IN
DEGREE PROJECT BIOTECHNOLOGY, SECOND CYCLE, 30 CREDITS
STOCKHOLM SWEDEN 2020,
Using bioaugmentation to enhance the denitrification process in a
treatment plant for landfill leachate
OSCAR SKIRFORS
KTH ROYAL INSTITUTE OF TECHNOLOGY
SCHOOL OF ENGINEERING SCIENCES IN CHEMISTRY, BIOTECHNOLOGY AND HEALTH
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Using bioaugmentation to enhance the denitrification process in a treatment plant for landfill leachate
A master thesis by Oscar Skirfors 2020-05-29, Stockholm
Main supervisor: Gunaratna Kuttuva Rajarao, department of Industrial Biotechnology CBH
External supervisor: Astrid Helmfrid, SÖRAB
Examiner: Qi Zhou
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Abstract
It has been illegal to deposit household waste in Swedish landfills since 2005. The large amount of waste deposited prior to this does however continue to pose an environmental concern, mainly in the form of leachate water. This study focused on enhancing the denitrification process in a leachate water treatment plant through bioaugmentation. The two strains Brachymonas denitrificans and Comamonas denitrificans as well a commercial seed mix from ClearBlu Environmental® (CBE-mix) containing amongst others, Pseudomonas putida AD 21 and Pseudomonas fluorescens, were investigated as candidates. Nitrite, nitrate, and ammonium concentrations were measured in laboratory-, and pilot-scale studies to follow the processes of nitrification and denitrification. The pilot study was conducted for 10 days in the middle of May 2020 with leachate from the treatment plant in an aerated and nonaerated setup in open field conditions. C. denitrificans and B. denitrificans were both shown to be able to adapt to growth in landfill leachate. The addition of these strains led to a higher rate of nitrate reduction compared to the control during the first days of the pilot experiment but showed no difference in the total amount of nitrate reduced. The combined nitrogen concentration of ammonium, nitrate, and nitrite was 6.7% lower than the control when using a culture augmented with the CBE-mix in the aerated setup. This could indicate aerated denitrification. The amount of nitrate reduced during the pilot experiment was increased with 32% when augmenting the community with the CBE-mix in a nonaerated setup. An explanation could be that certain strains in the mix were able to utilize hard to degrade organic carbon present in the leachate or that the mix had a higher ratio of reduced nitrate to consumed organic carbon than the indigenous community.
Keywords
Bioaugmentation Landfill leachate Wastewater treatment Biological nitrogen removal Denitrification
Waste management
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Sammanfattning
Det har varit olagligt att deponera hushållsavfall i Sverige sedan 2005, den stora mängd avfall som deponerats innan dess fortsätter dock att utgöra ett miljöproblem, främst genom genereringen av lakvatten. Den här studien fokuserade på möjligheten att förbättra denitrifikationen i ett reningsverk för lakvatten genom bioaugmentation. Två stammar tillhörande Brachymonas denitrificans, och Comamonas denitrificans, samt en kommersiell bakterieblandning från ClearBlu Environmental® innehållande bland andra Pseudomonas putida AD 21 och Psedomonas flourescens, undersöktes som möjliga kandidater. Ammonium- , nitrat- och nitritkoncentrationer mättes i odlingsstudier i labbskala och i en pilotstudie för att undersöka nitrifikation och denitrification. Pilotstudien utfördes i en luftad och en o luftad konfiguration utomhus i mitten av maj 2020, med lakvatten från reningsverket under en 10 dagars period. C. denitrificans och B. denitrificans klarade båda av att anpassa sig till tillväxt i lakvatten. Tillsats av dessa arter ledde till en ökning i nitratreduktionshastighet i början av pilotexperimentet men gav ingen total minskning av nitratmängden. Den sammanlagda slutkoncentrationen av ammonium-, nitrat- och nitritkväve var 6,7% lägre än i kontrollen när en kultur argumenterad med den kommersiella bakteriemixen användes i den luftade konfigurationen. Mängden reducerat nitrat ökade med 32% när en kultur augmenterad med den kommersiella mixen användes i den oluftade konfigurationen. En möjlig förklaring är att vissa stammar i mixen klarade av att tillgodogöra sig svårnedbrytbara kolföreningar i lakvattnet eller att ration mellan reducerat nitrat mot konsumerat organiskt kol var högre än i det ursprungliga microbsamhället.
Nyckelord
Bioaugmentation Deponilakvatten Vattenrening
Biologisk kväverening Denitrification
Avfallshantering
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Table of content
1 Introduction ...7
2. Background ...9
2.1. Nonbiological methods ...9
2.2. Biological nitrogen removal ...9
2.2.1. Nitrogen assimilation ...9
2.2.2. Nitrification ...10
2.2.3. Denitrification ...10
2.2.4. Annamox ...11
2.3. The treatment plant and the current treatment process ...12
2.5. The effects of inlet water quality for the treatment process ...14
2.5. Bioaugmentation in wastewater treatment ...14
2.6. Comamonas denitrificans ...16
2.7. Brachymonas denitrificans ...16
2.7. Janthinobacterium lividum ...16
2.8. ClearBlu Environmental® (CBE) Commercial seed mix ...17
3. Materials and Method ...18
3.1. Nutrients ...18
3.2. Water collection and sterilization ...18
3.3. Adaptation to leachate ...18
3.4. Isolation and selection ...18
3.5. Characterization of isolate ...18
3.6. Growth experiment on carriers ...18
3.7. Nitrite reduction...19
3.8. Denitrification profile in leachate water ...19
3.9. Nitrate reduction rates ...19
3.10. Metagenomic study ...20
3.11. Nitrification and Aerated denitrification experiment for isolate J. lividum ...20
3.12. Pilot study ...20
4. Results ...22
4.1. Adaptability of denitrifiers to leachate water ...22
4.2. Isolation and selection ...22
4.3. Characterization of isolate ...22
4.4. Metagenomic study ...22
4.5. Nitrite reduction...22
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4.6. Denitrification profile ...23
4.7. Biofilm formation on carriers ...24
4.8. Rate of nitrite reduction ...25
4.9. Nitrification and Aerated denitrification experiment for isolate J. lividum ...25
4.10. Pilot study ...26
5. Discussion ...29
6. Conclusion ...30
6. Future perspectives ...31
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1. Introduction
All waste produced in Sweden is supposed to be managed from the perspective of a waste management hierarchy as stated in article 4 of the current European waste management directive (2008/98/EC), namely: 1. Prevention, 2. Re-use, 3. Recycling, 4. Other forms of recovery i.e. energy, 5. Disposal. (1) The disposal at the bottom of the hierarchy refers to the practice of disposal in landfills. These are defined by the EU as storage areas for waste, either above or below ground, both temporary and permanent. (2)
The government has over the years in the spirit of the waste management hierarchy introduced legislation to minimize the use of landfills in the country. Big steps were the ban on the deposition of combustible waste in 2001 and the ban on the deposition of organic waste in 2005 (3), which in practice made the deposition of household waste in landfills illegal. All household waste deposited prior to this does however continue to pose an environmental concern. This concern comes mainly in the form of leachate water emitted by the older landfills. Landfill leachate water is defined as all water that has been in contact with deposited waste, this can either be naturally up-flowing groundwater but also rainwater that falls over the landfill.
