Introduction
The organic wastewater generated from the industrial sites is composed of biodegradable organics, volatile organic compounds, recalcitrant organics, suspended solids, and nutrient salts (nitrogen and phosphorus). The objective of wastewater treatment is to reduce organics in the wastewater and to remove nutrient salts to reduce the contamination of surface water and groundwater through wastewater treatment process. In the treatment of organic wastewater, a method of artificially increasing the efficiency of water self-cleaning is widely used, which is called a biological treatment. In general, the biological treatment for wastewater treatment can be categorized into aerobic process like activated sludge method and biofilm process, and anaerobic process like digestion method. In many of the biological treatments, various microorganisms such as bacteria, fungi, protozoa, and micrometazoa are involved in the purification, while dozens or more kinds of microorganisms constitute a mixed culture system. On the other hand, wastewater containing various kinds of miscellaneous components is a highly complex multicomponent system, and biological treatment is a process of removing mixed substrates by mixed cultured microorganisms [5, 11, 16].
From the industrial wastewater, non-biodegradable or bio-toxic materials like synthetic organic compounds are detected along with biodegradable organics, which pose major factors affecting the deterioration of wastewater treatment efficiency and limiting the biological treatment [9,19]. Removal of organic compounds by the biological treatment largely relies on the composition and treatment type of the wastewater and solid retention time. A typical aerobic biological treatment is known to remove soluble organic carbons until about 85%. Half of the organic compounds remained after biological treatment consists of humid acid, fulvic acid, and hymathomelanic acid. Meanwhile, easily degradable organics, like carbohydrates and protein, account for about 25% of the soluble organics [13].
Biological treatment, where microorganisms are used, is basically an economical method of treating wastewater by using metabolism; i.e., degradation, synthesis, and conversion of the organic matters presented in the wastewater. It is used with the objectives of removing organics, nitrogen, and phosphorus; reducing generated sludge; and collecting and treating natural resources. In the biological treatment, the physicochemical treatments such as precipitation, filtration, and coagulation are combined or used in parallel as a pretreatment or post-treatment to improve wastewater treatment efficiency.
Particularly, high concentrations of nitrogen included in the wastewater are not easily removed with general wastewater treatment, inflowing into the lakes and streams, thus inducing eutrophication to cause hyperplasia of hydrophytes, deficiency of dissolved oxygen in the water, bad order, and worsening water quality by accumulation of decomposed matter, thereby leading to serious environmental problems and ultimately making this nitrogen emerge as a new contaminant [10].
Physicochemical methods such as absorption and ion exchange, as well as the biological treatment method, are used to remove nitrogen in the wastewater. Though biological treatment method is friendlier to the environment, the efficiency is low in removing high concentrations of nitrogen; therefore, it is being used along with physicochemical method [2,20].
For the biological treatment used in the treatment of nitrogen containing wastewater, a considerable amount of research and development is continuing in terms of process. Researches are mainly advanced not only in the selection of new microorganisms that can treat high concentrations of nitrogen but also in improving wastewater treatment system, suiting the characteristics of microorganisms for process improvement and utilizing microorganisms in strain improvement and microorganism fixation [7-10].
In this study, microorganisms capable of degrading various organic materials and those that have excellent ammonium nitrogen removal capability were isolated and identified from the aeration tank which contained ammonium nitrogen in order to efficiently treat organic wastewater for environment purification. In addition, the isolated microorganisms were formulated and optimized in order to be utilized in treating the industrial wastewater that contains organic source and ammonium nitrogen.
Materials and Methods
Isolation of microorganisms in the aeration tanks
In order to isolate microorganisms codominant in the activated sludge in the aeration tank of food wastewater treatment plant, a sample from the aeration tank was set as an isolated sample and was diluted in the normal saline. The diluted wastewater was spread on the plate count agar (PCA) medium followed by culturing at 37℃ for 3 days. The population that formed the colony was isolated. The isolated strain was cultured in a liquid culture medium for the isolation of bacteria, while strain having exceptional cell growth was selected and used in the wastewater treatment efficiency test. The liquid culture medium for the isolation of bacteria was composed of 3.0 g glucose, 5.0 g yeast extract, and 3.0 g tryptone per 1 liter of medium. The pH of culture medium was adjusted to 7.0.
