil production, seafood processing, etc., such wastewater will usually contain high concentrations of organic pollutants, direct discharge will The environment causes serious pollution. Moreover, high-salt organic wastewater is not suitable for conventional biological treatment due to the toxic effects of salt on common microorganisms. This is mainly because: (1) When the salt concentration is too high, the osmotic pressure of the water body is high, which tends to dehydrate the microbial cells and cause the separation of the cell protoplasts; (2) in the case of a high salt concentration, the salting out will dehydrogenate The enzyme activity is reduced; 〔3) The high chloride ion concentration is toxic to bacteria; (4) Due to the increase of water density, the activated sludge is easy to float and it is difficult to achieve muddy water separation.

The existing high-salt wastewater treatment methods in China have multi-effect evaporation, reverse osmosis, electrodialysis, etc. However, these methods generally have the disadvantages of high energy consumption, poor economic efficiency and secondary pollution, which is related to the “energy saving reduction” actively advocated by the state. The principle of "recycling economy" and "circular economy" are contrary to each other. How to realize the resource management of high-salt organic wastewater has become a difficult point in the prevention and control of water pollution.

In recent years, domestic biological treatment of high-salt organic wastewater has been limited to laboratory tests and concentrated on the salinity of activated sludge. When the salinity of the high-salt wastewater is higher than 5%, the treatment effect begins to deteriorate. Halophilic bacteria are a kind of extreme microorganisms. Salt is a necessary condition for their growth and reproduction. The direct treatment of high-salt organic wastewater has attracted people's attention. However, at this stage, the field experiment of halophilic bacteria and purification of high-salt wastewater Reusing engineering cases, no reports have been reported.

He Jian, Li Shunpeng, Cui Zhongli et al. "In the domestication of salt-tolerant industrial wastewater with salt-tolerant sludge and its mechanism" (Chinese Journal of Environmental Science, 2002, 22 (6): 546~550): The microorganisms used in this study are common. Microbes, not halophiles. It is difficult to use ordinary microorganisms for high-salt wastewater. Microorganisms cannot be directly used for high-salt wastewater treatment. The salinity of wastewater can only be adapted to ordinary microorganisms, but this adaptation is very limited. 5%. Hu Xunjie, Yang Yunan, Liu Hong et al. In the treatment of different salinity production wastewater by halophilic bacteria-enhanced biological activated carbon

Research" (Environmental Science, 2007, 28 (10): 2213~2217): This experiment uses biological activated carbon treatment method, by adding halophilic bacteria (but not disclosed as the kind of halophilic bacteria) for bio-enhancement, research organisms Membrane (biofilm formed by halophilic bacteria immobilized on activated carbon) The adaptability and treatment effect of different water quality and different salt concentration of oil production wastewater, in order to provide a reliable reference for the treatment of oil production wastewater with different salt content in different areas of Shengli Oilfield 3%。 The sulphate sulphate sulphate sulphate.

Xie Qinglin, Li Yanhong, Zhu Yinian, etc. In the "Study on Biological Treatment Technology of High Salinity Wastewater" (Environmental Engineering 2004, 22 (2): 15~16), the characteristics of high salinity and good biodegradability of wastewater are firstly The municipal sewage treatment plant takes activated sludge and digested sludge for domestication, and then carries out biochemical treatment of wastewater. On the basis of laboratory experiments, the experimental device is transported to the sewage station for on-site testing, first using the UASB anaerobic reactor. The wastewater is treated separately with the SBR aerobic reactor, and the wastewater is treated with the MSB anaerobic reactor and the SBR aerobic reactor. The process flow refers to the description of the halophilic bacteria. In view, the halophilicity of halophilic bacteria is about 1% to 3%.

Wang Qianfu's "Separation of halophilic bacteria and its multi-phase taxonomy" (Lanzhou University School of Life Sciences, 2007) mainly describes the enrichment and isolation of halophilic bacteria, from the high in the Hexi Corridor area of Qingdao and Gansu. Samples were collected from the salt environment for separation of halophilic microorganisms and multi-phase classification studies. The salinity of the halophilic microorganisms was only 20%, but it was not applied to wastewater treatment.

