Dynamic comparison on the usage of probiotics in organic wastewater treatment under aerobic conditions in a diurnal environment

ABSTRACT

This study aims at evaluating and comparing pollution removal in wastewater treatment via the use of probiotics alone or in combination under aerobic conditions in diurnal cycles. Herein, 650 mL of organic wastewater was stored in 1-L conical flasks and then randomly divided into three treatment groups, each experiment was repeated three times. Group A was supplemented with 2% (v/v) photosynthetic bacteria (PSB; Rhodopseudomonas palustris) alone; group B was supplemented with 2% (v/v) B. subtilis alone; and group C was supplemented with 1% (v/v) PSB and 1% (v/v) B. subtilis. Results showed that the pH increases were in the order: group A < group C < group B. The performance of the probiotics in terms of ammonia nitrogen and total nitrogen (TN) removal was in the order: group A < group C < group B, whereas in terms of total organic matter (TOC) and total carbon (TC) removal, the order was group C < group B < group A. These results showed that the effect of probiotics combination treatment on ammonia nitrogen and TN removal was better than that of using B. subtilis alone, but worse than that of using PSB alone. The effect of B. subtilis alone treatment on TOC and TC removal was better than that of using PSB alone, but the combination of PSB and B. subtilis showed greater benefits on TOC and TC removal.

Implications: Photosynthetic bacteria and B. subtilis were used in this study to investigate carbon and nitrogen metabolism via the use of different probiotics and then study further on comparing and achieving the best pollution removal performance in probiotics alone or in combination treatment. To make observations realistic, the experiments were conducted under aerobic conditions in a diurnal cycle environment.

Introduction

People’s living standards are rising steadily, driven by speedy social and economic development. As a consequence, water pollution, especially contamination with organic matter and nutrients, is a well-known issue. Many studies have focused on using microorganisms to deal with this problem. Photosynthetic bacteria (Rhodopseudomonas palustris) and B. subtilis are two common effective microorganisms used in studies of wastewater treatment (Mekjinda and Ritchie, 2015; Deng et al., 2003).

Photosynthetic bacteria (PSB) are the oldest prokaryotes in the world and are widely distributed in water and wastewater (Okubo et al., 2006). PSB contain rich pigments such as carotenoid and bacteriochlorophyll, which play a dominant role in light harvesting and converting light energy into chemical energy through photosynthesis. Organic carbon can be used as an energy source to produce proteins, carotenoids, bacteriochlorophylls, biopolymers, antimicrobial agents, and pantothenic acid, and these materials are necessary for the survival and proliferation of PSB (Kuo et al., 2012). B. subtilis is a nonpathogenic gram-positive bacterium. Its spores are the dormant body, not its propagules. Meanwhile, many studies have demonstrated that B. subtilis can secrete digestive enzymes, such as protease, amylase, and lipase (Sonenshein, 2000; Kodama et al., 2007; Liu et al., 2014), and these enzymes can digest some organic matter.

In a previous study, Mekjinda and Ritchie (2015) illustrated that PSB can withstand high concentrations of organic wastewater and can effectively degrade highly resistant organic pollutants. Other studies have focused on the production of biosurfactants (Barros et al., 2008) and bioflocculants (Deng et al., 2003) by B. subtilis. High-concentration organic wastewater was used as the substrate in both studies, demonstrating that B. subtilis can survive and reproduce in high-organic-concentration wastewater and do well in organic pollution removal.

Nowadays, although much has been learned, studies have mainly focused on wastewater treatment systems. These systems require additional human and financial investments because of the large area occupied with a rather limited scope (Zhou et al., 2015). Small food factories or livestock farms do not have extra income to build wastewater treatment systems like that, but they are capable of producing large amounts of waste organic matter daily and pour them directly into rivers or lakes. A solution to this problem would be to purify such wastewater at the time of its production. Probiotics alone have shown high efficiency levels in performing organic pollution removal in relation to wastewater treatment systems. Few studies have focused on evaluating and comparing the effect and efficiency of probiotics alone or in combination in wastewater treatment under common conditions.