Isolation of ammonium nitrogen removal microorganisms
In order to isolate ammonium nitrogen (NH4-N) removal microorganisms, samples from soil, rivers, and wastewater around food, paper, and leather manufacturing plants nationwide were diluted in normal saline, and 0.7 g/l of (NH4)2SO4, as a single energy source, was spread on the solid medium wherein it was added. The strains were inoculated on the liquid culture medium for the isolation of bacteria and cultured at 30℃ for 3 days while the strain with excellent nitrogen removal capability was selected. The isolation medium was composed of 0.7 g (NH4)2SO4, 0.8 g K2HPO4, 0.2 g MgSO4 ·7H2O, 0.1 g FeSO4 ·7H2O, and 3.0 g CaCO3 per 1 liter of medium, with the pH adjusted to 7.0.
Identification and preservation of isolated strains
After investigating the morphological, physiological, and biochemical characteristics of the isolated strains, the identification of the strain was carried out according to Bergey's manual of determinative bacteriology [15] and biochemical tests for the identification of medical bacteria (2nd ed.) [12]. The DNA base composition of each strain (G+C contents) was analyzed by reversed-phase HPLC according to the method suggested by Tamaoka and Komagata [17]. The 16S ribosomal RNA gene is the most widely used marker gene in miocrobiology ecology. We therefor performed an additional experiment to demonstrate that sequencing of 16S rRNA gene for identification of bacteria. PCR amplification of the 16S rRNA genes was performed with primers containing universal primers amplifying the V4 variable region (515F: GTGCCAGCMGCCGCGGTAA and 806R: GGACTAC HVGGGTWTCTAAT) [3]. PCR products were pooled, column-purified, and size-selected through microfluidic DNA fractionation. Consolidated libraries were quantified by quantitative real-time PCR before loading into the sequencer. Sequencing was performed in a pair-end modality on the Illumina NextSeq 500 platform rendering 2×150 bp pair- end sequences. The isolated strains were preserved by subculture and kept in an ampoule after freeze-drying.
Preparation and characteristics of microbial augmentation
The strain with high ammonium nitrogen removal capabilities among the strains isolated from the samples of soil, stream, and wastewater around food, paper, and leather manufacturing plants was added into the strains which showed a high colony forming capability, total organic carbon (TOC), and chemical oxygen demand (COD) removal rate, among the microorganisms presented in the aeration tank of food wastewater treatment plant, in order to be used for microbial augmentation as a field application for industrial wastewater treatment. This complex microbial augmentation was named FIW-1, which was spread on the PCA medium to investigate the reproducibility of microorganisms and verify the viability of cells.
Measurement of cell mass
Cell mass was measured by washing the cell obtained by centrifugation of the culture solution of isolated strain twice, dried at 105℃ for eight hours, and then determined as dry cell weight (DCW).
Analysis of basic feature of wastewater
The total organic carbon concentration of the subjected wastewater was analyzed using TOC analyzer (Dohrman DC-180). NH4-N was analyzed by liquid analyzer. Meanwhile, chemical oxygen demand (COD) was analyzed according to standard methods for the examination of water [1].
Pilot test for treating industrial wastewater
A continuous culture test employing a lab scale pilot reactor with acryl was carried out in order to investigate the treatment efficiency of the food industrial wastewater. The wastewater was input into the reactor as continuous reaction was induced to the control group, into which only activated sludge was added, and the test group, into which microbial augmentation was applied to measure wastewater treatment efficiency.
The pilot test for food wastewater treatment efficiency was adjusted for its hydraulic retention time of 24 hr, while the treatment efficiency changes of the control with only activated sludge and treatment block and with microbial augmentation were observed over time. An inoculum of 200 mg/l was added daily.