Therefore, the prior art exists: existing general microorganisms or salt-tolerant bacteria have a salt tolerance of only 6% or less;

No application research, such as research on industrial wastewater treatment, has been carried out.

The key to the treatment of high-salt organic wastewater is: First, we must screen and isolate organic wastewater that can withstand high salinity.

Extreme halophilic bacteria; to select and isolate extreme halophilic bacteria for selection and combination culture; and to use extreme halophilic bacteria for the start-up and debugging of high-salt organic wastewater biological treatment system.

To date, there has not been an efficient and economical treatment method for treating high-salt organic wastewater.

Summary of the invention The inventors of the present invention have proposed and completed the present invention in order to solve the defects existing in the above-described high-salt wastewater treatment.

It is therefore an object of the invention to screen for extreme halophiles.

A further object of the present invention is to provide a composite halophilic fungus comprising the above extreme halophile.

It is still another object of the present invention to provide a method of preparing the above-described complex halophilic bacteria agent.

Another object of the present invention is to provide a method of treating high salt production wastewater.

Another object of the invention is to provide the use of the above extreme halophiles.

The extreme halophilic bacteria according to the present invention may be Salmonella iHalomonas sp), and its storage number is CGMCC No. 3081.

The extreme halophilic bacteria according to the present invention may be Pseudomonas sp., and its storage number is: CGMCC No. 3082.

The extreme halophilic bacteria according to the present invention may be Bacillus sp. 1⁄23ci77^ sp), and its storage number is CGMCC No. 3083.

The composite halophilic bacteria agent according to the present invention includes any two or three of the above extreme halophilic bacteria CGMCC No. 3081, CGMCC No. 3082, and CGMCC No. 3083.

Preferably, the composite halophilic bacteria agent according to the present invention comprises the above three extreme halophilic bacteria, wherein the halophilic bacteria CGMCC No. 3081: CGMCC No. 3082: CGMCC No. 3083 is 3:3:1.

The method for preparing the above composite halophilic bacteria agent according to the present invention comprises the following steps:

1) one or more of the above-mentioned halophilic bacteria CGMCC No. 3081, CGMCC No. 3082, CGMCC No. 3083 are cultured in a composite microbial culture medium at 25_35 ° C for 7 days - 10 days;

2) The above cultured compound extreme halophilic agent is expanded and cultured in an expansion medium to obtain a compound halophilic bacteria agent, wherein, in the steps 1) and 2), the medium formula used is: Yeast extract 10 g / L, protein halophilic 15g / L, MgS0 ₄ · 7H ₂ 0 20 g / L, KCL 2 g / L, NaCL 180 g / L, pH 7. 0.

The present invention also provides a method of treating high-salt production wastewater, the method comprising the step of adding the above-mentioned composite halophilic bacteria to a production wastewater treatment system, characterized in that the COD concentration of the production wastewater is 3000 rag/ L -3500 mg / L, cultured step by step according to the system's 1/10-1/20 volume ratio, and finally the treatment system is completely completed.

The invention also provides the use of the above extreme halophilic salt in the treatment of high salt production wastewater. As shown in Fig. 1, the process for treating the cyanuric chloride production wastewater using the combination of the three extreme halophilic bacteria of the present invention is mainly divided into two stages:

(1) Start-up phase

1. Preparation of compound extreme halophilic agent

Combining the above three extreme halophilic bacteria to prepare an extreme halophilic composite fungus;

2. Field application of extreme halophilic composite microbial agents

The extreme halophilic composite fungicide is expanded and cultured according to the needs of the site; then the extreme halophilic composite fungicide is added to the wastewater treatment system for treating cyanuric chloride, and the extreme halophilic composite fungicide is periodically added. The three extreme halophilic composite microbial agents were able to grow well in the treatment system and form a biofilm, completing the start-up phase.