Contaminated leachate from landfills can remain a pollution source emitting nitrogen, organic carbon, and heavy metals like lead for hundreds of years after the decommissioning of the landfill. (4)
One of these older landfills is situated at Löt waste management facility in Vallentuna municipality, managed by the company SÖRAB. This landfill was active between 1995 and 2005. The ammonium-rich leachate water emitted from the landfill is currently treated in a treatment plant for continuous biological treatment that was constructed in 2014. The ammonium is oxidized to nitrate by nitrifying bacteria, the nitrate is then reduced to nitrite and finally to nitrogen gas by denitrifying bacteria. The water is polished in a constructed wetland before its release to the recipient.
The treatment plant does however currently not operate satisfactorily. There are two major issues, the startup time of the plant in spring and the efficiency of the denitrifying step. The treatment plant is situated in the open field and is therefore exposed to the weather conditions.
There is an incorporated heating system utilizing the burning of locally produced landfill gas, but water temperatures do fall during winter with a fall in biological activity as a result. There is room for improvement regarding the time it takes for the biological activity to increase again in spring. There is also room for improvement regarding the efficiency of the denitrification step. All nitrate produced by oxidation of ammonium is not reduced to nitrogen gas and therefore leaves the treatment plant, resulting in a high nitrate load on the constructed wetland which in turn raises the need for longer than desired retention times there.
One way of improving the efficiency of a biological treatment step is through bioaugmentation.
Bioaugmentation is the process of adding a microbial strain or a consortium, or genetic material like a plasmid to i.e. a polluted area or a treatment plant. This is done when there is low microbial biomass present, i.e. polluted soils where the microbes have been wiped out by a high toxic load, or a new-started treatment plant. It can also be done when there is a specific, hard to degrade pollutant that the present community is unable to degrade, but there is a known strain, an engineered strain, or an adapted consortium capable of this phenomenon. It can also be applied to shift the composition of the present community to make it more efficient in removing
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For SÖRAB four different approaches of bioaugmentation would be interesting and were examined in this study.
1; If an organism can be added that can remove nitrate already in the aerated nitrification zone.
This would reduce the nitrate load to the nonaerated denitrification zone by adding a new microbial process, aerated denitrification.
2; The microbial community of the treatment plant could perhaps be shifted to increase the fraction of efficient denitrifiers. Which could lead to an increase in the rate of denitrification and improve the ratio of reduced nitrate and nitrite to added electron donor.
3; The Addition of new biomass in early spring could perhaps increase denitrification by reducing the startup time of the treatment plant after winter.
4; The addition of a microbial strain able to degrade complex carbon sources in the leachate would make more electron donor available in the treatment process. Which could reduce the need for an added external electron donor and reduce the emissions of total organic carbon (TOC) from the treatment plant.
Two strains in storage at KTH that have been isolated from wastewater, were investigated.
Comamonas denitrificans and Brachymonas denitrificans. A commercial bacterial seed mix from the company ClearBlu Environmental® consisting of 8 different strains and marketed for water treatment was also investigated. An attempt to isolate and characterize an efficient strain for bioaugmentation from the treatment plant was also conducted.
It is important to know the composition of a bacterial community to improve the denitrification process. Therefore, a metagenomic study was carried out on the microbial community in the treatment plant.
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2. Background
The treated leachate water is high in ammonia, but low in nutrients needed for bacterial growth, mainly phosphate and Chemical oxygen demand (COD). The leachate is also high in arsenic and chromium. (5–7). The composition of the ingoing wastewater during 2019 can be seen in table A1. The focus of this project is limited to biological nitrogen removal. There are however nonbiological methods useful for treating leachate that is high in ammonium but low in other nutrients. Two common methods of ammonium removal are air-striping and chemical precipitation.
2.1. Nonbiological methods
In air-striping, the alkalinity is raised to pH 10.8-11.5 which is the range that favors the conversion of ammoniumto ammonia gas. The gas can then be sparged from the treatment plant via an airflow. The Environmental Protection Agency (EPA) does however not recommend this method for waste streams with an ammonium content above 100 mg/l, as this would not be economically efficient. (8) There is a possibility that a fraction of the ammonium removed from the aerated nitrification basin at Löt, is striped.
Another method more suited for high strength ammonium wastewater is chemical precipitation.
Examples of chemicals that can be applied to precipitate ammonium is a combination of MgCl2
and Na2HPO4. Chemical precipitation is fast (the ammonium is removed in a matter of minutes), it reduces the need for aeration, temperature control, and biological startup times. It does however raise the salinity of the effluent, the addition of chemicals is costly, and the precipitate needs to be handled. (9)
2.2. Biological nitrogen removal
Several microbial processes are crucial for driving global nitrogen cycles. The function of this treatment plant is mainly to reduce the total amount of nitrogen by utilizing the microbial processes of nitrification to convert the ammonium to nitrate and denitrification to convert the nitrate to harmless nitrogen gas. There are however other pathways to the nitrogen cycle that could be utilized in biological nitrogen removal, such as anammox and nitrogen assimilation.
2.2.1 Nitrogen assimilation
Nitrogen makes up a large percentage of all biomass, mainly as building blocks for amino- and nucleic acids. It is believed that a 13% dry weight of an average bacterial cell consists of nitrogen. A major source for microbial nitrogen is ammonium which can be assimilated into biomass by all bacteria, several species can also assimilate nitrate. (10) Any bacterial growth in a treatment plant will thus reduce the content of ammonium and nitrate by binding it as biomass, which then can be sedimented. Assimilation is believed to play a major role in nitrogen treatment of municipal wastewater, one study approximated it to be responsible for 38-75% of the removed nitrogen of sewage wastewater depending on the COD:N ratio of the water. A higher COD content can sustain a higher growth rate and thus, more nitrogen is removed through assimilation. (11) Landfill leachate has a significantly lower COD:N ratio than sewage.
Nitrogen assimilation is thus believed to be a minor contributor to nitrogen removal in the treatment plant at Löt.
10 2.2.2 Nitrification
Nitrification is a two-step oxidative process; in the first step, ammonium is oxidized to nitrite using molecular oxygen as an electron acceptor. In the second step, nitrite is oxidized to nitrate, also with molecular oxygen as the electron acceptor.
