NL2028995B1 - Method for screening carbon sources for enhancing biofilm anaerobic denitrification for pond wastewater treatment - Google Patents

Abstract

Disclosed is a method for screening carbon sources for enhancing biofilm anaerobic denitrification for pond wastewater treatment. The method comprises the following steps: (1) separation and culture of biofilm anaerobes; (2) preparation of an anaerobe suspension; (3) measurement of metabolic characteristics of the anaerobes; and (4) measurement and analysis of carbon source use ratios of the anaerobes. In the present invention, carbon sources needed by the anaerobes are screened by a Biolog - ECO plate. Testing is rapid and time - saving, and experimental results are accurate, reliably and highly targeted. The nitrogen removal effects of the carbon sources screened by the method established in the present invention on aquaculture wastewater are obviously better than that of carbon sources screened by conventional methods.

Description

METHOD FOR SCREENING CARBON SOURCES FOR ENHANCING BIOFILM ANAEROBIC

DENITRIFICATION FOR POND WASTEWATER TREATMENT Technical Field The present invention belongs to the field of treatment of aquaculture wastewater, and relates to a method for screening carbon sources for enhancing biofilm anaerobic denitrification, in particular to a method for screening carbon sources for enhancing biofilm anaerobic denitrification for pond wastewater treatment. Background In recent years, aquaculture, and especially pond culture have become an important production mode for people to acquire high - quality animal protein. In China, the area of pond culture is 2.5669 million hectares. However, with increasing expansion of intensive culture, the culture density keeps increasing. The high - density high - input culture mode has led to serious pollution of nitrogen and phosphorus in ponds, bringing heavy pollution pressure to the water environment. Under the current dual pressures, namely shortage of water resources and continuous increase of culture water demands, it is urgent to conduct effective recovery and recycling of aquaculture wastewater to create a stable, controllable and healthy culture environment. As shown in studies, biofilms can effectively promote cycles of nitrogen and phosphorus. The biofilms almost exist everywhere in the nature if there is water (oceans, rivers, lakes and the like). With increasing density of the biofilms along the depth direction, the dissolved oxygen content decreases in a gradient manner. Due to gradient changes of dissolved oxygen inside the biofilms, an aerobiotic layer and an anaerobic layer of the biofilms are formed, wherein the aerobiotic layer is more suitable for survival of aerobes, and the anaerobic layer is more suitable for survival of anaerobes. Such differentiation of microbes on inner and outer layers leads to a unique microscopic ecological niche in the biofilms. Biofilm microbes can realize aerobiotic nitrification and anaerobic denitrification reactions at the same time in such environment with gradient distribution of dissolved oxygen. Degradation and nitrification reactions of organic matters are realized in the aerobiotic layer. NH" is converted to NO2° and NOs ~. The denitrification reaction is realized in the anaerobic layer, and NO, and NOs generated in the aerobiotic layer and the organic matters diffused to the anaerobic layer are converted to Na, thereby realizing the synchronous nitrification - denitrification nitrogen removal process of the biofilm. The biofilm method has drawn a lot of attention because of its advantages of being ecologically friendly, low in operation cost, stable in performance, easy to operate, etc. It has been widely applied in water treatment and in - situ remediation of environmental water body. However, the biofilm method has the disadvantage of insufficient supply of electron donors in actual application, leading to long time of biofilm culturing starting, low water treatment efficiency in the starting stage and slow increasing of a pollutant removal rate. It has become a restriction link in application of the biofilms in culture systems. The core approach to increase the biofilm nitrogen removal efficiency is to effectively supply enough electron donors for the biofilm. As shown in studies, as electron donors of nutrition and nitrogen removal denitrification of biofilm microbes, proper carbon sources can accelerate starting of biofilm culturing and effectively increase the denitrification nitrogen removal efficiency of the biofilm.

However, at present, most of the studies on carbon sources needed by microbe nitrogen removal in biofilm nitrogen removal reactions only focus on the use of the carbon sources by all the microbes attached to biofilms, but fail to screen the carbon sources needed by anaerobes in the biofilm anaerobic denitrification process. Some researchers have screened some carbon sources through experiments and contributed to starting of nitrogen removal biofilm culturing of biofilms to some extent, but these carbon sources are screened blindly, and the research is not conducted based on the denitrification demands of anaerobes on the biofilms. Only by enhancing the supply of effective electron donors in anaerobic denitrification of the biofilms, the nitrogen removal of the biofilms can be actually accelerated to yield twice the result with half the effort.