(ii) Domestication stage

After the start-up phase is completed, the acclimation phase is entered, namely: gradually reducing the dosage of the extreme halophilic composite microbial agents, and gradually increasing the amount of cyanuric cyanide production wastewater until the dosage of the extreme halophilic composite fungicide is 0, the amount of cyanuric acid production wastewater increased to 100%;

After the start-up and domestication are completed, the whole biological system and equipment are commissioned and operated, and the effluent of the high-salt cyanuric chloride wastewater is finally purified.

01%; The effect of the removal of total organic carbon (T0C) in the high-salt cyanide production wastewater is 80. 01%;

The effect of removing total organic carbon (T0C) in the treatment of high-salt cyanuric chloride production wastewater is 69.71%, according to the extreme halophilic bacteria CGMCC No. 3082 of the present invention;

The effect of removing total organic carbon (T0C) in the treatment of high-salt cyanuric chloride production wastewater is 74.66%, according to the extreme halophilic bacteria CGMCC No. 30823 of the present invention;

After the combination of the above three extreme halophilic bacteria, the effect of removing T0C in the high-salt cyanide production wastewater was 94.86%.

The composite extreme halophilic bacteria agent combined with the three extreme halophilic bacteria of the present invention can effectively treat the high-salt cyanuric chloride production wastewater, and the principle is as follows: The three extreme halophilic bacteria can be high High salt and organic matter in the wastewater of cyanuric chloride production as a nutrient. The more nutrients, the better the growth of extreme halophilic bacteria. When the amount of nutrients in the wastewater is less and less, and the amount is not available, Extremely halophilic bacteria are in a state of slow growth, no growth or even dormancy; that is, the high-salt cyanuric chloride production wastewater meets the qualified water discharge standard; once new high-salt cyanuric chloride production wastewater enters the treatment system, the third Extreme halophilic bacteria In a state of rapid growth. Therefore, according to the method of the present invention, the successful application of the aerobic pre-suspended biofilm solves the problem that the anaerobic reaction starts slowly and is difficult to be synchronized with the aerobic reaction, and the rapid start of the anaerobic reaction is realized; The muddy water is difficult to separate, and maintains the microbial concentration in the device, increasing the volumetric load.

The invention obtains three strains of extreme halophilic bacteria with salinity resistance of 15-25%, and the compound extreme halophilic bacteria agent after combination of three extreme halophilic bacteria can effectively treat high-salt cyanuric chloride production wastewater. Compared with other processing technologies, the method of the present invention is more in line with the national policy of "circular economy" and "energy saving and emission reduction", which can save a large amount of operating costs for enterprises and realize resource recycling treatment. DRAWINGS

Fig. 1 is a flow chart showing the process of treating the high-salt cyanuric chloride production wastewater by the three extreme halophilic bacteria combinations of the present invention.

Figure 2 is a fatty acid profile of Halomonas CGMCC No. 3081. Figure 3 is a fatty acid profile of Bacillus sp. G3⁄4ci7 i/s) CGMCC No. 3083.

Figure 4 shows the fatty acid profile of Pseudomonas (Malomonas CGMCC No. 3082. Extremely halophilic bacteria:

(1) Alomor s sp, deposited under the symbol CGMCC No. 3081, date of deposit: June 1, 2009;

( 2 ) Pseudomonas

Sp), deposit number CGMCC No. 3082, date of deposit: June 1, 2009;

(3) shown as Bacillus dc/^), deposit number CGMCC No. 3083, date of deposit: June 1, 2009, deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee, Address: Chaoyang District, Beijing Kushiro, Institute of Microbiology, Chinese Academy of Sciences, 100101. Specific real fe^:

Example 1 Extreme Extreme Halophilic Bacteria

1. Separation of extreme halophilic strains

1. 1) In-situ multi-point method for collecting sludge from the sludge tank of the salt chemical industry and sewage sludge at the sewage discharge port;

1. 2) The collected sludge is in a liquid enrichment medium containing 16wt%-26wtl of salt) Enrichment culture in medium 25-35 3 for 3-10 days;

