Sludge Treatment And Disposal Biology Essay


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The residue that accumulates in sewage treatment plants is called sludge (or biosolids). Treatment and disposal of sewage sludge are major factors in the design and operation of all wastewater treatment plants. Two basic goals of treating sludge before final disposal are to reduce its volume and to stabilize the organic materials. Stabilized sludge does not have an offensive odour and can be handled without causing a nuisance or health hazard. Smaller sludge volume reduces the costs of pumping and storage.

Treatment methods

Treatment of sewage sludge may include a combination of thickening, digestion, and dewatering processes.

Thickening is usually the first step in sludge treatment because it is impractical to handle thin sludge, a slurry of solids suspended in water. Thickening is usually accomplished in a tank called a gravity thickener. A thickener can reduce the total volume of sludge to less than half the original volume. An alternative to gravity thickening is dissolved-air flotation. In this method, air bubbles carry the solids to the surface, where a layer of thickened sludge forms.


Sludge digestion may also take place aerobically-that is, in the presence of oxygen. The sludge is vigorously aerated in an open tank for about 20 days. Methane gas is not formed in this process. Although aerobic systems are easier to operate than anaerobic systems, they usually cost more to operate because of the power needed for aeration. Aerobic digestion is often combined with small extended aeration or contact stabilization systems.

Both aerobic and anaerobic digestion convert about half of the organic sludge solids to liquids and gases.

During most of the 114 years since Winogradsky

first isolated N. europaea, microbiologists

considered these autotrophic, aerobic eubacteria

to be primarily responsible for global oxidation

of ammonia. Heterotrophic ammonia oxidizers,

which do not obtain their energy this

way, also contribute to nitrification, but at much

lower rates per unit biomass.

However, that notion changed during the past

decade with discovery of "anammox," an anaerobic

process for oxidizing ammonia that,

coupled to reduction of nitrite, produces nitrogen

gas. Anammox likely contributes significantly

to the global flux of ammonia. In 2005,

investigators described a Crenarchaeota, Nitrosopumilus

maritime, that oxidizes ammonia

to nitrite, adding an archaea to the eubacteria

that support aerobic growth on ammonia. The

Crenarchaeota are widely distributed in soils

and other environments containing ammonia,

suggesting that these organisms are major players

in nitrification.

Determining the genome sequences of representative

organisms that are involved in each of

these processes will provide useful details into

how each of them depends on oxidizing ammonia

to live. Several genome sequences for ammonia-

and nitrite-oxidizing eubacteria are in the

pipeline and could provide insights as to why

some AOB grow more efficiently when exposed

to high levels of ammonia, while others tolerate

only low concentrations; why specific genera of


Nitrogen, a major component of municipal wastewater, stormwater runoff, and industrial wastewater, is potentially toxic to aquatic organisms and plays a role in eutrophication. Nitrogen is an essential nutrient that may be removed through plant uptake. The ammonium and/or nitrate taken up by plants are stored in organic form in wetland vegetation. In addition to the physical translocation of nitrogen compounds in wetlands, the processes involved in nitrogen transformation are ammonification, nitrification, denitrification, nitrogen fixation, and nitrogen assimilation. Ammonification is the microbial conversion of organic nitrogen to ammonia. The energy released in this multistep, biochemical process is incorporated into the microbial biomass. Nitrification is a two-step, microbially mediated transformation of ammonia nitrogen to nitrate. Conversion of ammonium to nitrite by Nitrosomonas bacteria is followed by the oxidation of nitrite to nitrate by Nitrobacter bacteria. Removal of nitrate is by the biological process of denitrification by Bacillus, Enterobacter, Micrococcus, Pseudomonas, and Spirillum. This bioprocess involves the conversion of nitrate to nitrogen gas, thus providing complete removal of inorganic nitrogen from the wetland (ITRC, 2003).

The planted systems showed a constantly good nitrification throughout the whole test period. There was no significant difference between the different plant species (Figure 5). During summer the nitrate content of the effluent decreased because of plant uptake. Harvesting did not influence the N-uptake, but Miscanthus, Phragmites and Iris presented a removal percentage for total nitrogen between 59 to 66%. The other plant species only removed from 31 to 50% of the total nitrogen. The system filled with lightweight expanded clay performed significantly better than the other systems. The average total nitrogen removal was 70.5%, because nitrogen was removed over a much longer period (Figure 7).

