Effects affecting ammonia removal in synthetic wastewater by locally isolated Rhodobacter sp strain A1

. This research focused on the effects affecting ammonia removal in synthetic wastewater by Rhodobacter sp. strain A1 using one factor at a time method (OFAT). Rhodobacter sp. strain A1 are able to remove ammonia from synthetic wastewater due to its ability to assimilate ammonia. The ammonia removal experiment was conducted under different factors; Rhodobacter sp. strain A1 in-oculum size (2%, 4%, 6%), incubation temperature (20°C, 25°C, 30°C, 37°C, 40°C), initial pH of synthetic wastewater (5,6,7,8,9) and initial NH 4 Cl concentration (5 mg/L, 10 mg/L, 15 mg/L) for four days of incubation period. Then, the solution was tested using Nessler reagent which will produce yellow colour when it reacts with ammonia. The intensity of colour is proportional to the ammonia concentration. This experiment was followed by ammonia quantitative analysis via spectrophotometer at 425 nm. The results obtained were then calculated to get the percentage of ammonia removal by PNSB. The result revealed that the bacterium can achieved 97.90 % efficiency of total ammonia removal at optimum growth condition with 6% of inoculum size, incubation temperature at 30°C and initial pH 7. As a conclusion, this Rhodobacter sp. strain A1 can therefore serve as a good candidate in wastewater treatment for ammonia removal.


Introduction
Safe drinking water is a basic requirement for the living organism to survive.Each human requires at least 20 to 50 litres of safe water per day especially for drinking, washing, and cooking.Therefore, the demand access to clean and safe water has increased due to the rapid growth of the human population over these past few years.There are various water bodies that play an important ecological role in ecosystems such as lakes, streams, rivers, oceans, seas and glaciers.However, due to continuous increase in anthropogenic activities, the level of nitrogen content accumulates in the water bodies, especially ammonia (NH3) increase proportionally which leads to the pollution affecting the aquatic ecosystem (Hu et al. 2021).Moreover, increasing the level of nutrients pollutants is known as eutrophication.This condition can lead to bacterial and algae bloom, which lead to the decrease in water clarity followed by oxygen depletion causing biodiversity issues, habitat loss and disruption of the food chain.
Over 80% ammonia production is being used in fertilizer to grow plants while the other remaining will be used in industrial or chemical sectors.Ammonia is the key of basic building blocks for inorganic fertilizers which currently play a major role in sustaining the food production for half of human populations (Seruga et al. 2019).However, not all the ammonia that is used in agriculture will be absorbed by the plants but eventually flow into the landfills or wastewater (Adeniyi et al. 2023).In the mine industry, at least 6% of the excess ammonia and nitrate will dissolve in the mine runoff water.As a result, excess ammonia will accumulate in water, causing nutrient pollution of aquatic ecosystems (Hu et al., 2021).Besides, ammonia can cause irritation to eyes, nose and throat and higher concentration of ammonia might cause damage to the organs system which can lead to chronic disease (Padappayil and Borger, 2021) The excessive ammonia nitrogen (NH 4+-N) are also present in the groundwater pollutants and the increase in ammonia concentration deteriorate the quality of groundwater (Zhang and Okabe 2020).Nitrogen pollutants in the form of ammonia pose various health risks.If humans ingest drinking water with high concentration of ammonia, it may cause ammonia poisoning while the long-term ingestion can cause damage to the organ systems such as the liver (Morrissy et al. 2021).

@The Author(s). 2024. Published by CBIORE
The ammonia removal treatment is not cheap nor environmentally friendly.Mostly, ammonia removal from wastewater uses chemical such as chlorine which might lead to other problems such as chlorination.According to Zhang et al. (2015), the chlorination could inhibit the microorganism's growth and remove the majority of plankton in the water.Biological removal of ammonia from municipal wastewater is typically accomplished via nitrification and de-nitrification.However, the high concentration of ammonia can inhibit the nitrifying bacteria due to the high effect of toxicity (Liu et al., 2019).In this study, PNSB Rhodobacter sp.strain A1 will be used to optimize its efficiency in removing ammonia from wastewater.This is because Rhodobacter sp.strain A1 can assimilate ammonia and have a high tolerance toward high concentration of ammonia.Hence, making Rhodobacter sp strain A1 as an alternative method in bioremediation which is more cost effective and environmentally friendly to remediate ammonia from the environment.