Hence, PSB and B. subtilis were used in this study to investigate the best pollution removal performance in wastewater treatment. To make observations realistic, the experiments were conducted under aerobic conditions in a diurnal environment.

Moreover, this work investigates the metabolic characteristics of probiotics involved in the previously explained operations and situations.

Materials and methods

Materials

The PSB (Rhodopseudomonas palustris) cells were cultured in an improved PSB culture medium (purchased from Yichun Strong Microbial Technology Company, Jiangxi, China) by illuminating them with a 40-W incandescent lamp for 7 days. The resulting viable count was 3.0 × 10⁶ colony-forming units (cfu) mL⁻¹ and pH 6.27.

The B. subtilis cells (provided from the Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China) were activated, and cultured in a medium: 20 g glucose, 10 g tryptone, and 10 g yeast extract powder dissolved in 1 L distilled water, pH 7.5, and grown at 30 °C, shaken at 150 × g for 24 hr. The resulting viable count was 7.45 × 10⁶ cfu mL⁻¹ and pH 4.74.

To reduce interference, a volume of 6 L of organic wastewater was synthesized and used in this study. A detailed list of ingredients and the composition of the organic wastewater is shown in Table 1. All the ingredients ensure an organic wastewater with a high concentration of total organic matter (TOC), total phosphorus (TP), and ammonia nitrogen.

Table 1. Ingredients and composition of the organic wastewater.

Ingredient	Concentration (g L^−1)	Composition	Concentration (mg L^−1)
Saccharose	5	Total organic carbon	2025.25
Malic acide	3	Inorganic carbon	450.25
Sodium bicarbonate	1	Total nitrogen	466.5
Ammonium sulfate	2	Ammonia nitrogen	415.26
Potassium dihydrogen phosphate	0.4	Total phosphate	97.03
Magnesium sulfate heptahydrate	0.2	Dissolved oxygen	6.5
		pH	7.0
		C/N ratio	5.31

Experiments

The methodology of this study has been adopted to investigate effects and efficiency of wastewater treatment based on probiotics (alone or combined).

The reactors were 1-L conical flasks; 650 mL organic wastewater was added to the flask and then randomly divided into three treatment groups: group A was supplemented with 2% (v/v) PSB alone; group B was supplemented with 2% (v/v) B. subtilis alone; and group C was supplemented with 1% (v/v) PSB together with 1% (v/v) B. subtilis.

Other experimental conditions were controlled as follows:

1. Temperature: 28–30 °C and rotating speed: shaken at 110 × g were provided by a constant-temperature shaking table (DHZ-C; Beijing Meteorological Instrument Factory Co., Ltd., Beijing, China).

2. Aerobic conditions: all the experiments were exposed to air.

3. Twelve hours of artificial light a day (diurnal cycle) were provided by using two 40-W incandescent lamps (with a light intensity of 2000 lux).

Each experiment was repeated three times.

Analysis method

Dissolved oxygen (DO) concentration and pH were tested using a DO meter and a pH meter.

Supernatant of samples to be used for the analysis of ammonia nitrogen, total phosphorous (TP), total organic carbon (TOC), inorganic carbon (IC), and total nitrogen (TN) was collected by 9000 × g centrifugation for 15 min.

Ammonia nitrogen and TP were respectively tested by Nessler’s reagent spectrophotometry and ammonium molybdate spectrophotometric method (water quality determination of ammonia nitrogen by Nessler’s reagent spectrophotometry followed the People’s Republic of China publication HJ 535-2009). TOC, IC, and TN were tested by Multi N/C 3100, manufactured by Analytik Jena (N3-790010; Germany).

Statistical analysis

Three repetitions of each experiment were performed, and parallel measurements were conducted to ensure the accuracy of the data. Tukey’s test was adopted to analyze the significance of the values. The level of significance of the different groups exceeds 95% (p < 0.05).

