POTENTIAL USE OF DENITRIFYING BACTERIA IN REDUCTION OF

NITROGENOUS COMPOUNDS FROM THE EFFLUENT DISCHARGED INTO

LAKE VICTORIA, KENYA
Thesis presented by:

James M. Wachira (BSc)

Supervisors:

Prof. Romano Mwirichia

University of Embu
Dr. Cargele Masso
International institute of Tropical Agriculture/Icipe
Dr. Moses Thuita
International Institute of Tropical Agriculture/ Icipe
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 Background information

 Globally, human population/activities contribute to the rate at which nitrogen enters the terrestrial
ecosystem.
 Uncontrollable release of nitrogenous compounds- ecological damage-eutrophication.
 Coastal/marine ecosystem, eutrophication-production of water hyacinth, green macroalgae blooms,
toxin-producing algal blooms
 Bacteria play a central role in Nitrogen cycle: Nitrogen fixers, Nitrifying bacteria, Denitrifying
bacteria
 Bioremediation HN-AD process proposed as alternative process– efficient, space and cost, eco-
friendly, methods of nitrate removal

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 Statement of the problem

 Eutrophication in Lake Victoria basin-Kisumu-Nutrient enrichment

 Toxic cyanobacteria blooms in the lake- anoxia and hypoxia, harmful to aquatic life (fish)
and health risk.
 Aquatic weeds, water hyacinth
 Continuous nutrient loading- poor water quality- negative affects economic-aesthetic-
ecological functions
 Less treated through mechanical, physical or biological treatment techniques
 Research on the microbial relative abundance of potential aerobic denitrifiers

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 Justification

 New perception on total microbial diversity and potential aerobic denitrifiers

 Overcome the gap created by unsuitable traditional techniques by:
 Space and cost- single reactor-environmental friendly.
 HN-AD- effective and efficient for nitrogenous compounds utilization, NO3- &O2
 Reduction of fish mortality rate and improve water quality.

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General objective:
To assess the potential of aerobic denitrifying bacteria in the removal of nitrogenous compounds from
wastewater entering Lake Victoria

Specific objectives:
1. To assess the total diversity of microorganisms within wastewater discharged into Lake Victoria
2. To isolate and characterize aerobic denitrifying bacteria from the wastewater using cultural and
molecular approaches
3. To evaluate the nitrogenous compounds removal potential of different aerobic denitrifying
bacteria

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 Materials and Methods

1. To assess the total diversity of microorganisms within

wastewater discharged into Lake Victoria
 Study site Lake Victoria {0.7558° S, 33.4384° E}{Kisumu,
0.0917° S, 34.7680° E}
 Sample collection A total of eight samples of water collected
randomly
 Stored in ice-cooled boxes and transported to the lab
 Chemical analysis of the samples SGS Kenya Limited
Laboratory Services
• Nitrate-APHA-4500 Nitrite-APHA 4500.Ammonium-APHA-4500

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 Molecular diversity of the bacteria within the sample
 TMC DNA extraction, air dried, resuspended, gel, DNAstable® Biomatrica, air dried, shipped- MR DNA LAB,
Shallowater, TX, USA)
 PCR amplification-16S rRNA gene V4-V7 variable region-barcoded primers 515F/806R
 PCR products checked in 2% agarose gel-success/quality
 Illumina TruSeq DNA library Protocol- prep DNA library- pooled & purified PCR products
 Sequencing completed on Miseq 2*300 bp Version 3- Mnf’s Gdlns
 Q25 Sequence data processed-MR DNA ribosomal and functional gene analysis pipeline.
 OTUs picked at 3% divergence-97% similarity.
 Taxonomic assignment of OTU-BLASTn-curated database derived from NCBI

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 Analysis of sequence data

 R software version 3.6.2 and Microsoft Excel to generate plots

 Species richness-Ace, Chao1, Shannon and Simpson Alpha diversity indices-OTUs, using microbiomeSeq,
phyloseq and vegan R packages