Preparation of Purple Non-sulfur bacteria Enrichment Medium (PNSBEM)
PNSBEM was prepared with 1 g/L NH4Cl, 0.5 g/L Na2HPO4, 0.2 g/L MgCl2, 2 g/L NaCl, 2 g/L yeast extract and 6 mL of 80% sodium lactate.The pH of the medium was adjusted to pH 7 and sterilized by autoclaved.

Cultivation and Sub-cultivation of PNSB Rhodobacter sp. strain A1
The PNSB Rhodobacter sp.strain A1 which was obtained from the previous study (Shah et al., 2020) was cultured in PNSBEM at 30°C under anaerobic light conditions for 7 days in a 35 mL serum bottle.Then, 10% (V/V) of the bacteria culture was used as inoculum for ammonial removal experiments;

Ammonia Removal
The synthetic wastewater was prepared with 0.34 g/L CH3COONa, 0.01 g/L NH4Cl, 0.09 g/L K2HPO4, 0.05g/l MgSO4.7H2O and 0.34 g/l NaHCO3.The pH of the wastewater was adjusted to 7 by using NaOH.After that, 2 % (V/V) of Rhodobacter sp.strain A1 was added into the 35 mL synthetic wastewater.The incubation temperature was set up at 30°C.The samples were collected every 24 hours for 4 days.Then, the ammonia concentration was analyzed via Nessler method (Idi et al. 2015).The experiment was done in triplicate to obtain the average.In addition, different factors affecting Rhodobacter sp. in removing ammonia from synthetic wastewater was used on this study; inoculum size of the Rhodobacter sp.strain A1 (4% , 6%), temperature (20, 25, 37, 40°C), pH (5, 6, 8, 9) and NH4Cl concentration (5 mg/L, 15 mg/L).

2.5
Analysis Method

Nessler Method
The samples of synthetic wastewater with Rhodobacter sp.strain A1 was collected every 24 hours for 4 days and undergo centrifugation at 3500 rpm for 30 min to obtain the supernatant, which used to test the presence of ammonia.The ammonia was analysed via Nesslerization method where the Nessler reagent, Potassium mercuric iodide (K2HgI4) was added into the supernatant.According to (Jeong et al. 2013) the samples will turn into yellow colour due to the reaction between K2HgI4 and the ammonia present in the sample.The basic reaction is: The intensity of yellow colour is directly proportional to the ammonia concentration.After 20-30 minutes, the samples measured at absorbance 425 nm using a spectrophotometer.The percentage of removal was calculated using the formula (Ci -Cf)/Ci x 100%, where Ci = Initial concentration of ammonia and Cf = final concentration of ammonia in mg/L.

Statistical Analysis
Statistical analysis between the parameter and the factors affecting Rhodobacter sp.strain A1 in ammonia removal was carried out using Graphpad Prism Ver 8.0.One-way Analysis of variance (ANOVA) and post hoc Tukey's test was adopted to analyse the significance differences between the factors affecting Rhodobacter sp.strain A1 for ammonia removal in synthetic wastewater.