The use of hybrid systems seems to be very interesting to enhance nitrogen removal. The removal percentages increased from 70.5 to 80.8% (hybrid system 1) and from 47.2 to 77.0% (hybrid system 2). The removal of total nitrogen can be explained by plant uptake, denitrification or ammanox

Microorganisms Used in Water Treatment

Introduction: Briefly give Reasons to use microorganisms

3 Types of Microorganisms used in Bioremediation

Paracoccus denitrificans


Higher order taxa

Eubacteria (Kingdom); Bacteria (Domain); Proteobacteria; Alpha Proteobacteria (Class); Rhodobacterales (Order); Rhodobacteraceae (Family); Paracoccus (Genus)


Paracoccus denitrificans

Description and significance

Paracoccus denitrificans is a coccoid shaped gram-negative bacteria. They live in the soil in either aerobic or anaerobic environments. They also have the ability to live in many different kinds of media including C1 and sulfur. They can either use organic energy sources, such as methanol or methylamine, or act as chemolithotrophs, using inorganic energy sources with carbon dioxide as their carbon source. Paracoccus denitrificans was first isolate in 1910 by Martinus Beijerinck, a Dutch microbiologist, and was given the name Micrococcus denitrificans. In 1969, D.H. Davis changed the name of the bacteria to its present name because of the discovery that the bacteria contained many features known to be in mitochondria. It is possible that Paracoccus denitrificans is an ancestor to the eukaryotic mitochondria.

Image taken by Richard Evans-Gowing at the University of East Anglia, Norwich, UK.

Genome structure

The genome of Paracoccus denitrificans consists of two circular chromosomes and one plasmid. The first chromosome has 2,852,282 base pairs. The second chromosome has 1,730,097 base pairs and the plasmid has 653,815 base pairs. The plasmid encodes 611 known proteins such as Formyltetrahydrofolate deformylase and TonB-dependent siderophore receptor precursor. These proteins are not essential for the survival of the bacterium; however, the proteins transcribed and translated from the plasmid allow the bacterium to perform many of its metabolic functions. It is what gives Paracoccus denitrificans its unique features, such as the ability to metabolize ammonium to nitrogen gas.

Cell structure and metabolism

The cell structures of Paracoccus denitrificans is similar to those in an eukaryotic mitochondria. It is a gram-negative bacteria and therefore has all the properties typical of a gram-negative bacteria. This includes a double membrane with a cell wall. During the exponential growth phase, it is mainly rod-shaped; however, nearly spherical cells are observed when in the stationary phase. They are able to obtain energy from both organic, such as methanol and methylamine, and inorganic compounds, such as hydrogen and sulfur. A feature of this bacteria is its ability to single-handedly convert nitrate to dinitrogen in a process called denitrification.


Paracoccus denitrificans live primarily in the soil. They produce nitric oxide and nitrous oxide, which gives rise to atmospheric damage. They are also responsible for the loss of nitrogen fertilizer in agricultural soil. They do so by single-handedly converting nitrate into nitrogen gas.


There are no known pathological effects of this bacterium on humans.

Application to Biotechnology

Paracoccus denitrificans produces more than 5000 proteins. Many of these proteins and enzymes are useful in biotechnological applications. One such a application is the construction of a bioreactor, in this case, a tubular gel containing two bacteria, for the removal of nitrogen from wastewater. Paracoccus denitrificans has the unusual ability of reducing nitrite to nitrogen gas. In this bioreactor,Paracoccus denitrificans is paired up with Nitrosomonas europaea, which reduces ammonia to nitrite. This system simplifies the process of removing nitrogen from wastewater.

Current Research

A research study conducted in 2007 looked at the complex formed between cytochrome c and cytochrome c oxidase. The lab used multi-frequency pulse electron paramagnetic resonance spectroscopy to study the complex. It was concluded that there was no set orientation or distance between the two proteins that made up the complex. Another study, conducted in 2007, used Fourier transform infrared spectroscopy to study the effects of pH on the reduced-minus-oxidized FTIR spectra. This research study found two pH dependent processes. A third study on Paracoccus denitrificans, published in 2007, studied the mechanism that reduces NO to N2O. The study concluded that the protons used in these reactions are taken up from the periplasm and not due to a proton electrochemical gradient.