NH4+ +1.5 O2 → NO2- +H2O +2 H+ NO2- + 0.5 O2 → NO3-
The two steps are carried out by different groups of microbes. There are currently no known species that carry out both reactions. (10)
It is possible to inhibit the reduction of nitrite to nitrate, this is known as partial nitrification.
The operational requirements to inhibit nitrite oxidation are relatively simple. One study achieved almost complete nitrite accumulation by keeping the pH above 8.5 through the addition of sodium hydroxide. (12) The reduction potential of nitrite is lower than that of nitrate.
It is therefore possible to reduce the addition of organic electron donor by 40% by using partial nitrification instead of full nitrification. (13) Nitrite reduction has also been shown to be up to 4.3 times faster than nitrate reduction. (14) Inhibiting nitrite oxidation will also remove the nitrite oxidizers from the treatment plant, these compete with the other microbes for the scarce nutrients in the leachate without contributing to any meaningful reaction.
Nitrification is a relatively temperature-sensitive process with a linear range between 15 and 350 C. There is still some activity between 15 and 50 C. (15) This is problematic for treatment in open systems in colder regions, which applies to the treatment plant at Löt.
2.2.3 Denitrification
Denitrification is the process of reducing nitrate and nitrite to the gases nitrogen monoxide, nitrogen dioxide, and nitrogen gas. This process is carried out by microbes that utilize nitrite or nitrate as electron acceptors, usually with an organic electron donor. Microbes capable of denitrification are a nonhomogeneous and diverse group often capable of a range of other metabolic activities using different electron donors than nitric oxygen. Most of the known microbes that carry out denitrification are facultative aerobic heterotrophs that switch from oxygen respiration to denitrification under anoxic conditions since the redox couple molecular- oxygen:water is more electropositive than nitrate:nitrogen-gas. (10) It is therefore common in traditional waste-water treatment to have an anoxic reactor or an anoxic zone where denitrification takes place. There are however organisms capable of aerobic nitrification, such as Pseudomonas putida AD-21, showing a preference for nitric oxygen even in the presence of molecular oxygen (16).
Full denitrification from nitrate to nitrogen gas includes 4 reduction steps:
NO3- → NO2- → NO → N2O → N2
It is important for a system using denitrification as a means of nitrogen removal that most of the nitrogen is reduced completely to nitrogen gas to limit the release of the gases nitrogen monoxide and nitrogen dioxide. Nitrogen monoxide catalyzes the breakdown of stratospheric ozone, nitrogen dioxide does eventually also degrade nitrogen monoxide through photolysis.
(17) Nitrogen dioxide is also a potent greenhouse gas believed to have a global warming potential 265-298 times higher per mole than that of carbon dioxide. (18)
11 2.2.4 Anammox
Anammox is a process where microbes use ammonia as an electron donor and nitrite as an electron acceptor. This is currently only known to be carried out by a certain type of obligately anaerobic bacteria. (10)
The reaction can be summarized to the following:
NH4+ + NO2- → N2 + 2H2O
There are obvious benefits in utilizing the anammox process for the treatment of ammonium- rich leachate water. The C:N ratio of the leachate water at SÖRAB would be more in favor of the anammox process. The optimal Biological oxygen demand (BOD5):N ratio for conventional nitrogen treatment is believed to be between 10 and 20 (19), while the inlet water to the treatment plant at Löt had an average ratio of 0.23 in 2019. (7) Theanammox process functions optimally with a C:N ratio below 1 (20) as this reduces the competition from heterotrophic microbes capable of living in anoxic conditions. The total C:N ratio can be measured as TOC:total-N which was 0.86 on average in the treatment plant inlet during 2019 (7). This removes the need to add any external electron donor to the treatment plant which considerably reduces the cost of the process. The carbon dioxide emissions from the treatment plant would also be reduced as less organic carbon would be consumed. Anammox as the name suggests does not require molecular oxygen. This would remove the need for energy-expensive aeration within the treatment plant. The process has generally been carried out at higher temperatures, and an optimum has been shown to be achieved above 330 C. That study also concluded that there was activity in a temperature span of 15-430 C. The optimal pH-range was determined to be between 7.3 and 9, (21) but reasonable activity has been seen down do pH 6.5. (22) There are however different species with different temperature optimums, one cold-tolerant variant have been isolated from arctic marine sediments with anammox activity down to -20 C and an optimum at 150 C. (23) Any anammox treatment of leachate would have to be coupled with a partial nitrification process as the raw leachate contains undetectable amounts of nitrite. There is a possibility to achieve both partial nitrification and anammox in the same reactor but this would require substantial process monitoring and control. (13,20,24).
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2.3 The treatment plant and the current treatment process
A schematic of the treatment plant can be seen in figure 1 (a), and an aerial view in figure 1 (b).
The unit for continuous biological treatment consists of the ponds L2A, L2B, L2C, and L2D that are connected via overflow.
L2A is the nitrification zone. It is sparged with 3 11kw aerators coupled to 14 outlets to favor ammonium oxidizing bacteria. The zone is also heated by the combustion of locally produced landfill gas, to favor heat-sensitive nitrification even during spring and autumn. L2B is the denitrification zone. It is not aerated and equipped with two impellers to avoid sedimentation of suspended biomass. Brenta+ vp1 is added as an electron donor to a COD:nitrate-N ratio of 4. L2C is designed to remove any remaining COD, it is equipped with one 15kw sparger.
SÖRAB does currently not aerate L2C making this zone a continuation of the denitrification zone. There is no mixing in L2C, meaning that biomass will also start to sediment. L2D is the sedimentation zone. It is not aerated nor stirred. All suspended biomass is sedimented to the bottom of the zone and surface water is pumped to a constructed wetland for polishing. There is depending on the amount of rainfall some circulation of the water from L2D back to L2A.
The total hydraulic retention time of the treatment plant varies between one and four months.
Figure 1 (a). Schematic of the treatment plant at Löt. L2A is an aerated zone where ammonium is oxidized to nitrate during nitrification. L2B is the denitrification zone, nitrate and nitrite are reduced to nitrogen gas. This zone is equipped with slow- moving impellers to prevent sedimentation of the suspended biomass. An external electron donor/carbon source in the form of Brenta+ vp1 is also added to this zone. L2C is a post denitrification aeration zone and is equipped with an aerator to remove the rest of the electron donor added to L2B. This is not utilized by SÖRAB currently due to low COD levels coming from the L2B zone. L2D is a sedimentation zone, biomass is sedimented and the treated water is transferred to the wetland for polishing.
There is some recirculation from L2D to L2A. depending on the inlet water flow and biological activity.
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Figure 1b. An annotated aerial view of the treatment plant. Google Earth (2020-05-16).