Ammonia removal by Rhodobacter sp. strain A1
The locally isolated bacteria Rhodobacter sp.strain A1 is a purple non sulfur bacteria (PNSB) which is a photosynthetic bacterium that contain carotenoids and bacteriochlorophyls which we can observe in Figure 2a of their presence of red, pink colour due to the presence of pigments.PNSB is known as anoxygenic bacteria which undergo photosynthesis without producing any oxygen (Lu et al., 2021, Sakarika et al., 2019).PNSB is a metabolically versatile microorganism that can thrive in any type of environment.They are mainly photoheterotroph but can be interchangeable to be chemoheterotrophs or photo or chemo autotrophs.PNSB can switch between these modes depending on the composition of electron donors and acceptors that occur near them (Cerruti et al., 2020).Thus, this enables them to have a wide range of adaptation with various applicability and have a high tolerance when exposed toward the high concentration of nutrients and harmful compounds in wastewater under certain conditions (Chen et al., 2020).Their unique metabolism allowed them to grow in different cultivation conditions due to the ongoing and substantial changes in the ecological environment.
According to Yang et al. (2019), PNSB can assimilate the ammonia in wastewater into protein components and other cellular components for growth purpose under anaerobic conditions.Among the various forms of nitrogen source, bacteria prefer ammonia/ammonium more as a nitrogen source because they can support rapid growth rate in bacteria compared to certain amino acids that are regarded as poor nitrogen sources because they support much slower growth rate in bacteria (Wang et al., 2016;Maeda et al., 2018).

Effect of Inoculum Size in Ammonia Removal
Figure 3 showed that 6% inoculum size has the highest amount of ammonia removal from the synthetic wastewater during the four days of analysis.Huang et al. ( 2018) stated that higher inoculation quantity is better because it can remove more ammonia in a short time which indicate faster nutrient consumption.Therefore, during day 1 and 2, ammonia removal is highest for 6% inoculum size in comparison to lower inoculum size with about 78% removal.The ammonia removal is higher during day 3 and 4 in comparison to day 1 and 2 for all of the inoculum sizes.However, the removal of ammonia for all of the inoculum sizes during day 3 and 4 showed no significant difference between all of the inoculum sizes.It can be concluded that, 6% inoculum size showed higher efficiency in removing ammonia due to its high ammonia removal from day 1 until day 4 incubation in the range of 96 to 98% removal of ammonia.This showed that higher inoculum size or biomass has the effects on ammonia removal which is in agreement with Zheng et al. (2023) due to the transformation pathway of ammonia requires a large proportion of assimilation in the cell.

Figure 3
Effect of Inoculum Size in Ammonia Removal by Rhodobacter sp.strain A1

Effect of Temperature in Ammonia Removal
PNSBs are adaptable to temperature between 10°C and 40°C (Chen et al. 2020).Incubation at 30°C showed the highest percentage of ammonia removal of 97.94%.Using ANOVA, the effect of temperature for ammonia removal showed a significant difference at 30°C between all tested temperatures with low probability values.This study showed that ideal ammonia removal was achieved using the optimum temperature of 30°C for Rhodobacter sp strain A1.This is in agreement with Zhang et al. (2020) for Pseudomonas aeuroginosa and Huang et al. (2018) for Rhodospeudomonas sp.Smobiisys501 with highest ammonia removal at 30°C.This showed that Rhodobacter sp strain A1 is a mesophilic bacteria that thrive at temperature between 30°C to 35°C which showed high ammonia removal from the synthetic wastewater due to its ability to assimilate the ammonia from the wastewater.Ammonia could be directly assimilated through the synthesis of glutamate (Bolay et al. 2018).According to Batista et al (2019), this process is catalysed via two alternative pathways, GDH pathways which involve Glutamate dehydrogenases (GDHs) or GS-GOGAT pathways which involve glutamine synthetase (GS) and glutamate synthase (GOGAT).When there is an excess of energy, assimilation of ammonia is carried out by GS as the enzyme consume ATP (Reitzer et al, 2014).In contrast, the GDH-dependent ammonia assimilation does not require ATP as the enzyme operate well during low carbon and limiting energy.The higher temperatures may interfere with the normal function of photosynthetic reactions, resulting in photosynthesis machinery damage (Kaftan et al. 2019).Then, ATP production will decrease and lead to the disruption of GS-dependent assimilation.According to Reitzer et al ( 2014), the GOGAT pathway begins with GS, so it may reflect the relative activities of GS and GDH in assimilating ammonia into glutamate.This could be one of the reasons why the efficiency of ammonia removal reduces after 30°C with 40°C removed least amount of ammonia among all the temperatures with 62.91% efficiency.On the other hand, lower temperature might lower the enzymatic reaction direct or indirectly.Therefore, incubation temperature is one of the factors that is affecting Rhodobacter sp.strain A1 in ammonia removal.