1. "Paracoccus denitrificans". 4 June 2007. <>

2. "CP000491" 4 June 2007 <>

3. Reimann, J., Flock, U., Lepp, H., Honigmann, A., Adelroth, P. "A pathway for protons in nitric oxide reductase from Paracoccus denitrificans." Elsevier 1767.5 (2007), 362-373. 4 June 2007 <>

4. Gorbikova, E., Belevich, N., Wikström, M., Verkhovsky, M. "Protolytic reactions on reduction of cytochrome c oxidase studied by ATR-FTIR spectroscopy": Biochemistry. 46.13 (2007), 4177 - 4183. 4 June 2007 <>

5. Lyubenova, S., Siddiqui, M., Vries, M., Ludwig, B., Prisner, T., "Protein-protein interactions studied by EPR relaxation measurements: cytochrome c and cytochrome c oxidase." J. Phys. Chem. B. 111.14 (2007), 3839 - 3846. 4 June 2007 <>

6. "Paracoccus denitrificans". 4 June 2007. <>

7. Uemoto, H., Saiki, H. "Nitrogen Removal by Tubular Gel Containing Nitrosomonas europaea and Paracoccus denitrificans." Applied and Environmental Microbiology. 62.11 (1996), 4224-4228. 4 June 2007 <>

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Nitrobacter hamburgenesis


Bacteria; Proteobacteria; Alphaproteobacteria; Rhizobiales; Bradyrhizobiaceae; Nitrobacter; Nitrobacter hamburgensis (1)

Description and significance

Nitrobacter hamburgensis, a gram-negative bacteria, was isolated from soil of the Old Botanic Garden in Hamburg and of a corn field in Yucatan. The main types of environments they inhabit are soil, building sandstone, and sewage sludge. Its cells are 0.5-0.8 x 1.2-2.0 m in size. They are mostly pear-shaped and motile via one subpolar to lateral flagellum. Intracytoplasmic membranes appear as caps of flattened vesicles or membrane vesicles in the central region of the cell. (2) The bacteria have an enzyme capable of oxidizing nitrite (3). This is why it is important to sequence the genome of N. hamburgensis.

Genome structure

There is one circular DNA chromosome and three circular DNA plasmids. The chromosome has 4,406,967 nucleotides. Plasmid 1 has 294,829 nucleotides, 2 has 188,318 nucleotides, and 3 has 121,408 nucleotides. (1)

Cell structure and metabolism

N. hamburgensis gains energy from oxidation of nitrite to nitrate via the enzyme nitrite oxidoreductase (NOR). It has a maximum doubling time of 10 to 18 hours. (3)


Nitrobacter hamburgensis is an example of nitrite-oxidizing bacteria. This bacteria has the capability of metabolizing nitrogen in nitrite form in its environment. It is found mainly in soil and freshwater. (4,6,7) (See Current Research section for a summary of the work of Nitrobacter hamburgensis and nitrobacter.)


As of present, there is no evidence for Nitrobacter hambugensis having pathological characteristics.

Application to Biotechnology

The nitrification feature of Nitrobacter hamburgensis has been appreciated. The bacteria has provided a solution to removing high levels of nitrogen from municipal effluents of wastewater treatment plants. Biofilms with different nitrifying bacteria including N. hamburgensis have been constructed. Before the invention of these biofilms very large and expensive reactors were used for this purpose. (4)

(See Current Research section for more on this topic and other specific examples of Nitrobacter hamburgensis' involvement in biotechnology)

Current Research

This section summarizes some of the current research on Nitrobacter hamburgensis. While this organism has long-been described, this species in particular has not sparked current research interest. After exhausting my resources I was only able to find two articles about Nitrobacter hamburgensis that were recently published. The other two articles are about nitrobacter species in general.

Recent research has investigated the benefits of using nitrifying bacteria in neutralizing wastewater. Researchers have constructed biofilms with different nitrifying bacteria including N. hamburgensis. They were successful in removing high levels of nitrogen in a short amount of time from municipal effluents from wastewater treatment plants. The biofilms are sufficient alternatives for the treatment of industrial wastewater that otherwise requires very large and expensive reactors for efficient bioremediation of effluents. (4)

Other current research has identified evidence that the previously published sequence of norX in N. hamburgensis X14(T) contains an invalid base "insertion," which resulted in a frameshift and a misidentified start codon. (5)

Going along the lines of neutralizing wastewater, nitrobacter and another nitrifying bacteria have been found in the Seine River in France. Agricultural and urban pollution result in high concentrations of nitrogen in the Seine River and hence in the waster water treatment plants downstream of the river. Scientists have identified nitrobacter as one of the bacteria responsible for oxidizing nitrite products upstream of the plant (in the freshwater). Nitrobacter was also found as the main bacteria in the waste water effluents. The overall result is nitrified waste water that flows into the sea. (6)