1. 3) The sludge-like mud after enrichment and cultivation is separated and cultured in a separation medium at 25-35 Torr to obtain a single strain; the isolated single strain is then subjected to purification and separation medium. 2-6 purification and isolation cultures were selected to select 23 strains of extreme halophilic bacteria with a salt tolerance of 15-25%; the purified separation medium used was the same as the components and components of the above separation medium;

The components of the liquid enriched medium and the contents of the components are as follows: peptone 15 g / L, MgS0 ₄ · 7H ₂ 0 20 g / L, KCL 2 g / L, NaCL 150 - 250 g / L, pH 7. 0 ;

The components of the separation medium and the content of each component are the same as the components of the purified separation culture medium and the components, and are: casein hydrolysate 5 g/L, yeast extract 10 g/L, peptone 15g / L, MgS0 ₄ · 7H ₂ 0 20 g / L, KCL 2 g / L,, NaCL 150-250 g / L, pH 7. 0;

2. Separation and screening of extreme halophiles

The isolated strains of 23 extreme halophilic strains with a salt tolerance of 15-25% were screened one by one:

The treatment effect of each strain of extreme halophilic bacteria in high-salt cyanuric chloride wastewater containing 18wt%-20% of salt:

2. 1) Select a strain with a T0C removal effect of 80. 01% and measure it as Halomonas sp) (preserved by the General Microbiology Center of the China Microbial Culture Collection Management Committee, with the accession number CGMCC No 3081 );

Its identification technology is: Identification of microbial physiological and biochemical reactions combined with MIDI Sherlock Mi s; MIDI Sherlock Mi s identification system software can control Agi lent's 6850, 6890 and 7890 GCs, through the short chain obtained by gas chromatography A map of the type and content of fatty acids is compared to quickly and accurately identify microbial species.

Figure 2 is a fatty acid profile of Halomos sinensis CGMCC No. 3081, which has a salt tolerance of 15%-25%.

2. 2) Select a strain of strain that has a T0C removal effect of 74.66%, and determine it as Bacillus (^ci ^ sp) (deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, deposit number CGMCC No. 3083);

The method of identification is the same as the method for identifying nas nas iHal nas; Figure 3 For the fatty acid profile of Bacillusus Us77/s) CGMCC No. 3083, the Bacillus sp. has a salt tolerance of 15% to 25%.

2. 3) Select a strain of strain that has a T0C removal effect of 69.71%, and determine it as Pseudomonas (3⁄4ew i3⁄4^a3s sp) (deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee, No. CGMCC No. 3082);

The identification method is the same as the method for identifying M. monosyllium IMomonas; Figure 4 shows the fatty acid profile of Pseudomonas alomo s CGMCC No. 3082, the salt tolerance of the Pseudomonas { Halomonas) is 15% - 25% .

01%; The effect of removing the total organic carbon (T0C) is 80. 01%; The total halophilic bacteria CGMCC No. 3082 had a total organic carbon (T0C) removal effect of 69.71%; the extreme halophilic bacteria CGMCC No. 30823 had a total organic carbon (T0C) removal effect of 74.66%; 86%; After removing the T0C effect of the high-salt cyanuric chloride wastewater after the combination of the bacteria was 94.86%;

Example ² Preparation of Compound Halophilic Agent

1) The above three extreme halophilic bacteria are cultured in a compound culture medium at 25-35 7 for 7 days to 10 days, wherein the strain CGMCC No. 3081: CGMCC No. 3082: CGMCC No. 3083 is 3:3 : 1, a compound extreme halophilic agent;

The composition of the composite agent medium and the content of each component are: yeast extract 10 g / L, protein halophilic 15 g / L, MgS0 ₄ · 7H ₂ 0 20 g / L, KCL 2 g / L, NaCL 180 g/L , _P H 7. 0.