Therefore, it is an urgent problem to be solved in this field to screen the carbon sources of biofilm anaerobes in a pond culture system and construct a matching map of the carbon sources needed by the biofilm anaerobes.

Summary In view of this, the present invention provides a method for screening carbon sources for enhancing biofilm anaerobic denitrification for pond wastewater treatment, and aims to enhance the anaerobic denitrification rate of biofilms, better enhance nitrogen removal capacities of the biofilms and realize efficient treatment of aquaculture wastewater.

In order to realize above purposes, the present invention provides a method for screening carbon sources for enhancing biofilm anaerobic denitrification for pond wastewater treatment. The method comprises the following steps: (1) separation and culture of biofilm anaerobes: separating the anaerobes from a biofilm to a normal saline culture solution to obtain a culture solution containing the anaerobes; (2) preparation of an anaerobe suspension: mixing the anaerobe culture solution in (1) with normal saline to obtain an anaerobe suspension; (3) measurement of metabolic characteristics of the anaerobes: dripping the anaerobe suspension prepared in (2) onto a Biolog - ECO plate and starting culture; (4) measurement and analysis of carbon source use ratios of the anaerobes: detecting the Biolog - ECO plate in (3) on a microplate reader and selecting carbon sources with strong metabolism as carbon sources for enhancing biofilm anaerobic denitrification.

The present invention has the beneficial effects:

1. Different from previous studies (focusing on metabolic characteristics of microbes in water bodies and on biofilms) in which metabolic characteristics of other microbes are measured in an aerobic environment, the method obtains metabolic characteristics of the anaerobes in a targeted manner under an anaerobic environment and the metabolic characteristics can be used for screening carbon sources for anaerobic denitrification, so that nitrogen removal demands of the anaerobes can be measured more accurately.

2. The present invention can avoid blind screening of the carbon for enhancing biofilm nitrogen removal. Based on demands of the biofilm anaerobes, a carbon source map is used to screen the carbon sources needed by denitrification of biofilm anaerobes in a targeted manner, so that the adverse result of secondary water pollution of the biofilm caused by the blindly screened carbon sources can be avoided. The present invention especially provides a method for screening carbon sources needed by biofilm anaerobes for pond wastewater treatment. The carbon sources for enhancing denitrification, screened by the method, can achieve the purpose of enhancing biofilm nitrogen removal.

Further, in step (1), a method for separation and culture of the biofilm anaerobes comprises: culturing the biofilm in a pond culture wastewater treatment system, putting a biofilm sample under a sterile condition into normal saline, conducting anaerobic culture, shaking fully, and separating the anaerobes attached to the biofilm into the normal saline culture solution to obtain the separated anaerobe culture solution.

Preferably, culture time of the biofilm in the pond culture wastewater treatment system is 15 - 30 days; 3 g of the biofilm sample is weighed quantitatively; the normal saline is 150 ml of sterile normal saline with a mass fraction of 0.9%, and the normal saline is pre - filled with pure nitrogen to keep an anaerobic environment; and the time of anaerobic culture is more than 2 h.

The further beneficial effects are that: the normal saline is pre - filled with the pure nitrogen and the anaerobic environment can be kept, so that a proper survival environment is provided for the anaerobes to avoid affecting biological characteristics.

Further, in step (2), a method for preparation of the anaerobe suspension comprises: transferring the separated anaerobe culture solution in step (1) into an anaerobic incubator, and mixing uniformly with the sterile normal saline to obtain the anaerobe suspension.

Preferably, a mass ratio of the anaerobe culture solution to the sterile normal saline is 1: 3 -

4.

The further beneficial effects are that: the preparation process of the anaerobe suspension is conducted in the anaerobic incubator, so that the anaerobic environment is kept during operation.