Results and Discussion
Isolation of microorganism in the aeration tank
The codominant microorganisms in the aeration tank degrade organic sources symbiotically or competitively to use a self-proliferation process and to discharge water and carbon dioxide as a final decomposition product. In order to select a codominant microorganism in the activated sludge in the aeration tank in the food wastewater treatment plant, a sample was spread on the PCA medium and cultured for 3 days. The 7 dominant strains that formed the colony were selected.
Among the strains isolated from the aeration tank, AT2, AT9, and AT12 strains were isolated as dominant microorganisms. When the AT2, AT9, and AT12 strains were added, the treatment efficiency for the total organics in the influent water of the subjected wastewater was higher in the TOC removal activities compared with those of the control group wherein only an activated sludge was added as shown in Table 1. Particularly, in the case of the isolated AT12, the TOC removal rate was 81%, which was the highest treatment efficiency as well as the highest COD removal efficiency. These isolated strains (AT2, AT9, and AT12) were used to prepare the microbial augmentation for biological wastewater treatment.
Table 1. Selection of microorganisms for application test in the wastewater treatment site
*Control: Only activated sludge was added. TOC: total organic carbon.
Isolation of nitrogen removal microorganisms
In order to isolate the ammonium nitrogen (NH4-N) removal microorganisms, the strains having excellent nitrogen removal capability among the 92 strains were isolated from the soil, stream, and wastewater around food, paper, and leather manufacturing plants nationwide. First, 12 strains with excellent colony forming capabilities when cultured on the solid culture medium containing ammonium nitrogen for isolation of bacteria were selected. Among the isolated strains, strain FN47, which has exceptional strain growth and ammonium nitrogen removal capability, was finally selected. Isolated FN47 was found to be excellent not only for its ammonium nitrogen removal capability but also for its organic removal activity.
Identification of isolated strains
The microorganisms in the aeration tanks of the wastewater treatment plant were examined, isolated, and investigated for their morphological, physiological, and biochemical characteristics for each strain (AT2, AT9, and AT12) (data not shown). The isolated strain AT2 was a gram-negative bacillus and facultative aerobic bacteria that had mobility. It showed a positive response to the catalase and oxidase, producing acid in the glucose, but showed a negative response toward Voges-proskauer (VP) test. It likewise showed a positive reaction to urease. The G+C content was 60%. These results exhibited similar characteristics with Pseudomonas species. It was found that the strain AT2 was a species which was very close to Pseudomonas sagittaria, when 16S rRNA of the isolated strain AT2 was analyzed further for more accurate identification (Table 2). Therefore, the isolated strain AT2 was finally named Pseudomonas sp. AT2.
Table 2. BLAST search results of isolated strains
*BLAST : basic local alignment search tool
The AT9 strain was a gram-negative bacillus and had mobility. It showed a positive response to catalase and negative response to oxidase, forming acid by reacting with glucose. When strain AT9 was used as a carbon source, it could not be fermented. Meanwhile, the strain AT9 could not use nitrate and showed a negative response to urease. The G+C content of DNA was 67%, showing similar characteristics with the Acinetobacter species. It was found that the strain AT9 was a species which was very close to Acinetobacter baumannii, when 16S rRNA of the isolated strain AT9 was analyzed further for more accurate identification. Therefore, the isolated strain AT9 was finally named Acinetobacter sp. AT9.
Meanwhile, the isolated strain AT12 was a gram-positive bacillus and had mobility. The strain AT12 showed a positive response towards both catalase and oxidase. However, AT12 could not produce acid with the glucose. AT12 used nitrate and showed a negative response to urease. The G+C content of AT12 was 58%. These results exhibited similar characteristics with Alcaligenes species. It was found that the strain AT12 was a species which was very close to Alcaligenes aquatilis, when 16S rRNA of the isolated strain AT12 was analyzed further for more accurate identification. Therefore, the isolated strain AT12 was finally named Alcaligenes sp. AT12.