The final research I will summarize describes the quorum sensing of nitrobacter bacteria. Quorum sensing is a term used to define a feature of bacteria that requires a certain number of them for something to happen. For example, researchers have discovered that nitrobacter can oxidize nitrite in soil which has been exposed to diesel fuel for a long period of time. They found that a large population of the bacteria is required for the nitrification to take place. (7)



2. E. Bock et al. 1983. "New facultative lithoautotrophic nitrite-oxidizing bacteria." Archives of Microbiology, vol. 136, no.4. (281-284)

3. Jens Aamand, Thomas Ahl, and Eva Spieck. 1996. "Monoclonal Antibodies Recognizing Nitirite Oxidoreductase fo Nitrobacter hamburgensis, N. winogradskyi, and N. vulgaris." Applied and Environmental Microbiology, vol. 67, no. 7. (2352-5)

4. Franco-Rivera A, Paniaqua-Michel S, Zamora-Castro J. 2007. "Characterization and performance of constructed nitrifying biofilms during nitrogen bioremediation of a wastewater effluent." Journal of industrial microbiology and biotechnology, vol. 34, no. 4. (279-287)

5. Maron PA, Coeur C, Pink C, Clays-Josserand A, Lensi R, Richaume-A Potier. 2006. "Validation of the correct start codon of norX/nxrX and universality of the norAXB/nxrAXB gene cluster in nitrobacter species." Current Microbiology, vol 53, no 3. (255-257)

6. Aurelie Cebron and Josette Garnier. 2005. "Nitrobacter and Nitrospira genera as representatives of nitrite-oxidizing bacteria: Detection, quantification and growth along the lower Seine River (France)." Water Research, vol 39, no 20. (4979-92)

7. Deni J and Penninckx MJ. 2004. "Influence of long-term diesel fuel pollution on nitrite-oxidizing activity and population size of nitrobacter spp in soil." Microbiol Res, vol 159, no 4. (323-329)

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Nitrosomonas europea


Higher order taxa

Domain; Phylum; Class; Order; family [Others may be used. Use NCBI link to find]

Domain: Prokaryota

Phylum: Proteobacteria

Class: Beta Proteobacteria

Order: Nitrosomonadales

Family: Nitrosomonadaceae

Genus: Nitrosomonas

Species: N. europea


NCBI: Taxonomy

Nitrosomonas europaea

Description and significance

Picture of Nitrosomonas europaea. Photo by Stan Watson, Woods Hole Oceanographic Institute.

Nitrosomonas europaea is a Gram-negative chemolithoautroph with the shape of bacillus. It is a nitrite oxidizing bacteria that lives in places rich in ammonia and inorganic salt, such as soil, sewage, freshwater, the walls of buildings and on the surface of monuments. It's often found in the polluted areas where the air contains high levels of nitrogen compounds.

It is important enough to have its genome sequenced because this organism plays a central role in the availability of nitrogen to plants and hence in limiting C02 fixation. These bacteria are important players in the treatment of industrial and sewage waste in the first step of oxidizing ammonia to nitrate. N. europaea is also capable of degrading a variety of halogenated organic compounds, including trichloroethylene , benzene and vinyl chloride, which may make it an attractive organism for bioremediation.

Genome structure

Its genome consists of a single circular chromosome of 2,812,094 bp. The GC skew analysis indicates that the genome is divided into two unequal replichores. Genes are distributed evenly around the genome, with approximately 47% transcribed from one strand and approximately 53% transcribed from the complementary strand. A total of 2,460 protein-encoding genes emerged from the modeling effort, averaging 1,011 bp in length, with intergenic regions averaging 117 bp. Genes necessary for the catabolism of ammonia, energy and reductant generation, biosynthesis, and CO(2) and NH(3) assimilation were identified. In contrast, genes for catabolism of organic compounds are limited. Genes encoding transporters for inorganic ions were plentiful, whereas genes encoding transporters for organic molecules were scant. Complex repetitive elements constitute ca. 5% of the genome. Among these are 85 predicted insertion sequence elements in eight different families. The strategy of N. europaea to accumulate Fe from the environment involves several classes of Fe receptors with more than 20 genes devoted to these receptors. However, genes for the synthesis of only one siderophore, citrate, were identified in the genome. This genome has provided new insights into the growth and metabolism of ammonia-oxidizing bacteria.