2) Expanding the above-mentioned compound extreme halophilic agent

The above compound extreme halophilic agent is expanded and cultured in a compound halophilic agent expansion medium at 25-30 ° C for 7-10 days to obtain a compound halophilic agent;

The composition of the composite agent and the content of each component are: yeast extract 10 g / L, peptone 15 g / L, MgS0 ₄ - 7H ₂ 0 20 g / L, KCL 2 g / L, NaCL 180 g /L, pH 7. 0. Example 3 Using the three extreme halophilic bacteria combinations of the present invention to treat the simulated production of cyanuric chloride production wastewater of "Degusai Sanzheng (Yingkou) Fine Chemical Co., Ltd."

The use of the three extreme halophilic bacteria combinations of the present invention is used to treat 18% by weight of high-salt cyanuric chloride production wastewater containing "Degussa Sanzheng (Yingkou) Fine Chemicals Co., Ltd.": 1. First prepare a composite test agent:

The above three extreme halophilic bacteria of the present invention are cultured in a composite bacterial culture medium at 25 ° C for 10 days to obtain a composite halophilic agent;

The composition of the composite microbial culture medium is: yeast extract 10 g/L, protein halophilic 15 g/L, MgS0 ₄ · 7H ₂ 0 20 g/L, KCL 2 g/L, NaCL 180 g/L, pH 7. 0 ;

The above composite halophilic agent is expanded and cultured:

The above-mentioned compound extreme halophilic agent is expanded and cultured in a composite microbial expansion medium at 25 ° C for 10 days to obtain a compound halophilic agent after expanding the culture;

The composite agent expansion medium components are: yeast extract 10 g / L, peptone 15 g / L, MgS0 ₄ · 7H ₂ 0 20 g / L, KCL 2 g / L, NaCL 180 g / L, pH 7 . 0;

2. Add the above compound bacteria to the treatment system of high-salt cyanuric chloride production wastewater, and add an extreme halophilic agent according to 1/10 of the volume of the biological treatment system. After pretreating the company's salt-containing 18 wt% high-salt cyanuric chloride production wastewater (the pretreatment is a well-known technique in the art), it enters the treatment system and is subjected to anaerobic treatment and aerobic treatment. The T0C content can reach below 10mg/L, the ammonia nitrogen content is below 4mg/L, and the cyanide content is below 0. 054mg/L, which basically meets the wastewater reuse standard of the enterprise.

Example 4 Simulation experiment of the wastewater of cyanuric chloride production of "Hebei Lingang Chemical Co., Ltd." using the three extreme halophilic bacteria combinations of the present invention

The three extreme halophilic bacteria combinations of the present invention are used for treating high-salt cyanuric chloride production wastewater containing "salt of 18 wt% to 20 wt%" of "Hebei Lingang Chemical Co., Ltd."; the process is basically the same as that of the embodiment 1.

1. First prepare a composite test agent:

The above three extreme halophilic bacteria of the present invention are cultured for 3 days in 3CTC in a composite bacterial culture medium to obtain a composite halophilic agent;

The above composite halophilic agent is expanded and cultured:

The above-mentioned compound extreme halophilic agent is expanded and cultured in a composite microbial expansion medium for 3 days, and a compound halophilic agent is obtained after expanding the culture;

The composite agent expansion medium components are: yeast extract 10 g / L, peptone 15 g / L, MgS0 ₄ · 7H ₂ 0 20 g / L, KCL 2 g / L, NaCL 180 g / L, pH 7 . 0; 2. Add the above compound bacteria to the treatment system of high-salt cyanuric chloride production wastewater, and add the extreme halophilic agent according to the volume of the biological treatment system of 1Z10-1/20. After pretreating the company's salt-containing 18wt%-20wt% high-salt cyanuric chloride production wastewater, it enters the biological treatment system of the high-salt cyanuric chloride production wastewater of the present embodiment, in which anaerobic and anaerobic Aerobic treatment, after treatment by biological system, T0C content can reach below 10mg/L, ammonia nitrogen content is below 4mg/L, cyanide content reaches below 0. 054mg/L, basic high-salt cyanuric chloride production wastewater reaches enterprise back With requirements.