 Heatmaps comparing bacterial abundance at the genus level in different sampling sites using the gplots package

 Beta diversity-PCA done- investigate ecological distance of sampling sites AmpViz2 package

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 2. To isolate and characterize aerobic denitrifying bacteria from the wastewater using
cultural and molecular approaches
Isolation and culturing

 Effluent samples (50 ml), screening medium(100 ml) sterilized 500 ml Erlenmeyer flask

 Shaken at 150 rpm, 30 ̊C, 72 hours to enrich denitrifying bacteria

 The enrichment samples were serially diluted up to 10 folds.

 100 µl from 108, 109, and 1010 spread plated on solid BTB plates, incubated, 30 ̊C for 48 hours.

 Colonies picked ,purified by repeated streaking on fresh solid Bromothymol blue plates -P isolates

 Incubated at 30 ̊C for 24 hours

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 Screening for nitrogenous compounds utilization

 Single colony of each bacteria inoculated into 100 ml of SM-150 rpm, 30 ̊ C, 48 hours
Nitrification, (NH4)2SO4
Denitrification, NaNO3- Nitrogen sole source

 Ammonium oxidation-Phenate method. Phenol solution+nitroprusside catalyst and hypochlorite. Indophenol+ammonia.

WL640 nm

 Nitrite-N-(1-naphthyl)-ethylene diamine photometry(NEDA) method. Nitrite and sulphanilamide-diazo

compound+NEDA-red azo dye. WL543 nm

 Nitrate-Phenol Disulphonic Acid (PDA) method. Nitrate+PDA=nitro derivative, yellow color in alkaline solution.
WL410 nm.

 Each treatment performed in triplicates-one-way ANOVA- measure average mean of absorbance. SSl p<0.05.

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 Molecular characterization of the isolates

 DNA extraction- Phenol chloroform protocol (Sambrook et al., 1989).

 DNA amplification- 8F and 1492R primers

 Purification of PCR products- Purified via QIAquick PCR purification kit protocol- Gel

 DNA Sequencing- Inqaba Biotech, South Africa-8F and 1492R to sequence PCR products

 Similarity level to existing type strains was done on EzBioCloud Server (Yoon et al., 2017)

 Phylogenetic analysis-Molecular Evolutionary Genetics Analysis (MEGA) version X (Kumar et al., 2018)

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 3. To evaluate the nitrogenous compounds removal potential of different aerobic denitrifying bacteria

Assessment of nitrogenous compounds utilization from Lake and sewage wastewater

 A single colony inoculated in 20 ml sterilized wastewater, 50 ml falcon tubes

 Incubation done at 150 rpm at 30 ̊ C for 7 days

 Ammonium oxidation-Phenate method. WL640 nm

 Nitrite-N-(1-naphthyl)-ethylene diamine photometry(NEDA) method. WL543 nm
 Nitrate-Phenol Disulphonic Acid (PDA) method. WL410 nm.

 Each treatment performed in triplicates- One-way ANOVA- measure average mean of absorbance. SSl p<0.05.

12
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 Results
Chemical Analysis
level of pollution/nutrient loading-nitrogen

Sample origin     Rivers   Treated Untreated   Lake     Safe/Normal Range

WWTP  
WWTPs

Parameters Method Units (J1) (J8) (J7) (J2) (J4) (J3) (J5) (J6)
         