Results and discussion

Effects of probiotics on carbon metabolism in organic wastewater treatment

Total organic carbon

As Figure 1a shows, the concentration of TOC was obviously lower with the B. subtilis alone and probiotics combined treatments than in groups with the PSB alone. In particular, the concentrations of TOC in group A reached the level of 2000 mg L⁻¹. However, TOC was quickly reduced in the B. subtilis alone and probiotics combined treatments after the 1st day, and then reached a similar TOC level; the TOC curve obtained via the use of probiotics combination treatment was above the TOC curve belonging to the B. subtilis alone treatment, with the treatment time increased by 3–11 days.

Figure 1. Effects of probiotics on TOC removal (a), IC removal (b), and TC removal (c) in wastewater treatment.

The TOC removal rate was obviously higher in the B. subtilis alone and probiotics combined treatments than in the groups with PSB alone treatment after the 1st day (Tukey’s test: p < 0.05). The TOC removal rates of groups A, B, and C respectively were 31.37%, 55.73%, and 46.67% at the 2nd day. At the 6th day, the TOC removal rates of groups A, B, and C were 11.84%, 85.86%, and 91.93%, respectively. In addition, the TOC removal rate was obviously higher with the probiotics combination treatment than with the B. subtilis alone treatment, at the 3th, 5th, and 7th days (Tukey’s test: p < 0.05).

Obviously, the effects of probiotics on TOC metabolism ranked in the following order, from the best to the poorest performance: probiotics combination treatment > B. subtilis alone treatment > PSB alone treatment.

Nagadomi et al. (2000) studied the simultaneous removal of chemical oxygen demand (COD), phosphate, nitrate, and H₂S in synthetic sewage wastewater using porous ceramic immobilized photosynthetic bacteria, and with dissolved oxygen greater than 3 mg L⁻¹. A high efficiency in COD removal (up to 89%), far from these results, was obtained through the use of PSB alone. In this study, the efficiency of TOC removal through the use of B. subtilis alone or probiotics combined treatments reached a good level.

Inorganic carbon

As Figure 1b illustrates, the three IC curves show similar trends; the concentrations of IC showed a decrease the 1st day and an increase the 2nd day, but the inorganic carbon levels were all reduced afterwards and finally reached a similar level of 300 mg L⁻¹ (removal rate: 33.33%).

Total carbon

As Figure 1c shows, the three TC curves have trends similar to those of TOC mentioned above. Following the analysis of TOC and IC, it is possible to determine TC through the relationship

Figure 1c illustrates that the resulting main trend was a decrease in the level of TC after the treatment with B. subtilis alone or probiotics combined, but little effect on the concentrations of TC in the treatment with PSB alone.

Izu et al. (2001) studied the effects of aeration conditions on the growth of purple nonsulfur bacteria in a biological wastewater treatment process via denaturing gradient gel electrophoresis and fluorescence in situ hybridization. Their studies illustrated that PSB survival was little affected by aeration conditions. The maximum PSB ratio (up to 80%) was obtained without aeration. Their conclusion may be related to the lowest TOC removal in the PSB alone treatment with aeration, in a diurnal cycle environment. Zhou et al. (2015) studied the effect of light sources on PSB in wastewater treatment; their findings also illustrated that PSB can improve COD removal.

Effects of probiotics on nitrogen metabolism in organic wastewater treatment

Ammonia nitrogen

Ammoniac nitrogen pollution has been identified in the Jiang He lacustrine water system as one of main factors of eutrophication. Furthermore, it can be transformed into nitrite, a human carcinogen.

As Figure 2a shows, the concentrations of ammonia nitrogen in organic wastewater were all decreased by increasing the treatment time, both in the case of using probiotics alone and probiotics combined. The concentrations of ammonia nitrogen were obviously lower with the PSB alone treatment than in groups B and C after the 5th day (Tukey’s test: p < 0.05). Results illustrated that the ammonia nitrogen removal rate was increasing in time.

Figure 2. Effects of probiotics on ammonia nitrogen removal (a), TN removal (b), and C/N ratio (c) in wastewater treatment.

Obviously, the effects of probiotics on ammonia nitrogen metabolism ranked in the following order, from the best to the poorest performance: PSB alone treatment > probiotics combination treatment > B. subtilis alone treatment.