The ammonium nitrogen degrading strain FN47 was a gram-positive bacillus having the size of 0.5~0.6×1.2~2.7 μm and possessing mobility. The FN47 colony had a light-yellow color and it was facultative aerobic bacteria that could grow well under aerobic, as well as anaerobic condition. The strain FN47 showed a negative response to urease and well-degraded starch and cellulose. The strain FN47 produced acids by reacting with saccharides like glucose, fructose, and lactose. Its G+G contents of DNA were 71%. These results exhibited similar characteristics with Microbacterium species. It was found that the strain FN47 was a species which was very close to Microbacterium diaminobutyricum, when 16S rRNA of the isolated strain FN47 was analyzed further for more accurate identification (Table 2). Therefore, the isolated strain FN47 was finally named Microbacterium sp. FN47.
Incubation characteristics of isolated strains in the aeration tank
In order to investigate the incubation characteristics of isolated strains Pseudomonas sp. AT2, Acinetobacter sp. AT9, and Alcaligenes sp. AT12, the wastewater itself was used as a culture medium. After adding nitrogen, phosphorus, and micronutrients of 3.0 g (NH4)2SO4, 0.3 g K2HPO4, 0.02 g FeSO4, 0.02 g MgSO4, and 0.01 g MnSO4 per 1 liter of medium, the growth activities of the isolated strains were investigated. The wastewater was used without sterilization so that the isolated strains could be used in actual wastewater generation sites. Therefore, wastewater had a contamination rate at around 105 -106 CFU/ml in the influent wastewater. Still, the isolated strains occupied most of the wastewater after culture so that affinity for substrate by the isolated strains was elevated. Particularly, when the culture test was conducted according to the nutrient source for isolated strain Alcaligenes sp. AT12, high culture characteristics were exhibited in the test group wherein nitrogen, phosphorus, and micronutrients were added compared with the test group with organic nitrogen. The optimum activity of the strain was exhibited after 30 hours of culture (Fig. 1).
Fig. 1. Culture test using the isolated strain AT12 in the aeration tank of food wastewater. The symbols ; (■) activated sludge, (■) isolate AT12+organic nitrogen source (0.1 g yeast extract), (■) isolate AT12+3.0 g (NH4)2SO4, 0.3 g K2HPO4, 0.02 g FeSO4, 0.02 g MgSO4, and 0.01 g MnSO4 per 1 liter of medium.
Ammonium nitrogen removal efficiency in the industrial wastewater
In order to remove ammonium nitrogen contained in the industrial wastewater, the food wastewater having a high nitrogen concentration was adopted for the test (Table 3). The ammonium nitrogen removal rate was depleted in the control where only activated sludge was added, suggesting that the active microorganisms inside the aeration tank suppressed dominance of the nitrification bacteria, which ultimately dropped total nitrogen removal efficiency. However, the ammonium nitrogen removal rate reached 71% in the treatment group with Microbacterium sp. FN47, which was excellent compared with that of the control. From the above results, it can be inferred that Microbacterium sp. FN47 is an effective ammonium nitrogen removal strain in the wastewater treatment site and is expected to improve water quality.
Table 3. Treatment efficiency of Microbacterium FN47 in the food wastewater containing ammonium nitrogen
*Control: only activated sludge was added. T-N: total nitrogen, TOC: total organic carbon.
Preparation of microbial augmentations and their characteristics
In order to investigate the wastewater treatment efficiency of the strains isolated from the aeration tank, the culture broth of the isolated strains Pseudomonas sp. AT2, Acinetobacter sp. AT9, and Acaligenes sp. AT12, as well as the culture broth of ammonium nitrogen degrading strain Microbacterium sp. FN47 with the defatted rice bran as a medium, were used to prepare the microbial augmentation FIW-1 for food wastewater treatment.