Further, in step (3), a method for measurement of the metabolic characteristics of the anaerobes comprises: dripping the anaerobe suspension prepared in step (2) onto the Biolog - ECO plate and then culturing the Biolog - ECO plate.

The Biolog - ECO plate is a product ECO plate exclusively researched and developed by American BIOLOG Company. It is designed specially for microflora analysis and ecological research. The Eco plate contains 31 carbon sources most commonly used in microflora analysis, which were selected by international soil microbiologists. It is one of the most typical and authoritative methods for studying the microbial ecological function diversity in the world. Each Biolog - ECO plate has 96 holes. There are 3 parallel groups on the ECO plate, with 31 carbon sources in each group and 3 negative controls. After addition of the same sample, 3 groups of parallel data can be obtained. The characteristic utilization of the 31 carbon sources by a microflora is called as a metabolic fingerprint map of the microflora. A lot of metabolic information can be obtained from the fingerprint map of each micro - plate, which can be applied to research of microbe functional diversity of pure and mixed florae (e.g., soil samples) Preferably, the culture is conducted under an anaerobic condition of 25 - 28°C and the culture time is 48 h; and whole processes of dripping the anaerobe suspension and culturing the Biolog - ECO plate are conducted in the anaerobic incubator under the anaerobic environment.

The further beneficial effects are that: the carbon sources needed by the anaerobes are screened by the Biolog - ECO plate; the Biolog - ECO plate has abundant carbon sources, and parallel and control tests are conducted, so that testing is quick and time - saving, and experimental results are accurate, reliable and highly targeted.

Further, in step (4), a method for measurement and analysis of the carbon source use ratios of the anaerobes comprises: taking the Biolog - ECO plate quickly from the anaerobic incubator after culture in (3), conducting detection on the microplate reader immediately, recording readings of optical density values, calculating average well colour development (AWCD) values of measured light absorption values to obtain the AWCD values of each type of carbon sources, and selecting the carbon sources with strong metabolism as the carbon sources for enhancing biofilm anaerobic denitrification.

Preferably, a detection wavelength is 590 nm.

Preferably, the carbon sources with the AWCD values of 1.2 - 2.5 are selected as the carbon sources for enhancing biofilm anaerobic denitrification.

The further beneficial effects are that: through calculation of the AWCD values of each type of carbon sources and establishment of a method for the matching map of the carbon sources needed by the biofilm anaerobes, the carbon sources of the biofilm anaerobes in the culture system are screened, thereby increasing the supply of electron donors in biofilm anaerobic denitrification and actually increasing the nitrogen removal rate of the biofilm.

Detailed Description The technical solutions in the embodiments of the present invention will be described below clearly and completely. Apparently, the described embodiments are only a part of embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those ordinary skilled in the art without making creative work belong to the protection scope of the present invention.

A Biolog - ECO plate involved in the following embodiments is manufactured by American BIOLOG Company, with a model of ECO Micro Plate No. 1506; 5 A microplate reader is manufactured by Thermo, with a model of Multiskan FC mk3.

A calculation method of AWCD: a capacity of utilizing carbon sources by a water body microflora is expressed by AWCD. The calculation formula is AWCD = 3 (Ci—R) /n, wherein Ci denotes measured light absorption values of 31 carbon source holes; R denotes a light absorption value of a reference hole; and n is the total number of medium carbon sources, which is equal to 31 in the research.

Embodiment 1 A method for screening carbon sources for enhancing biofilm anaerobic denitrification for pond wastewater treatment comprises the following steps: (1) separation and culture of biofilm anaerobes: culturing a biofilm in a pond culture wastewater treatment system for 15 days, weighing a biofilm sample of 3 g quantitatively under a sterile condition and putting the biofilm sample into 150 ml of 0.9% sterile normal saline which is pre - filled with pure nitrogen for keeping an anaerobic environment, conducting anaerobic culture for 2 h, shaking fully, and separating the anaerobes attached to the biofilm into a normal saline culture solution to obtain a separated anaerobe culture solution; (2) preparation of an anaerobe suspension: transferring the separated anaerobe culture solution in step (1) into an anaerobic incubator, and mixing uniformly with the sterile normal saline according to a mass ratio of 1 : 3 to obtain the anaerobe suspension; (3) measurement of metabolic characteristics of the anaerobes: dripping the anaerobe suspension prepared in step (2) onto a Biolog - ECO plate in the anaerobic incubator and then culturing for 48 h at 25°C in the anaerobic incubator; (4) measurement and analysis of carbon source use ratios of the anaerobes: taking the Biolog - ECO plate quickly from the anaerobic incubator after culture for 48 h in (3), conducting detection on a microplate reader immediately with a detection wavelength of 580 nm, recording readings of optical density values, calculating AWCD values of measured light absorption values to obtain the AWCD values of each type of carbon sources, and selecting the carbon sources with the AWCD values of 1.2 - 2.5 as the carbon sources for enhancing biofilm anaerobic denitrification.