River River Kisat Lagoon Homabay Homabay Dunga A Dunga B
Nyando WWTP B
Sondu WWTP WWTP
    Latitude 0° 22' 46" 0° 0' 57" 0° 4' 57" 0° 28' 35" 0° 31' 27" 0° 30' 41" 0° 8' 43" 0° 8' 20"
Sampling GPS                  
points Longitud 34°49‘ 21" 35° 16' 32" 34° 44' 58" 34°30'42" 34°26'53" 34° 28' 34" 34°44'13.2" 34°44'11"
e
pH APHA 4500 H+   6.27 6.78 6.76 6.71 7.46 7.42 7 6.44
TN- 0.3 mg·L rivers/ 0.1
Total Nitrogen Calculation mg/l 1.23 1.34 24.64 18 45.43 1.57 3.36 3.92 mg/l in lakes (US EPA)
TDS APHA 2540 C mg/l 45 144 661 553 1308 264 276 242
Nitrite as N APHA-4500-NO2 mg/l <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 Ammonia-0.02 mg/L-Some
fish(US EPA)
Chloride APHA-4500-CL mg/l 7.88 12.32 62.09 60.12 146.35 10.35 9.36 11.83
B
BOD
Ammonia as N APHA-4500- mg/l <0.02 <0.02 19.49 10.77 38.69 <0.02 2.84 2.55 1-2 mg/L-Clean water
NH3 F
3-5 moderate clean
Nitrate as N APHA-4500-NO3 mg/l 0.11 0.22 <0.01 0.08 0.07 0.45 <0.01 <0.01 6-9 slightly polluted.
100 or greater-very polluted
BOD5 @ 20º C APHA 5210 mg/l 45 30 153 60 126 63 73 183
with organic waste (CIESE)

13
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 Alpha Diversity Index Analysis

 Boxplots showing Alpha diversity comparisons between

three sample types.
 Diversity increase from WWTPs to Lake
• Lake-richest bacterial density
• WWTPs-least bacterial density- elevated levels of
pollutants than rivers and lake waters (Wang et al., 2012)

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 Alpha Diversity Index Analysis
Alpha-diversity comparisons between samples using 4 Alpha diversity indices

Sample ID Site No. of sequences OTUs Chao1 Ace Simpson Shannon

J1 River Sondu 23,730 1,202 1361.56 1355.19 0.99 8.53

J2 Lagoon WWTP 29,531 883 1097.03 1055.39 0.96 6.94

(Untreated)
J3 Homabay Beach 23,596 1,241 1456.78 1401.15 0.99 8.30
J4 Homabay WWTP 29,354 732 1022.38 1050.56 0.96 6.28
(Untreated)

J5 Dunga site A 29,380 995 1199.48 1191.98 0.97 6.50

J6 Dunga site B 18,205 1,033 1368.41 1392.83 1.00 8.53
J7 Kisat WWTP 29,518 702 1032.31 945.88 0.98 7.11
(Treated)
J8 River Nyando 24,610 1,138 1343.11 1301.36 0.99 8.03

Sequence: OTUS
Min: 18,205 Min: 702
Max: 29,531 Max: 1,241

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 Beta diversity analysis

 Similarity and dissimilarity of different microbial

communities composition-PCA
 Similar color codes-correspondence
 Lake, untreated WWTPs, and rivers samples clustered
together, separately.
 Untreated WWTP ecologically far apart from the treated
WWTP

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 Microbial community diversity

 A total of 1763 OTUs spread across 26 bacterial phyla

 Proteobacteria(B)- accounted for almost 59% of all
bacterial sequences
 Bacteroidetes(O)
 Firmicutes(G)
 Detected in all the samples (Paiva et al., (2015)

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 Microbial community diversity