Zhang et al. (2012) studied the effect of B. subtilis on metabolic characteristics. Their findings were consistent with this study and also illustrated that B. subtilis can improve ammonia nitrogen removal.

Total nitrogen

As Figure 2b shows, the concentrations of TN in organic wastewater were all decreased by increasing the treatment time both in the case of using probiotics alone and probiotics combined. Moreover, the TN trend was very similar to that of ammonia nitrogen. These kinds of treatments also succeeded in lowering the concentrations of TN in wastewater. Such concentrations were obviously lower with the PSB alone treatment than in the treatment groups of B. subtilis alone and together with PSB, at the 4th, 5th, 7th, 8th, 10th, and 11th days (Tukey’s test: p < 0.05). The concentrations of TN were obviously lower with the probiotics combination treatment than in the treatment groups of B. subtilis alone, at the 3th, 4th, 5th, 7th, 8th, 10th, and 11th days (Tukey’s test: p < 0.05).

Obviously, the effects of probiotics on total nitrogen metabolism ranked in the following order, from the best to the poorest performance: PSB alone treatment > probiotics combination treatment > B. subtilis alone treatment.

Nagadomi et al. (2000) studied the effect of PSB on nitrite metabolism: their findings also illustrated that nitrite can be removed through the use of PSB.

Effects of probiotics on C/N ratio in organic wastewater treatment

The C/N ratio is a crucial factor in microorganism metabolism. A low C/N ratio and a high concentration of ammonia is the main problem in nitrogen removal from fecal wastewater or other organic wastewater.

As Figure 2c illustrates, the C/N ratio curves obtained by treating wastewater with B. subtilis alone or with probiotics combined have similar trends and at the lowest ratio (C/N ratio: 2–5), but group A’s curve looks different from those of the other two groups and the C/N ratio grows higher with increasing treatment time.

This strange phenomenon can be explained as follows: due to the simultaneous reduction of TC (TOC and IC) and TN, in groups B and C, C/N ratios remain within the range of 2–5. Obviously, group A follows a different trend because of the infinitesimal change of the TOC and a significant reduction of the TN, resulting in a big change of the C/N ratio.

Effects of probiotics on the pH value in organic wastewater treatment

Figure 3a shows the comparison between the pH levels of the three treatment groups. The figure suggests that the pH values increased with increasing treatment time and finally reached a similar level (pH 9.10–9.28). The pH curve obtained from the treatment with PSB alone was always above the other two curves from the 0th to the 7th day. The pH curve obtained from the treatment with B. subtilis alone was similar to the pH curve obtained from the treatment with PSB and B. subtilis combined, except at the 3th day.

Figure 3. Effects of probiotics on pH values (a) and DO values (b) in wastewater treatment.

Szymanski and Patterson (2003) studied the function of effective microorganisms in on-site wastewater treatment systems (septic tanks). Their studies showed that the pH tended to drop after completion of the trail. The likely explanation for the differences in the pH variation among the two studies is that they utilized different microorganisms with differing metabolisms as well as different cultivating conditions.

In addition, Zhang et al. (2012) studied the effects of the optimal conditions for heterotrophic nitrification via the use of an aerobic denitrification bacterium (Bacillus methylotrophicus, strain L7). Their studies suggested that the optimal conditions were C/N 6, pH 7–8, and a wide range of ammonia nitrogen concentration, from 80 to 1000 mg L⁻¹. Their conclusion was consistent with ours, in relation to TOC, IC, ammonia nitrogen, TN; the metabolic rate was very high at a pH level of 7–9. A few small distinctions that appeared may be related to the use of different strains of bacillus.