As shown in results, the carbon sources for enhancing biofilm denitrification in the culture treatment system, screened under the anaerobic condition include: D - xylose, Tween 80, a- D - glucose - 1 - phosphate and phenylethylamine.

Embodiment 2

A method for screening carbon sources for enhancing biofilm anaerobic denitrification for pond wastewater treatment comprises the following steps: (1) separation and culture of biofilm anaerobes: culturing a biofilm in a pond culture wastewater treatment system for 30 days, weighing a biofilm sample of 3 g quantitatively under a sterile condition and putting the biofilm sample into 150 ml of 0.9% sterile normal saline which is pre - filled with pure nitrogen for keeping an anaerobic environment, conducting anaerobic culture for 3h, shaking fully, and separating the anaerobes attached to the biofilm into a normal saline culture solution to obtain a separated anaerobe culture solution; (2) preparation of an anaerobe suspension: transferring the separated anaerobe culture solution in step (1) into an anaerobic incubator, and mixing uniformly with the sterile normal saline according to a mass ratio of 1 : 4 to obtain the anaerobe suspension; (3) measurement of metabolic characteristics of the anaerobes: dripping the anaerobe suspension prepared in step (2) onto a Biolog - ECO plate in the anaerobic incubator and then culturing for 48 h at 28°C in the anaerobic incubator; (4) measurement and analysis of carbon source use ratios of the anaerobes: taking the Biolog - ECO plate quickly from the anaerobic incubator after culture for 48 h in (3), conducting detection on a microplate reader immediately with a detection wavelength of 590 nm, recording readings of optical density values, calculating AWCD values of measured light absorption values to obtain the AWCD values of each type of carbon sources, and selecting the carbon sources with the AWCD values of 1.2 - 2.5 as the carbon sources for enhancing biofilm anaerobic denitrification.

As shown in results, the carbon sources for enhancing biofilm denitrification in the culture treatment system, screened under the anaerobic condition include: D - xylose, Tween 80, a-D - glucose - 1 - phosphate and phenylethylamine.

Embodiment 3 A method for screening carbon sources for enhancing biofilm anaerobic denitrification for pond wastewater treatment comprises the following steps: (1) separation and culture of biofilm anaerobes: culturing a biofilm in a pond culture wastewater treatment system for 20 days, weighing a biofilm sample of 3 g quantitatively under a sterile condition and putting the biofilm sample into 150 ml of 0.8% sterile normal saline which is pre - filled with pure nitrogen for keeping an anaerobic environment, conducting anaerobic culture for 5h, shaking fully, and separating the anaerobes attached to the biofilm into a normal saline culture solution to obtain a separated anaerobe culture solution; (2) preparation of an anaerobe suspension: transferring the separated anaerobe culture solution in step (1) into an anaerobic incubator, and mixing uniformly with the sterile normal saline according to a mass ratio of 1 : 3.5 to obtain the anaerobe suspension;

(3) measurement of metabolic characteristics of the anaerobes: dripping the anaerobe suspension prepared in step (2) onto a Biolog - ECO plate in the anaerobic incubator and then culturing for 48 h at 26°C in the anaerobic incubator; (4) measurement and analysis of carbon source use ratios of the anaerobes: taking the Biolog - ECO plate quickly from the anaerobic incubator after culture for 48 h in (3), conducting detection on a microplate reader immediately with a detection wavelength of 590 nm, recording readings of optical density values, calculating AWCD values of measured light absorption values to obtain the AWCD values of each type of carbon sources, and selecting the carbon sources with the AWCD values of 1.2 - 2.5 as the carbon sources for enhancing biofilm anaerobic denitrification.