The efficiency of the nitrogen treatment process can be evaluated by looking at the concentration differences between the incoming water and water in the sedimentation basin L2D from which water is pumped out of the treatment plant. The main objective of the treatment plant is to reduce total nitrogen. Figure 2 (a) depicts the ingoing concentrations and the concentration in the sedimentation basin during 2018. The average difference in ingoing and outgoing concentrations to the treatment plant is 71.2% meaning that 71.2% of the ingoing nitrogen is removed in the treatment process. (6)
The nitrogen removal as discussed takes place through two major processes, nitrification in L2A and denitrification in L2B. The efficiency of the nitrification is assessed by the concentration of ingoing ammonium and the concentration of ammonium in the L2D zone. The ammonium as can be seen in figure 2 (b) for 2018 is almost completely removed except for April when the treatment plant was still in its start-up stage.
If all of the removed ammonium is assumed to have been oxidized to nitrate, then the efficiency of the denitrification could be estimated as: 𝑁𝑖𝑡𝑟𝑎𝑡𝑒+𝑁𝑖𝑡𝑟𝑖𝑡𝑒
𝐼𝑛𝑙𝑒𝑡𝑡 𝑎𝑚𝑚𝑜𝑛𝑖𝑢𝑚−𝑎𝑚𝑚𝑜𝑛𝑖𝑢𝑚 𝑟𝑒𝑚𝑎𝑖𝑛𝑖𝑛𝑔 𝑖𝑛 𝐿2𝐷 . This can be calculated as 71.4%. This value of denitrification corresponds well to the value of removed total nitrogen. (6)
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Figure 2 (a). The concentration of total nitrogen in the influent to the treatment plant and the sedimentation zone during 2018. The numbers depict the 12 months of the year (6)
Figure 2 (b). The concentration of ammonium nitrogen in the influent to the treatment plant and the sedimentation zone during 2018. The numbers depict the 12 months of the year (6)
Figure 2 (c). The concentration of nitrate and nitrite in the sedimentation zone during 2018. The numbers depict the months of a year. (6)
2.4 The effect of the inlet water quality on the biological treatment
It is known that both chromium and arsenic are inhibitors of microbial denitrification. A drop in denitrification activity of approximately 20% has been recorded when increasing the metals separately from 54 to 1260 mg/kg in soil systems. (25) The average concentrations of arsenic and chromium in the leachate water were 2019 measured to 38 µg/l and 147 µg/l respectively.
(7) This is thousands of times lower than the levels considered non-contaminated in the study
0 100 200 300 400 500 600 700 800
1 2 3 4 5 6 7 8 9 10 11 12
mg/l
month
Total - N in the treatment plant at Löt during 2018
Inlett L2D
0 100 200 300 400 500 600
1 2 3 4 5 6 7 8 9 10 11 12
mg/l
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Ammonium -N in the treatment plant at Löt during 2018
L2D Inlett
0 50 100 150 200 250
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Nitrate and Nitrite - N in the L2D sedimentation zone at the treatment plant at Löt during 2018
Nitrate Nitrite
15 and it can thus be assumed that the arsenic and chromium levels are non-inhibitory to denitrification activity in the treatment plant.
The low phosphate to nitrogen ratio in the inlet is concerning and could lead to nitrite accumulation in L2B, where only the first step of the denitrification is carried out due to phosphate deficiency. A study concluded that the nitrate-N:phosphate-P ratio could not be higher than 100 to achieve full denitrification without nitrite accumulation. (26) The nitrate- N:phosphate-P ratio at the L2A measuring point in the treatment plant which is the influent to L2B, did during 2018 not go below this ratio at a single point with an average ratio of 1455. (6) Optimal nitrogen removal requires an even higher amount of phosphate and the optimal ratio is believed to be between 10:1 to 5:1 for Ntot – Ptot. (19) The 2019 average for the inlet water to the treatment plant was 503:1.2 (7), the phosphate content is thus much lower than what would be considered necessary for optimal nitrogen removal. SÖRAB have previously experimented with phosphate additions to the treatment plant, this was concluded to be problematic as the addition of phosphate also led to an unacceptable increase of phosphate concentrations in the treatment plant effluent.
Optimal removal of both organic carbon and nitrogen is believed to be achieved when the ratio is between 10:1 and 20:1. (19) When looking specifically at denitrification, a study, using acetate as a carbon source, found that optimal denitrification occurred with a C:N ratio of 3.9- 4:1 of acetate-C to nitrate-N. (27) One gram of carbon from acetate equals approximately 2 g BOD5 or 2.7 g of COD. (28) reworked this would mean a BOD5:N ratio of 8:1 and a COD ratio of 10.8:1 for optimal denitrification.
The average BOD7:N ratio in the inlet to the treatment plant was 115:503 during 2019. (7) Note that SÖRAB measures its BOD content as BOD7 instead of BOD5. Measuring BOD7 gives a slightly higher BOD value than BOD5. This still means that the BOD:N ratio is much lower than what would be required for optimal treatment. The configuration chosen for the treatment plant with the aerated zone preceding the nonaerated zone further increases this problem, as much of the organic carbon will be consumed in the aerated zone while the total nitrogen content will only decrease slightly. This necessitates the addition of large amounts of external electron donor to the L2B zone, currently in the form of Brenta+ vp1. SÖRAB does currently base the addition of electron donor on the concentration of nitrate-N in the influent to the L2B zone. This is dosed to a COD:nitrate-N ratio of 4:1. This is considerably lower than what some consider as necessary. (16,19,27) SÖRAB optimized the addition of electron donor in 2014 when the treatment plant was constructed. It was then concluded that the ratio of 4:1 was sufficient to achieve complete denitrification. It is worth noting however that the addition of the electron donor was combined with a phosphate addition during the optimization period. (29)
The leachate at SÖRAB has a low BOD:TOC ratio. This ratio describes the relationship between biodegradable and non-degradable carbons. The ratio depends on the individual wastewater composition but it is usually around 3 in municipal wastewater. (30) The 2019 average at SÖRAB was however as low as 0.26. (7) This poses a dual-issue, there is limited carbon available for the microbes in the treatment plant, but the non-degradable TOC also pass through the treatment plant, increasing the load on the constructed wetland. A fraction of this can also reach the recipient.
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2.5 Bioaugmentation in wastewater treatment
There is a lot of precedent of bioaugmentation in wastewater treatment, and different strains, consortium, and genes have been successfully added to remove general eutrophication agents such as COD and total nitrogen but also more specific hard to degrade substances such as dyes and phenols. (31) Newly constructed or renovated treatment plants are often seeded with biomass from another treatment plant to help build a good treatment community from the beginning. This is also a form of bioaugmentation. SÖRAB did not add any microbes when they started the treatment plant at Löt avfallsanläggning in 2014 but relied on naturally present microbes. This makes a bioaugmentation approach interesting when it comes to improving the efficiency of the treatment plant as key strains might be missing from the community.
Below is a description of the microbes considered for augmentation of the leachate-water treatment plant.