100%
90%
80%  Proteobacteria-Betaproteobacteria (B)
70%
Alphaproteobacteria (R)- favored by pH and nutrients
60%
(Newton et al., 2011)
50%
40%
30%
 Gammaproteobacteria (P)- perform denitrification process
20%
in biological wastewater treatment plant (Lu H et al.,
10% 2014)
0%
d u) d o) TP
)
TP
)
TP
)
_B
) A) _B
)
 Bacteriodetes, Bacteroidia (Gn) and Sphingobacteria
on an W W W y y_ y
S y ba Ba Ba
e r rN n _W t _W y _W m
a
ga ga (Dg)-Treatment of the wastewater. (Niestępski et al.,2020)
iv iv
e o isa a o un un
(R (R ago (K ab (H (D (D
J1 J8 (L J7 o m J3 J5 J6
J2 (H
J4  Nitrospira (N cycle)-0.6% in sample J6 to 0.0% in samples
Acidobacteria Acidobacteriia Holophagae Actinobacteria J2, J4, J5 and J7- nitrifying bacterium(Mehran et al.,
Armatimonadia Bacteroidia Cytophagia Flavobacteriia 2020).
Sphingobacteriia Chlamydiia Chlorobia Anaerolineae
Caldilineae Chloroflexia Dehalococcoidia Cloacimonetes
Cyanobacteria Oscillatoriophycideae Deferribacteres Deinococci  Nitrospira-Negatively correlated to high ammonia
Elusimicrobia Endomicrobia Fibrobacteria Bacilli concentration (Sun et al., 2020)
Clostridia Erysipelotrichia Negativicutes Fusobacteriia
Gemmatimonadetes Ignavibacteria Lentisphaeria Nitrospira
Phycisphaerae Planctomycetia Alphaproteobacteria Betaproteobacteria
Deltaproteobacteria Epsilonproteobacteria Gammaproteobacteria Oligoflexia
Spirochaetia Synergistia Mollicutes Opitutae
Verrucomicrobiae
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 Microbial community diversity

Heat map showing the relative abundance of the major genera

• Proteobacteria- Dechloromonas spp detected in all

samples-denitrification (Coates et al., 2001)
• Bacteriodetes-Bacteroides spp-nitrates reduction
capabilities (Jewell et al., 2017).
• Planktothrix agardhii toxin (Lagoon WWTP)-producing
strain of Cyanobacteria- nutrient loading/ environmental
parameters- high BOD (Monteagudo et al., 2016). .
• Cyanobacteria-bioindicator for monitoring eutrophic
ecosystem (Soltani et al., 2012).

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 Screening for Nitrogen removal Percentage mean of nitrogenous compounds utilizatio
basal media
Each isolate was screened for the removal of nitrogenous
compounds from basal media

Average mean of absorbance

  Nitrate Nitrite
Sample ID Ammonium
1
JM2 0.0243 0.0240 0.0227
2
JM4 0.078 0.0923 0.0567
3
JM5 0.0753 0.0747 0.0933
4
JM7 0.1050 0.07030 0.0850
5
JM10 0.0553 0.0827 0.0950
6
JM11 0.1063 0.0643 0.0133
7
JM14 0.0446 0.0533 0.0757
8
JM15 0.0530 0.0853 0.0333
9
JM19 0.0370 0.0263 0.0720
10
JM22 0.0333 0.0417 0.0337

Control
Ammonium: 0.52
Nitrate: 0.352
Ammonium 95.32% (JM2) to 79.55% (JM11)
Nitrite: 0.428
Nitrate 93.18% (JM2) to 73.78% (JM4)
Nitrite 94.69% (JM2) to 77.8% (JM10)
20
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 Assessment of nitrogenous compounds Percentage mean of Nitrogenous compounds utilization
utilization from the wastewater in wastewater

Average mean of absorbance

  Nitrate Nitrite
Sample ID Ammonium
1
JM2-HB 0.0377 0.3337 0.1857
2 JM4-HB 0.0547 0.2743 0.1757
3
JM5-HB 0.0687 0.2653 0.1650
4
JM7-HS1 0.0660 0.3843 0.1860
5 JM10-HS 0.0750 0.2647 0.1453
6 JM11-KS 0.0600 0.0643 0.2033
7
JM14-LS 0.0540 0.3253 0.2863
8
JM15-LS 0.0753 0.3140 0.3743
9 JM19-LS 0.0327 0.3477 0.2733
10
JM22-DB 0.0643 0.2363 0.2270