Optimum media were developed for growing cells of the isolated strains in the batch liquid culture. Each cell culture liquid was mixed in the defatted rice bran at a ratio of 2:8 and then solid phase fermentation was induced, followed by air drying at room temperature to prepare the microbial augmentation with the four strains. These four types of microbial augmentations were mixed at an equal ratio, with a complex microbial augmentation finally prepared. The complex microbial augmentation was prepared by the mixing of each strain in the defatted rice bran at a similar ratio. The nutrient solution of 0.5% yeast extract was added at 5% (v/w) in the rice bran, and combined with nitrogen, phosphorous source, and mineral nutrients Ca2+ and Fe2+. Ca2+ was added to fill alkalinity and Fe2+ was added to relax the absorption of microorganisms in the form of CaCl2 and FeSO4. AMP, ADP, CaCl2, and FeSO4 were also added at a weight ratio of 10% (W/W) by combining 1:1:2.0:0.5, respectively. The combined complex microbial augmentation was named FIW-1, and in the subsequent experiments, FIW-1 was added to the experimental group to examine its activity. The probiotic activity of the strains contained in the complex microbial augmentation FIW-1 was maintained at about 109 cells without significant change within 45 days of room temperature storage. The moisture content of the complex microbial augmentation containing four types of strains was measured 28.5%.
For the isolated strains, an optimum medium composition was established for cell growth in the batch liquid culture, and each culture broth was mixed with the defatted rice bran at a ratio of 2:8, followed by solid phase fermentation for 2 days. The fermented cell was air-dried to make each microbial preparation with five types of strains. These five strains were mixed for the microbial augmentation. All the five strains for the microbial augmentation maintained a cell mass higher than 109 CFU/g. In the case of a microbial augmentation FIW-1 where five microbial strains were mixed, the cell mass was 2.4×1010 CFU/g, the density was 0.305 g/cm3 , and the moisture content was 28.5% (Table 4). There was difficulty in wastewater treatment in the actual wastewater treatment plants due to the mixing of various materials including recalcitrant organics. The microorganisms could be made as a formulation or immobilized, and then inoculated so that the density of the population can be raised for a biodegradation process in the wastewater treatment plants [4].
Table 4. Characteristics of microbial augmentation FIW-1
Pilot test for the effectiveness of industrial wastewater by microbial augmentation FIW-1
The wastewater treatment test for the subjected wastewater from the food industry was conducted in the lab scale reactor to confirm the applicability of the microbial augmentation FIW-1 for industrial wastewater treatment.
The treatment efficiency test was carried out for the control in which only activated sludge from the test site was added and for test group into which microbial augmentation FIW-1 was added in the pilot for continuous treatment. The 1st treated water was set as an influent during five days before the inputting of the microbial augmentation FIW-1 and before the treated supernatant of aeration tank was set as an effluent. The TOC, COD, and ammonium nitrogen concentration were measured after making the reaction continue for 10 days in the culture.
In the case of control wherein only activated sludge was added, the TOC was removed at a rate of 63%, while the COD removal rate was 64%. However, when microbial augmentation FIW-1 was applied, the TOC and COD removal rates were increased to 92% and 85%, respectively. In the case of ammonium nitrogen removal rate, it was 37% in the control, and 82% of removal rate was observed from the test group with microbial augmentation FIW-1 (Table 5).
Table 5. Effectiveness of wastewater treatment using microbial augmentation FIW-1 on bench scale pilot
*TOC: total organic carbon, COD: chemical oxygen demand.
Field Application Test
A test was carried out to ascertain if the microbial augmentation of FIW-1 can be applied in the wastewater treatment site. The average COD in the influent water for 5 days before field test was 985 mg/l and in the effluent water 621 mg/l, showing that the COD removal rate of the aeration tank was merely 37%. However, during 3 days after inputting of the microbial augmentation FIW-1, a constant treatment rate (43%) was observed, followed by 62% of the treatment rate with a COD value of 374 mg/l after an elapse of 5 days.
These results indicated that the microbial augmentation FIW-1 was composed of a dominant microorganism, and ammonium nitrogen removal microorganism can establish an effective biological treatment system not only in the food wastewater which was the subjected wastewater in the study but also in the paper mill wastewater and leather wastewater containing ammonium nitrogen if it is used through a site adaption process.
Acknowledgement
This work was supported by a Research Grant of Gyeongnam National University of Science and Technology in 2018 year.
The Conflict of Interest Statement
The authors declare that they have no conflicts of interest with the contents of this article.
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