2.6 Comamonas denitrificans
C. denitrificans is a gram-negative, rod-shaped bacteria belonging to the class of betaproteobacteria. The species was first described after isolation from a municipal wastewater treatment site by the department of industrial biotechnology KTH, due to its outstanding denitrification capabilities. The species can switch from aerobic to nitrate respiration without a lag-phase. The confirmed temperature range of the species is 20-370 C without the capability for growth at 40 C. (32) The species could based on these figures be assumed to be mesophilic which would mean capabilities of growth down to approximately 100 C. Studies have indicated that species of the Comamonas genus are favored by glycerol and that they come to dominate in wastewater treatment reactors where glycerol is used as the electron donor. (33) This presents the interesting option to combine augmentation measures with a switch to glycerol as a new electron donor to favor the added cultures, giving them a possibility to become dominating within the treatment plant community.
2.7 Brachymonas denitrificans
B. denitrificans is like C. denitrificans a gram-negative, rod-shaped bacteria belonging to the class of betaproteobacteria. The strain used was isolated by the department of industrial biotechnology from tannery wastewater (34). B. denitrificans has a strict respiratory metabolism capable of aerobic respiration and complete denitrification. The species grow in a pH range of 5-9 and a temperature range of 10-400 C. (35)
2.8 Janthinobacterium lividum
J. lividum is a gram-negative, rod-shaped bacteria belonging to the class of betaproteobacteria.
The species has a strict respiratory metabolism and grows in a temperature range of 4-300 C. J.
lividum is distinguishable through its dark violet color due to the pigment violacein. (36) It is a known denitrifier. (37) It is also known to produce a series of antibiotics as well as forming a very rigid biofilm, with a high extracellular polysaccharide (EPS) secretion. (38,39) The species Janthinobacterium svalbardensis, from the same genus as J. lividum has shown the capability of simultaneous nitrification and denitrification under aerated conditions. It would be very beneficial if the isolated strain showed similar potential. (40)
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2.9 ClearBlu Environmental
®(CBE) Commercial seed mix
There are commercial seed mixes sold by companies to be able to improve or start-up wastewater treatment. One mix developed and sold by ClearBlu Environmental® uses a combination of Pseudomonas putida AD-21, Pseudomonas fluorescens, and six different strains of Bacillus. It is the two Pseudomonas species that make the mix interesting. This mix is claimed by the company to be able to lower COD, surfactants, Hydrogen sulfide, suspended solids, and nitrate. As well as controlling odor and reducing sludge volumes. It is recommended to be used to increase startup times of treatment plants. (41)
P. putida AD-21 is a gram-negative, rod-shaped bacteria from the class gammaproteobacteria.
(42) It is a known aerobic denitrifier capable of performing denitrification at oxygen levels as high as 6 mg/l. The strain has an optimal denitrification activity when the C:N ratio is 8, it does however not reduce nitrate effectively when using ethanol as the electron donor. (16) P. putida grows in a temperature range of 4-370 C. (43,44) The species is popular in bioaugmentation to remediate various pollutants due to its ability to degrade vastly different compounds. (45–47) P. putida can utilize highly complex carbons in its metabolism including benzene, toluene, and xylene (48). P. putida has also been shown to secrete enzymes capable of reducing highly toxic hexavalent chromium to trivalent chromium which is a less toxic form of the heavy metal. (49) Note that much of this bioremediation research is made on other strains than AD-21, it does however show that P. putida has a complex metabolism overall, and the capability to degrade complex human-produced substances should exist to some extent in most strains.
P. fluorescens is a gram-negative, rod-shaped bacteria from the class of gammaproteobacteria.
It has an optimal growth temperature range between 25 and 300 C, (42) but it has been shown to grow with denitrification activity down to 50 C. (50) P. fluorescence is known as one of the most numerically present denitrifies in the soil microbiome. (51) P. fluorescens is however not an aerobic denitrifier and studies have shown the denitrification to be inhibited by oxygen. (52) This species is like P. putida, popular in bioaugmentation approaches and strains are capable of utilizing hard to degrade carbons such as crude oil. (53)
Bacillus is a group of gram-positive, rod-shaped species belonging to the class bacilli. The different species vary greatly, one defining feature is that all Bacillus form endospores. (54) The company states that the mix operates well in a pH range of 6-10 and a temperature range of 5-550 C. (55)
18
3 Materials and Method 3.1. Nutrients
All nutrient broth (NB) and nutrient agar (NA) used in the study has a composition of 5.0 g/l peptone and 3.0 g/l meat extract.
3.2. Water collection and sterilization
Water collection was carried out on the 5/2-2020 at Löt avfallsanläggning . Water was collected from approximately 1.5 m depth at SÖRABs sampling points: L2A and L2B. The water was brought to KTH and centrifuged at 5000 g for 10 min. The supernatant was collected and filtered through three steps of syringe filters: 1.2, 0.45, and 0.2 µl. Water was applied to Nutrient-agar (NA) after each filtration step to monitor sterility.
3.3. Adaptation to leachate
C. denitrificans and B. denitrificans were inoculated from NA to filter-sterilized L2B water in flasks at 250 C and 63 rpm. Growth was monitored through OD600 measurements. The CBE- mix was inoculated through the addition of freeze-dried bacterial powder to sterile filtered L2A.
3.4. Isolation and selection
Water from L2B as well as a sediment sample collected from L2B on the 5/2-2020 was plated on NA. Twenty-five colonies from the water and 25 colonies from the sediments were isolated based on differences in colony morphology. Ten of these were investigated for denitrifying capability. The colonies were amplified by inoculation in NB at 250 C and 63 rpm overnight.
Biomass was diluted to OD600 ~0.5 in NB. Sodium nitritewas added yielding a nitrite—N concentration of 50 mg/l. The cultures were incubated for 5 h in 10 ml lidded glass vials. Nitrite concentration was measured at hours 2 and 5 using Hach® test strips.
3.5 . Characterization of isolate
Determining of gram state: cultivation on selective MacConkey and phenylethal (FE) agar.
Oxidase test: cells were smeared onto paper, one drop of ethyl-p-phenylenediamine was added and an eventual color shift observed. Catalase test: One drop of hydrogen peroxide was applied to a glass slide, cells were applied to the drop and the possible creation of bubbles was observed.
Fermentation: lactose fermentation was investigated by determining the color on the MacConkey colonies. 16S-rRNA sequencing: DNA was extracted and purified using the Invitrogen DNA extraction kit. Purified DNA was amplified using PCR with Phusion polymerase. See table A2 and A3 for primers and PCR specifications.
3.6. Growth experiment on carrier materials
Different microbes were tested for biofilm growth on a carrier material.