As shown in results, the carbon sources for enhancing biofilm denitrification in the culture treatment system, screened under the anaerobic condition include: D - xylose, Tween 80, a-D - glucose - 1 - phosphate and phenylethylamine.

Reference Embodiment (1) separation and culture of biofilm aerobes: culturing a biofilm in a pond culture wastewater treatment system for 15 days, weighing a biofilm sample of 3 g quantitatively under a sterile condition and putting into 150 ml of 0.9% sterile normal saline, conducting aerobic culture for 2 h, shaking fully, and separating the aerobes attached to the biofilm into a normal saline culture solution to obtain a separated aerobe culture solution; (2) preparation of an aerobe suspension: mixing the separated aerobe culture solution in step (1) uniformly with the sterile normal saline according to a mass ratio of 1 : 3 under an aerobic condition to obtain the aerobe suspension; (3) measurement of metabolic characteristics of the aerobes: dripping the aerobe suspension prepared in step (2) onto a Biolog - ECO plate under the aerobic condition and then culturing indoors for 48 h at 25°C under the aerobic condition; (4) measurement and analysis of carbon source use ratios of the aerobes: after culture of the Biolog - ECO plate for 48 h in (3), conducting detection on a microplate reader immediately with a detection wavelength of 590 nm, recording readings of optical density values, calculating AWCD values of measured light absorption values to obtain the AWCD values of each type of carbon sources, and selecting the carbon sources with the AWCD values of 1.2 - 2.5 as the carbon sources for enhancing biofilm anaerobic denitrification.

As shown in results, the carbon sources for enhancing biofilm denitrification in the culture treatment system, screened under the aerobic condition include: cellobiose, N - acetyl - D - glucosamine, mannose, glycogen and xylan.

Performance test: nitrogen removal enhancement effects of the screened carbon sources are verified.

Conventional biofilm reactors used in the following experiment include: 100L culture water tank, 3 biofilm reactors, an air charging device, a heating rod and a water pump. Through air charging, DO (dissolved oxygen) in the water tank is kept at 4 - 5 mg/L, and DO of the biofilm reactors is 3.5 mg/L. Three parallel up - flow biofilters are disposed in the reactors and are connected in parallel.

Two types of carbon sources screened by the above two methods are applied to the biofilm reactors. The biofilm was cultured for 15 d in the biofilm reactor and reserved in water for 6 h. Ammonia - nitrogen removal rates and nitrate removal rates of the two types of carbon sources are calculated respectively to verify effects of the screened carbon sources in enhancing nitrogen removal in culture wastewater, wherein ammonia - nitrogen removal rate = (influent ammonia - nitrogen amount — effluent ammonia - nitrogen amount)/influent ammonia - nitrogen amount, and nitrate removal rate = (influent nitrate amount — effluent nitrate amount)/influent nitrate amount.

Records of the results: the ammonia - nitrogen removal rates of the carbon sources screened under the aerobic condition are respectively: 56% for cellobiose, 63% for N - acetyl - D - glucosamine, 40% for mannose, 51% for glycogen and 38% for xylan; and nitrate removal rates are respectively: 38% for cellobiose, 26% for N - acetyl - D - glucosamine, 27% for mannose, 19% for glycogen and 23% for xylan.

The ammonia - nitrogen removal rates of the enhancing carbon sources screened under the anaerobic condition are respectively: 53% for D - xylose, 45% for Tween 80, 63% fora - D - glucose - 1 - phosphate and 49% for phenylethylamine; and nitrate removal rates are respectively: 78% for D - xylose, 72% for Tween 80, 81% for a - D - glucose - 1 - phosphate and 63% for phenylethylamine.

It is thus clear that, the nitrogen removal effects (ammonia - nitrogen removal rate and nitrate removal rate) of the carbon sources screened by the method established in the present invention on aquaculture wastewater are obviously better than that of carbon sources screened by conventional methods.