  Control    
Sample ID Ammonium Nitrate Nitrite
Ammonium: 93.03% (JM2) to 66.67% (JM10)
        Nitrate: 41.51% (JM22) to 11.15% (JM11)
Kisat_WWTP(KS) 0.214 0.4333 0.347
Nitrite: 89.09% (JM11) to 8.41% (JM15)
Homabay_Beach(HB) 0.22467 0.4097 0.38667
Homabay_WWTP(HS) 0.22567 0.44 0.224
Lagoon_WWTP(LS) 0.28667 0.4287 0.40867
Dunga_Beach(DB) 0.21533 0.404 0.319 21
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 Molecular characterization Phylogenetic analysis of 16S rRNA gene libraries
Sample Sample Related Type Strain Type Strain Similarity to Sequence
Name Accession Type Length
Accession
Number Number Strain (bp).

JM2 ON227428 Klebsiella quasivariicola KPN1705 99.84 670

JM4 ON227429 Pseudomonas citronellolis NBRC 100 457
103043
JM5 ON227430 Pseudomonas mosselii CIP 105259 100 468
JM6 ON227431 Pseudomonas mosselii CIP 105259 100 531
JM7 ON227432 Enterobacter cloacae subsp. LMG 2683 99.86 715
dissolvens
JM8 ON227433 Enterobacter cloacae subsp. LMG 2683 100 850
dissolvens
JM10 ON227417 Pseudomonas monteilii NBRC 100 788
103158
JM11 ON227418 Klebsiella aerogenes KCTC 2190 100 834
JM12 ON227419 Enterobacter cloacae subsp. LMG 2683 100 751
dissolvens
JM13 ON227420 ATKM_s P818 100 759
JM14 ON227421 Klebsiella variicola subsp. SB5531 99.84 626
tropica
JM15 ON227422 Enterobacter roggenkampii EN-117 100 660
JM16 ON227423 ATKM_s P818 99.11 803
JM17 ON227424 Pseudomonas mosselii CIP 105259 100 568
JM18 ON227425 Klebsiella variicola subsp. SB5531 99.87 801
tropica
JM19 ON227426 Pseudomonas mendocina NBRC 14162 100 710

JM22 ON227427 Pseudomonas asiatica RYU5 100 748

Isolates obtained through culture dependent methods and their Two clusters: 1st: Enterobacter and Klebsiella
similarities to known type strains 2nd Pseudomonas members
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 CONCLUSION

 Freshwaters had higher bacterial densities than WWTP, and this could be attributed to elevated levels of organic and inorganic
pollutants present in WWTPs

 Proteobacteria was the richest phylum that was largely represented by Betaproteobacteria, Alphaproteobacteria and
Gammaproteobacteria

 Based on culture-independent 16S rRNA gene sequencing, all isolated bacteria were closest matches to Gammaproteobacteria.

 Members of Gammaproteobacteria are well adapted to high environmental stressors and possess high biodegradation capacity.

 This unique feature makes Gammaproteobacteria more efficient in the bioremediation of contaminated sites.

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 RECOMEDATIONS/Take-Home Message

 Further investigation is necessary to identify and characterize more promising strains that are competent to carry
out the SND process.
 Several eutrophic coastal and marine waterbodies should be sampled at different seasons of the year to comprehend
the microbial density and activity in various water reservoirs in a given period of time.
 Empirical investigation on all potential microorganisms isolated from eutrophic ecosystems, including whole
genome sequencing and amplification of nitrogen cycle genes.
 In situ bioremediation techniques such as biostimulation remediation technique can be applied to boost aerobic
degradation of microbes involved in SND process.
 Potential isolates should be preserved and utilized to remove extra nitrogen from wastewater effluents.

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 Acknowledgement

Prof. Moses Thuita

Prof. Romano Mwirichia
Dr. Cargele Masso

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 THANK YOU
FOR YOUR
ATTENTION!!

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