B. denitrificans and C. denitrificans were cultivated at 180 rpm in 370 C overnight (ON) in NB.
J. lividum was cultivated at 250 C in 63 rpm, ON in NB. Plastic carriers were added to flasks, NB was added to three flasks, one flask was only inoculated with unfiltered L2B leachate.
According to the specifications in table 1. All flasks were inoculated in 250 C at 63 rpm and monitored for 14 days. Fresh NB was added after 4 days. Carriers without detectable biofilm formation were sonicated in deionized water which was then investigated for dissociated biofilm.
19 Table 1. The specifications regarding the four different setups for the biofilm formation experiment.
Added biomass Media
B. denitrificans + C.
denitrificans NB
B. denitrificans + C.
denitrificans + J. lividum NB
J. lividum NB
- Unfiltered L2B
3.7. Nitrite reduction
Separate cell suspensions of J. lividum, B. denitrificans, and C. denitrificans in NB were diluted to OD600 ~0.5 in NB. Sodium nitritewas added yielding a nitrite—N concentration of 50 mg/l.
nitrite was measured after 24h and 48h with Hach® nitrite and nitrate test strips.
3.8. Denitrification profile in leachate water
A general denitrification profile was investigated for each strain as well as for combinations of amplified L2B community and the strain.
The L2B community was inoculated by adding unfiltered leachate water to NB and the strains were inoculated from NA to NB. Cultures were inoculated for 48h in 250 C at 63 rpm. The cell suspensions were centrifuged at 2000 rpm for 20 min. The supernatant was decanted. The pellets were washed with 20 ml of unfiltered L2B water with the same centrifuge specifications.
L2B unfiltered water was added to a total volume of 50 ml. 14 µl Brenta + was added. Nitrate and nitrite were measured after 24h and 48h using Hach® test strips.
3.9. Nitrate reduction rates
Rates of nitrate reduction were compiled from different experiments used to optimize and test the process to avoid spending unnecessary time in the lab during the Covid-19 pandemic.
C. denitrificans was taken from the denitrification profile experiment previously described.
(3.7) See table 1 for specifications.
The L2B-community, J. lividum, and B. denitrificans: The experiment was carried out like the previously described experiment (3.7) but with 10 min centrifuge times. Nitrate was monitored using Hach® spectrophotometric nitrate test kit LCK 339, giving more exact values. Nitrite was monitored using test strips see table 2.
B. denitrificans + C. denitrificans. The experiment was carried out like the previously described experiment (3.7) but with 10 min centrifuge times and without the washing step leading to more nutrients remaining from the NB. Nitrate was monitored using Hach® spectrophotometric nitrate test kits LCK 339. Nitrite was monitored using test strips, see table 2.
20 Table 2. Setup for the determination of the rate of nitrate reduction. The time indicates the duration of the experiment.
3.10. Metagenomic study
A metagenomic study was carried out to characterize the microbial communities of L2A, L2B, and L2B sediment. DNA was extracted and treated using the DNeasy® Powerwater® kit (Quiagen). DNA was amplified using PCR with the HotStart polymerase. See table A4 and A5 for PCR specifications. Dynabeads MyOne Carboxylic Acid beads, Invitrogen kit was used for DNA cleanup. The sample was sent to NGI at SciLife labs for sequencing.
3.11. Nitrification and Aerated denitrification experiment for isolate J. lividum
Biomass was cultivated in NB during 48h, in 250 C at 63 rpm in baffled flasks. Ten ml of cell suspension was centrifuged at 2000 rpm for 20 min. The supernatant was decanted. The pellets were washed with 20 ml of unfiltered L2B water with the same centrifuge specifications. The biomass was diluted to OD600 1.438, in unfiltered L2A leachate in a 250 ml shake flask. The culture was incubated for 24 h in 250 C at 63 rpm. L2A with no added biomass was used as control. Nitrate and ammonia were monitored using Hach® spectrophotometric test kits LCK 339 and TNT 830, nitrite was measured after 24 h using Hach® test strips.
3.12. Pilot study
C. denitrificans and B. denitrificans were cultivated separately in 200 ml for 72 h in 250 C at 63 rpm, in NB, final OD of 1.170 for B. denitrificans and 0.55 for C. denitrificans corresponding to 1.04 g wet weight (WW) and 0.77 g WW respectively. Cell suspensions were centrifuged 20 min 2000g + 20 min 2500 g.
6 5 l plastic containers were set up next to the treatment plant at SÖRAB with the specification in table 1. Aeration was applied using one Airset 540 pond-sparger with two air stones attached.
The setup with the 6 containers was covered with black plastic to increase temperature. The experiment was conducted for 10 days. The amount of CBE-mix added was higher than recommended by the company dosing sheet, to see any positive effect, if there were any, during the limited time of the experiment. Brenta+ vp1 and glycerol which were used as electron donors were added two times at days 0 and 7, which combined would give a COD:Nitrate-N0
ratio of 4, which is the same as used by SÖRAB currently.
Nitrate and ammonium were measured using Hach® spectrophotometric test kits LCK 339 and
1 The starting biomass for L2B, B. denitrificans and J. lividum is estimated based on following measurements and might therefore not be completely accurate.
Species Starting OD1 Starting NO3- (mg/l) Time (h) Temp (0C)
L2B 0.365 80.4 24 25
C. denitrificans 0.312 90 20.5 25
B. denitrificans 0.594 80.4 24 25
J. lividum 0.496 80.4 24 25
C. denitrificans + B. denitrificans 0.733 71.6 24 RT
21 TNT 830, nitrite was monitored using Hach® nitrite and nitrate test strips. Temperature and pH were monitored using a HANNA HI 98811-5 field measurer. Measurements were done on days 3, 7, and 10. Ammonium was not measured in the nonaerated setup as previous experiments had shown that the ammonium concentrations remained constant without aeration.
Table 3. The 6 different experimental setups for the pilot study. The first addition of Brenta+
vp1 and Glycerol was rounded to 1.5 ml due to a lack of accurate measuring devices on hand.
It should have been 1.4 ml Brenta+ vp1 and 1.55 ml glycerol.
Setup nr 1 2 3 4 5 6
Water L2A L2A L2B L2B L2B L2B
Electron donor/carbon
source2
- - Brenta+ vp1
1.5 ml Glycerol day
0, 1.1 ml Glycerol day 7
1.5 ml Brenta+
vp1 day 0, 1.0 ml Brenta+
vp1 day 7
1.5 ml Brenta+
vp1 day 0, 1.0 ml
Brenta+
vp1 day 7
Microbes 4.5 g of
CBE-mix - 4.5 g of CBE-mix
0.77g WW C.
denitrificans + 1.04 g WW B.
denitrificans
0.77g WW C.
denitrificans + 1.04 g WW B.
denitrificans
-
Aeration Yes Yes No No No No
22
4. Results
4.1. Adaptability of denitrifiers to landfill leachate
Both B. denitrificans, C. denitrificans, and the CBE-mix clearly showed growth in sterile- filtered leachate water without additives. B. denitrificans showed the highest growth rate. See figure 3.