Effects of probiotics on DO in organic wastewater treatment

At the same time, as Figure 3b shows, the DO concentrations in organic wastewater were also influenced through the treatment with probiotics alone or probiotics combined. After being influenced by the probiotics use, the DO was thoroughly consumed at the 24th hr. Although DO finally reached a similar level (6.1–6.6 mg L⁻¹), the recovery process was not the same. The DO concentrations in PSB alone gradually increased with increasing treatment time from the 1st to the 7th day. The DO concentrations in B. subtilis alone and probiotics combination treatments were recovered at the 2nd day, but then consumed from the 3rd to the 4th day and gradually increased after that. The likely explanation for this phenomenon is rapid growth of bacteria because of the high initial concentration of nutrients in the previous 2 days. But we have seen that the concentrations of TOC and TN were respectively reduced 50% and 30% during this period. So we may assume that the rapid decline in nutrition and DO concentration made the B. subtilis adaptively change. This adaptive change might be a reduction in reproduction rate, which then caused the peak in DO concentration at the 2nd day. But the gradual increase in the B. subtilis population caused the DO consumption from the 2nd to the 4th day. The gradually increased DO concentration from the 4th to the 7th day also can be explained by the slowing of bacterial reproductive processes as nutrients were consumed, and finally the DO concentration was more or less unchanged (6.1–6.6 mg L⁻¹) during the last 5 days.

As we can see from Figure 3b, the DO was basically consumed from the 1st to the 6th day, especially from the 1st to the 4th day. At the same time, considering from Figure 1 to Figure 2, the pH variation and TOC, IC, TN, and ammonia nitrogen metabolic rates were extremely fast. It is possible to state that oxygen is a crucial factor for B. subtilis to survive and proliferate. This fact also illustrated that PSB can survive and proliferate under aerobic environments in 12:12-hr light:dark cycles. Nevertheless, the TOC removal rate stayed at a very low level in PSB alone treatment groups.

Conclusions

In this study, the pH and DO values were greatly affected by probiotics alone or in combination in organic wastewater treatment, under aerobic conditions in a diurnal cycle environment. PSB and B. subtilis alone could improve the treatment effect on ammonia nitrogen, TN, TOC, and TC removal, but the effect of probiotics combination treatment on ammonia nitrogen and TN removal was better than that of using B. subtilis alone, but worse than that of using PSB alone. The effect of B. subtilis alone treatment on TOC and TC removal was better than that of using PSB alone, but the combination of PSB and B. subtilis showed greater benefits on TOC and TC removal.

Funding

This work was supported by the National Waterfowl Industry Technology System (CARS43), the New Variety Breeding of Livestock and Poultry (2012C12906-14), the National Science & Technology pillar program during the 12th Five-Year Plan period (2012BAD39B04), and the International Science & Technology Cooperation Program of China (2013DFR30980).

Additional information

Funding

This work was supported by the National Waterfowl Industry Technology System (CARS43), the New Variety Breeding of Livestock and Poultry (2012C12906-14), the National Science & Technology pillar program during the 12th Five-Year Plan period (2012BAD39B04), and the International Science & Technology Cooperation Program of China (2013DFR30980).

Notes on contributors

Jun Liu

Jun Liu is a Master of Agriculture at the College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, People’s Republic of China, and the Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Science, Hangzhou, People’s Republic of China.

Yali Liu

Yali Liu is a Doctor of Agriculture at the Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Science, Hangzhou, People’s Republic of China.

Guoqin Li

Guoqin Li, Junda Shen, Zhengrong Tao, Yong Tian, and Li Chen are Researchers at the Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Science, Hangzhou, People's Republic of China.

Junda Shen

Guoqin Li, Junda Shen, Zhengrong Tao, Yong Tian, and Li Chen are Researchers at the Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Science, Hangzhou, People's Republic of China.

Zhengrong Tao

Guoqin Li, Junda Shen, Zhengrong Tao, Yong Tian, and Li Chen are Researchers at the Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Science, Hangzhou, People's Republic of China.

Yong Tian

Guoqin Li, Junda Shen, Zhengrong Tao, Yong Tian, and Li Chen are Researchers at the Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Science, Hangzhou, People's Republic of China.

Li Chen

Guoqin Li, Junda Shen, Zhengrong Tao, Yong Tian, and Li Chen are Researchers at the Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Science, Hangzhou, People's Republic of China.

Chunmei Li

Chunmei Li is a Professor at the College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, People’s Republic of China.

Lizhi Lu

Lizhi Lu is a Professor at the Institute of Animal Husbandry and Veterinary Science, Zhejiang Academy of Agricultural Science, Hangzhou, People’s Republic of China.