The above description of the disclosed embodiments enables those skilled in the art to realize or use the present invention. Many modifications to these embodiments will be apparent to those skilled in the art. The general principle defined herein can be realized in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principle and novel features disclosed herein.

Claims (10)

CONCLUSIONS 1. A method for screening carbon sources for enhancing biofilm - anaerobic denitrification for treatment of pond wastewater, which method comprises the following steps: (1) separating and culturing biofilm - anaerobes: separating the anaerobes from a biofilm in normal saline to obtain a culture solution containing the anaerobes; (2) preparation of an anaerobe suspension: mixing the anaerobe culture solution in (1) with normal saline to obtain an anaerobe suspension; (3) measurement of the metabolic characteristics of the anaerobes: the anaerobes suspension prepared in (2) is dropped onto a Biolog - ECO plate and the culture is initiated (4) measurement and analysis of the relationship between the use of carbon sources by the anaerobes: detection of the Biolog - ECO plate in (3) on a microplate reader and selection of highly metabolised carbon sources as carbon sources for the enhancement of the biofilm - anaerobic denitrification. The method of screening carbon sources for enhancing biofilm - anaerobic denitrification for pond waste water treatment according to claim 1, wherein in step (1), the method of separating and culturing the biofilm - anaerobes comprises: culturing of the biofilm in a pond culture wastewater treatment system, introducing a biofilm sample in a sterile state into normal saline, culturing under anaerobic conditions, shaking completely, and separating the anaerobes attached to the biofilm in the normal saline to remove the separated anaerobic culture solution. The method for screening carbon sources for enhancing biofilm - anaerobic denitrification for treatment of pond wastewater according to claim 2, wherein the culture time of the biofilm in the wastewater treatment system of the pond culture is 15 - 30 days; 3 g of the biofilm sample is quantitatively weighed; the normal saline is 150 ml of sterile normal saline with a mass fraction of 0.9%, and the normal saline is pre-charged with pure nitrogen to maintain an anaerobic environment; The method for screening carbon sources for enhancing biofilm - anaerobic denitrification for pond waste water treatment according to claim 1, wherein in step (2), the method for preparing the anaerobic suspension comprises: transferring the into step (1) separated anaerobic culture solution in an anaerobic incubator, and mixing evenly with the sterile normal saline to obtain the anaerobic suspension. The method for screening carbon sources for enhancing biofilm - anaerobic denitrification for pond waste water treatment according to claim 4, wherein the mass ratio between the anaerobic culture solution and the sterile normal saline solution is 1:3-4. The method of screening carbon sources for enhancing biofilm - anaerobic denitrification for pond waste water treatment according to claim 1, wherein in step (3), the method of measuring the metabolic characteristics of the anaerobes comprises: dripping the anaerobic suspension prepared in step (2) onto the Biolog - ECO plate and then culturing the Biolog - ECO plate. The method for screening carbon sources for enhancing biofilm - anaerobic denitrification for pond wastewater treatment according to claim 6, wherein the culture is conducted under an anaerobic condition of 25-28°C and the culture time is 48 hours; and the entire processes of dripping the anaerobic suspension and culturing the Biolog - ECO plate in the anaerobic incubator are performed under anaerobic conditions. The carbon source screening method for enhancing biofilm - anaerobic denitrification for pond waste water treatment according to claim 1, wherein in step (4), the method of measuring and analyzing the ratio of carbon source utilization by the anaerobes includes: quickly removing the Biolog ECO plate from the anaerobic incubator after culture in (3}, immediately performing a detection on the microplate reader, recording optical density readings, calculating mean color development values (AWCD) in the wells of measured light absorption values to obtain the AWCD values of each type of carbon source, and selecting the high metaboliser carbon sources as the carbon sources for enhancing the anaerobic denitrification of the biofilm. The method of screening carbon sources for enhancing biofilm - anaerobic denitrification for pond waste water treatment according to claim 8, wherein the detection wavelength is 590 nm. The carbon source screening method for enhancing biofilm - anaerobic denitrification for pond wastewater treatment according to claim 8, wherein the carbon sources with AWCD values of 1.2 - 2.5 are selected as carbon sources for enhancing of biofilm - anaerobic denitrification