Figure 3. Growth curves of C. denitrificans, B. denitrificans, and the CBE-mix in filter-sterilized leachate water.
4.2. Isolation and selection
None of the 10 isolates tested were shown to have any measurable nitrite reduction within the 5 h measurement period. A violet pigmented strain was found to have denitrifying capability in a parallel study working on leachate from Löt avfallsanläggning conducted by Isac Ingfeldt 2020. It was incubated in sterile-filtered L2B water over a weekend with full nitrate and nitrite reduction.
4.3. Characterization of isolate
The biochemical assessment showed that the violet isolate was catalase positive, oxidase negative, gram-negative, and incapable of lactose fermentation. The 16S-rRNA sequencing gave a 99% match with Janthinobacterium lividum spp.
4.4. Metagenomic study
The first round of results from NGI was inconclusive. It is possible that a large amount of DNA was lost during the DNA cleanup step. A second round of DNA was prepared but NGI has yet to be able to accept this, in part due to reduced staff because of the Covid-19 pandemic.
4.5. Nitrite reduction
The test showed that J. lividum, C. denitrificans and the community present in the treatment plant can reduce nitrite while B. denitrificans cannot, which can be seen in table 4. The OD600
measurement and thus the added biomass of J. lividum is hard to assess since the strain grows through rigid biofilms that are not homogenizable through vortexing. No conclusions could, therefore, be made regarding nitrite reduction rates in J. lividum.
-0,05 0 0,05 0,1 0,15 0,2 0,25 0,3
0 20 40 60 80 100 120 140
OD600
t (h)
Growth curves of C.denitrificans, B.denitrificans and the CBE-mix
C. denitrificans B. denitrificans CBE-mix
23 Table 4. The concentration of nitrite in NB after the addition of different microbes to a concentration of OD600 ~0.5.
Time (h) 0 24 48
J. lividum 50 mg/l 30 mg/l 0 mg/l
C. denitrificans 50 mg/l 0.3 mg/l -
B. denitrificans 50 mg/l 50 mg/l 50 mg/l
L2B - community 50 mg/l 0 mg/l -
4.6. Denitrification profile
Three different profiles are showed in figure 4 (a) since these profiles where identical when using the semiquantitative measurement strips. These are the L2B amplified community, the amplified community + J. lividum, and the amplified community + B. denitrificans. These showed a rapid reduction of nitrate with nitrite accumulation. The nitrite is then reduced once all nitrate is gone. This is a profile shared with J. lividum, figure 4 (c). The particular strain of B. denitrificans examined, was shown to be unable to reduce nitrite, its denitrification profile is thus a rapid reduction of nitrate with an accumulation of nitrite that is not reduced even when all of the nitrate has been reduced, figure 4 (f). C. denitrificans does not seem to favor nitrate over nitrite reduction. Its profile is thus a slower reduction of nitrate with only a small accumulation of nitrite, figure 4 (e). The combination of C. denitrificans and the L2B community gives a rapid nitrate reduction with simultaneous nitrite reduction leading to a lower nitrite accumulation, which is then reduced once all the nitrate has been reduced, figure 4 (b).
A combination of B. denitrificans and C. denitrificans shows similarly a denitrification profile with simultaneous nitrate and nitrite reduction without nitrite accumulation, figure 4 (d).
24
Figure 4. General denitrification profiles of the (a) amplified L2B community, (a) the community + J. lividum, (a) the community + B. denitrificans, these three different profiles are showed in window (a) since they were identical. (b) the community + C.
denitrificans, (c) J. lividum, (d) C. denitrificans, (f) B. denitrificans, and (d) a combination of C. denitrificans and B. denitrificans.
Nitrate + nitrite – N is showed in blue; nitrate in gray, and nitrite in orange.
4.7. Biofilm formation on carriers
Bright violet biofilm was detectable as dots on the carriers after three days in both the J. lividum culture and the J. lividum + C. denitrificans + B. denitrificans combined culture. The biofilm from the combined cultured appeared homogeneous when examined under microscope and there was no sign of C. denitrificans or B. denitrificans incorporated into the biofilm. There was no detectable biofilm formation when examining the C. denitrificans + B. denitrificans culture. Nor was any biofilm detected upon sonication of a carrier after three days. There was rust-colored biofilm formation on the carriers inoculated in unfiltered L2B water after three days. No additional biofilm was formed in any of the experiments after the addition of fresh NB on day 4, and the biofilm dissociated after an additional week.
25
4.8. Rates of nitrate reduction
The rate of nitrate reduction per hour normalized after added biomass is illustrated in figure 5.
The data shows that the amplified L2B community is the fastest nitrate reducer, however with nitrite accumulation. J. lividum was not only the slowest nitrate reducer but did also have nitrite accumulation. C. denitrificans and B. denitrificans have similar rates with B. denitrificans being slightly higher but with nitrite accumulation, while C. denitrificans have no accumulation of nitrite. The combination of the two strains have as would be expected a rate between the two, without nitrite accumulation.
Figure 5. The rate of nitrate reduction normalized after added biomass
4.9. Nitrification and Aerated denitrification experiment for isolate J. lividum
J. lividum performed poorer regarding nitrification and denitrification during aerated conditions, as can be seen in table 5.
Table 5. The measured concentrations of ammonium, nitrate, and nitrite nitrogen when cultivating J. lividum under aerated conditions.
Ammonium (mg/l) Nitrate (mg/l) Nitrite (mg/l)
Start 141 73.4 -
J. lividum 89.8 78.6 10-15
Control 79.4 79.4 10-15
0 1 2 3 4 5 6
L2B C. denitrificans B. denitrificans J. lividum C. denitrificans + B.
denitrificans mg/l/ODt0/h
Nitrate reduction rates from the laboratory study
26
4.10. Pilot study
The different setups of the study are referred to by the numbers assigned to them in table 3.
All data collected from the pilot study can be seen in appendix table A6. A figure displaying the geographical setup can be seen in figure A1. This could be interesting from a heat distribution perspective.
Relative biomass was measured as a comparison to L2B water collected on the 5th - Feb 2020 when there was low biological activity in the treatment plant. The change in relative biomass can be seen in figure 6. The L2B setups, 3-6, have a higher biomass concentration in general than the L2A setups, 1-2. The two L2A setups have converged on one biomass concentration regardless of the initially added biomass. The biomass is still increasing in all L2B setups, 3-6, after 10 days. Setup 3 had the highest biomass concentration, at the end of the experiment while setup, 4 had the lowest.
Figure 6. The biomass in the different setups on the third day of the experiment, measured as a difference in OD600 from L2B water collected in February.