Figure 4
Effect of Temperature in Ammonia Removal by Rhodobacter sp.strain A1

Effect of pH in Ammonia Removal
Figure 5 showed that Rhodobacter sp.strain A1 have the highest ammonia removal at pH 7. From the result obtain, statistical analysis showed significant differences between all pH for ammonia removal with pH 7.This is because the majority of PNSB are grown in a pH range between 6.8 and 8.5 and perform best assimilation of ammonia when the initial pH is around 7.0 (Chen et al., 2020).This is in agreement with bacterial studies in ammonia removal conducted by Zhang et al., (2020), Huang et al. (2018), Rout et al., (2017) and Zheng et al., (2023) showed pH 7 has the highest ammonia removal due to the enzymatic activities being at its optimal at the neutral pH.However, the percentage of ammonia removal started to decrease as pH further from 7. The decrease of ammonia removal percentage due to the inability of Rhodobacter sp.strain A1 to survive in extreme pH conditions (Huang et al, 2018).This indicate that this bacteria thrive in neutral pH and can only survive at weak acid and weak alkaline conditions.

Effect of NH4Cl Concentration in Ammonia Removal
The NH4Cl concentrations of 5, 10 and 15 mg/L were used as initial concentrations.As shown in Figure 7, the lowest percentage of ammonia removal occurs at the highest concentration of 15 mg/L with 35.4% on day 1.However, the percentage removal of ammonia increased sharply to 92.12% on day 2 and then remained above 90 % through the following days.According to previous study, low initial NH4Cl concentration showed higher ammonia removal compared to high initial NH4Cl concentration (Idi et al., 2015).However, in this experiment, ANOVA showed no significant differences between all NH4Cl concentrations for ammonia removal.NH4Cl concentration does affect ammonia removal at the beginning but showed almost similar percentage of ammonia removal after reaching the fourth day which is above 90% removal.Furthermore, in this study it showed that Rhodobacter sp strain A1 has a high tolerance towards concentration of ammonia ranging from 5 mg/L to 15 mg/L and could assimilate and remove ammonia efficiently from the synthetic wastewater.According to previous study, PNSB could withstand up to 42 mg/ L to 170 mg/L of ammonia but has lower ammonia removal of only 72% to 42% (Idi et al., 2015).Thus, this study showed that Rhodobacter sp strain A1 ability to remove ammonia efficiently at lower ammonia concentration ranging from 5 mg/L to 15 mg/L. .

Conclusion
This study analysed the potential of PNSB Rhodobacter sp.strain A1 in treating ammonia synthetic wastewater via assimilation process.The removal efficiency reached 97.94% under anaerobic light with optimum growth condition.Light enables Rhodobacter sp strain A1 to undergo photosynthesis to produce energy and allow them to perform their biological functions.The 6% of inoculum size was the better inoculation rate for ammonia removal than among all inoculation size as it can consume nutrient faster than lower inoculation level.The suitable pH and incubation temperature for ammonia removal is pH 7 and 30°C because Rhodobacter sp.strain A1 can growth optimally at this condition.Lastly, high NH4Cl concentration led to the slower ammonia consumption by Rhodobacter sp.strain A1.Thus, the treatment of PNSB Rhodobacter sp.strain A1 shows great potential to be extended on real wastewaters that contain excessive ammonia concentration.

Figure 2
Figure 2 a) Rhodobacter sp.before addition of synthetic wastewater; b) Rhodobacter sp. with addition of synthetic wastewater.

Figure 5 Figure 6
Figure 5Effect of pH in Ammonia Removal by Rhodobacter sp.strain A1