Example 5 Simulation experiment of the wastewater of cyanuric chloride production of "Degussa Sanzheng (Chongqing) Fine Chemical Co., Ltd." using the three extreme halophilic bacteria combinations of the present invention

The use of the three extreme halophilic bacteria combinations of the present invention for treating "Degussa Sanzheng (Chongqing) Fine Chemical Co., Ltd." salt-containing 13wt%-15wt% high-salt cyanuric chloride production wastewater, the process and the above two The examples are basically the same.

1. First prepare a composite test agent:

The above three extreme halophilic bacteria of the present invention are cultured in a composite bacterial culture medium at 35 ° C for 7 days to obtain a composite halophilic agent;

The composition of the composite bacteria medium is: yeast extract 10 g / L, protein halophilic 15 g / L, MgS0 ₄ · 7H ₂ 0 20 g / L, KCL 2 g / L, NaCL 180 g / L, pH 7. 0 ;

The above composite halophilic agent is expanded and cultured:

The above-mentioned compound extreme halophilic agent is cultured in a combined microbial expansion medium for 35 days, and expanded for 7 days to obtain a composite halophilic agent after expanding the culture;

The composite agent expansion medium components are: yeast extract 10 g / L, peptone 15 g / L, MgS0 ₄ - 7H ₂ 0 20 g / L, KCL 2 g / L, NaCL 180 g / L, pH 7 . 0;

2. Add the above-mentioned composite microbial agent to the treatment system of high-salt cyanuric chloride production wastewater, and add an extreme halophilic agent according to 1/20 of the volume of the biological treatment system. After pretreating the company's salt-containing 18wt%_2 (1⁄4t% high-salt cyanuric chloride production wastewater), it enters the biological treatment system of the high-salt cyanuric chloride production wastewater of this embodiment, and there is anaerobic in this system. And aerobic treatment, after treatment by biological system, T0C content can reach below 30-40mg / L, ammonia nitrogen content below 10mg / L, cyanide content of less than 0. 05mg / L, to achieve high-salt cyanuric chloride production wastewater Meet the enterprise recycling requirements.

Claims

Rights request

An extreme halophilic bacterium, characterized in that the halophilic bacterium is a salt bacterium, and the preservation number is: CGMCC No. 3081 ο

2. An extreme halophilic bacterium, wherein the halophilic bacterium is Pseudomonas, and the storage number is CGMCC No. 3082.

3. An extreme halophilic bacterium, wherein the halophilic bacterium is Bacillus, and the storage number is CGMCC No. 3083.

A complex halophilic bacterial agent, characterized in that the composite halophilic bacterial agent comprises any two or three of the extreme halophilic bacteria according to claim 1, 2 or 3.

The composite halophilic bacteria agent according to claim 4, wherein the complex halophilic bacteria agent comprises the extreme halophilic bacteria according to claims 1, 2 and 3, wherein the strain CGMCC No. 3081: CGMCC No. 3082: CGMCC No. 3083 is 3: 3: 1.

A method of producing a composite halophilic bacterial agent according to claim 4, wherein the method comprises the steps of:

1) cultivating one or more of the halophilic bacteria according to claim 1, 2, 3 in a composite microbial culture medium at 25-35 7 for 7 days to 10 days;

2) The above-mentioned cultured compound extreme halophilic agent is expanded and cultured in an expanded medium to obtain a compound halophilic bacteria agent.

The method according to claim 6, characterized in that in the steps 1) and 2), the medium used is: yeast extract 10 g/L, protein halophilic 15 g/L, MgS0 ₄ · 7H ₂ 0 20 g/L , KCL 2 g/L, NaCL 180 g/L, pH 7. 0.

A method for treating high-salt production wastewater, characterized in that the method comprises the step of adding the complex halophilic bacterial agent according to claim 4 to a production wastewater treatment system, wherein the COD concentration of the production wastewater It is 3000 mg/L -3500 rag/L and is added in a 1/10-1/20 volume ratio of the system.

9. The use of extreme halophilicity as claimed in claim 1, 2 or 3 for the treatment of high salt production wastewater.