The concentrations of nitrate and ammonium for the aerated setups,1-2, can be seen in figure 7. The rate of nitrate increase is initially lower in setup 2 compared to the aerated control 1, but there is no clear rate difference between the two setups after the day 3 sample point.
Ammonium has been depleted after 3 days in both setups. Nitrite is not shown in the figure but was depleted at day 7 for both setups, see table A6. The final nitrate concentration is 6.7%
lower in setup 2 compared to the aerated control, 1, after 10 days.
-0,3 -0,2 -0,1 0 0,1 0,2 0,3 0,4 0,5 0,6
0 2 4 6 8 10 12
OD600
days
Relative biomass concentrations in the pilot-experiment
L2A, Aerated [1]
L2A, Aerated, CBE-mix [2]
L2B, Brenta+, CBE-mix [3]
L2B, Glycerol, C/B. denitrificans [4]
L2B, Brenta+, C/B. denitrificans [5]
L2B, Brenta+ [6]
27
Figure 7. The concentration of ammonium and nitrate – N in the two aerated setups. The setup with the CBE-mix has a lower initial increase in nitrate concentration but the rates converge during the second measurement period.
The concentration of nitrate-N for the nonaerated, 3-6, setups can be seen in figure 8, the rates of nitrate reduction can be seen in figure 9. The nitrate concentration in setup 3 is lower than the other nonaerated setups, 4-6, while there is no major difference between the three other nonaerated setups after 10 days. The average rate of nitrate reduction is 32% higher for setup 3 compared to the other nonaerated setups.
Figure 8. The reduction of nitrate – N for the different nonaerated setups.
New electron donor was added to the nonaerated setups, 3-6, on day 7. The rate of nitrate reduction drops for setups 5 and 6 after day 3. It then increases again once new Brenta+ vp1 is added on day 7. Setup 4 has a slight decrease in nitrate reduction rate during the experiment, the rate does not increase when new glycerol is added on day 7. Setup 3 is the only setup that shows an increase in reduction rate towards the end of the first week, but there is no
0 2 4 6 8 10 12 14
100 110 120 130 140 150 160 170 180 190
0 2 4 6 8 10 12
Axeltitel
mg/l
day
Ammonium and Nitrate - N for the aerated setups [1] and [2] in the pilot experiment
L2A, Aerated [1]
L2A, Aerated, CBE-mix [2]
Ammonium for both setups
0 20 40 60 80 100 120 140
0 2 4 6 8 10 12
mg/l -N
Days
Nitrat - N concentration for the nonaerated setups, [3-6] in the pilot experiment
L2B, Brenta+, CBE-mix [3] L2B, glycerol, C/B. denitrificans [4]
L2B, Brenta+, C/B. denitrificans [5] L2B, Brenta+[6]
28 additional increase but instead a decrease in rate after the addition of Brenta+ vp1 on day 7.
Setup 5 has a higher rate of nitrate reduction during the first three days of the experiment compared to the nonaerated control, 6, and then a lower rate between days 3 and 7 with the same average rate for setups 5 and 6 during the first week.
Figure 9. The rates of nitrate reduction between sampling points for the nonaerated setups, 3-6 0
2 4 6 8 10 12
L2B, Brenta+, CBE-mix [3]
L2B, Glycerol, C/B.
denitrificans [4]
L2B, Brenta+, C/B.
denitrificans [5]
L2B, Brenta+ [6]
mg/l/day
Nitrate reduction rates for the nonaerated setups in the pilot experiment
day 0-3 Day 3-7 Day 7-10
29
5. Discussion
The present study focused on enhancing the denitrification process in the leachate water treatment plant at Löt avfallsanläggning . C. denitrificans and B. denitrificans were both shown to be able to adapt to and proliferate in sterile-filtered landfill leachate without additives. These are interesting results as the strains have been isolated from sewage and tannery wastewater, respectively, (32,34) and have to the knowledge of the author never been attempted for adaptation to landfill leachate before, which is a media vastly different in nutrient composition.
The CBE-mix also showed the ability to adapt to and proliferate in the sterile leachate water.
These results show that both C. denitrificans, B. denitrificans, and the CBE-mix potentially could be added to the treatment plant where they then would have a chance of adaptation and growth.
C. denitrificans, J. lividum, and an amplified L2B community were all shown to perform complete denitrification in leachate water with Brenta+ vp1 as an electron donor without any additional nutrient additives. B. denitrificans was under these conditions able to reduce nitrate.
The amplified L2B community and J. lividum showed a preference for nitrate reduction over nitrite reduction leading to nitrite accumulation. C. denitrificans instead suggested a preference for nitrite reduction with almost no nitrite accumulation. Combinations of C. denitrificans + the amplified L2B community, as well as a combination of C. denitrificans + B. denitrificans, led to reduced nitrite accumulation compared to the amplified L2B community and B.
denitrificans on their own. The L2B community had the fastest rate of nitrate reduction, followed by B. denitrificans and C. denitrificans, with the mix of the two strains having a rate right in between. J. lividum had the lowest rate of nitrate reduction of the investigated cultures.
The nitrite accumulation in the L2B community could be a result of the experimental setup.
The amplification of the community by aerated cultivation in NB does not select for denitrifies but fast or otherwise competitive growth. The amplified community in the lab could therefore not be directly comparable to the actual treatment plant community. The fact that both C.
denitrificans and B. denitrificans can perform nitrate reduction in leachate water open for the possibility that sewage sludge could be used to augment leachate treatment plants.
It would have been interesting from a bioaugmentation perspective if the isolated strain of J.
lividum had shown similar capabilities of simultaneous nitrification and denitrification proven in J. svalbardensis (40). That was however not the case as the culture augmented with J. lividum had lower rates of ammonium and nitrate removal compared to the control under aerated conditions. This could be due to J. lividum competing with nitrifiers and denitrifiers in the indigenous community for nutrients or that the antibiotics expressed by the species impact the leachate community. The low rate of nitrate reduction in J. lividum was the reason for it not being tested in the pilot study.
The test strips used to measure nitrite could be considered a semiquantitative method with which it was difficult to calculate the exact rate of denitrification. Denitrification rate has thus been described as the rate or nitrate reduction with or without nitrite accumulation. The reduction of nitrate is still a meaningful metric since nitrate reduction is slower than nitrite reduction and thus often the rate-determining step.
The carrier experiment can be considered a pre-study for any further experiment SÖRAB wants to proceed with regarding carrier materials. The community present can form visible biofilm on plastic carriers. The composition of this biofilm and the effect that using carriers would have on nitrogen removal was not investigated. If the combined culture of C. denitrificans + B.
denitrificans had formed biofilm on carriers then that could have been a suitable method of biomass application. Biofilm could be grown onto the carriers during winter. The carriers could then be applied to the treatment plant in spring, this is however not a viable strategy since none