Cell structure and metabolism

Nitrosomonas europaea is an autotroph. It can obtain the carbon that it needs to grow by getting it from the atmosphere in a process known as "carbon fixation". Carbon fixation is the process of converting carbon in a gaseous form into carbon bound up in organic molecules. This bacterium contains "carboxysomes" (dark spots which can be seen scattered throughout the cell), which store the enzymes used to fix carbon dioxide for cell carbon.


This nitrifying bacterium is the most studied of the ammonia-oxidizing bacteria that are participants in the biogeochemical N cycle. Nitrifying bacteria play a central role in the availability of nitrogen to plants and hence in limiting CO2 fixation. The reaction catalyzed by these bacteria is the first step in the oxidation of ammonia to nitrate. N.europaea also is capable of degrading a variety of halogenated organic compounds, including trichloroethylene, benzene, and vinyl chloride. The ability of nitrifying organisms to degrade some pollutants may make these organisms attractive for controlled bioremediation in nitrifying soils and waters.

Application to Biotechnology

Conversion of ammonia to dinitrogen in wastewater was tried by Nitrosomonas europaea because Nitrosomonas europaea contains ammonia-oxidizing enzyme, nitrite reductase, and nitrous oxide reductase.

Current Research

The dynamics of growth and death of immobilized Nitrosomonas

europaea were studied. For this, the death rate of suspended

cells was determined in the absence of ammonium or oxygen by following the loss of respiration activity and by fluorescein-diacetate (FDA)/ lissamine-green staining techniques. The death rates obtained in the absence of oxygen or ammonium were incorporated in a dynamic growth model and the effects on the performance of the immobilized-cell process was illustrated by model simulations. These model simulations and experimental validation show that if decay of biomass occurs, the biomass concentration in the center of the bead decreases. As a result, the systems react slower to changes in substrate concentrations than if all cells remain viable.


Patrick Chain et al. (May 2003). "Complete Genome Sequence of the Ammonia-Oxidizing Bacterium and Obligate Chemolithoautotroph Nitrosomonas europaea". Journal of Bacteriology 185: 2759-2773.

John G. Holt, Noel R. Krieg, editor, editor-in-chief (1984) Bergey's manual of systematic bacteriology volume2 page1809- 1825

DOE JOINT GENOME INSTITUTE - US Department of Energy Office of Science

DJW, Microbial of the Week 1999, Nirosomonas europae.

Nagatsuta, Midori-ku, Yokohama "Conversion of ammonia to dinitrogen in wastewater by Nitrosomonas europaea". Department of Bioengineering, Tokyo Institute of Technology


Stacy Heather "Nitrosomonas europaea"

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Psuedomonas putida

Pseudomonas putida 

are Gram-negative rod-shaped bacteria. They are classified as Group 1 in Pseudomona. Other Pseudomonads are being re-evaluated to see if they truly fall into this category, while P. putida is firmly place in this group. P. putida are flourescent, aerobic, non sporeforming, oxidase positive bacteria. Having one or more polarflagella, they are motile organisms. They can be found in moist environments, such as soil and water, and grow optimally at room temperature. Certain strains have the ability to grow on and break down many dangerous pollutants and aromatic hydrocarbons such as toluene, benzene, and ethylbenzene. P. putida can also be used in petroleum plants to purify fuel. This bacterium is also capable of promoting plant growth after root colonization as well as simultaneously providing protection for the plant from pests and other harmful bacteria.

n genetic terms, Pseudomonas putida is very similar to strains of Pseudomonas aeruginosa, an opportunistic human pathogen. Although there is a considerable amount of genome conservation, P. putida seems to be missing the key virulent segments that P. aeroginosa has. Being a non-pathogenic bacteria, there has been only a handful of episodes where P. putida has infected humans. For the most part, it has been with immunocompromised patients, causing septicaemia, pneumonia, urinary tract infections, nosocomial bacteremia, septic arthritis, or peritonitis. P. putida is also closely related to Pseudomonas syringae, an abundant plant pathogen, but again it lacks the gene that causes such disease.

Several cases of disease caused by Pseudomonas putida have been investigated, being that the bacterium rarely colonizes mucosal surfaces or skin. One case was a 43-year-old female who was receiving nightly peritoneal dialysis treatments following a laparoscopic ovarian cyst operation. She developed peritonitis due to infection by Pseudomonas putida. Through this case and others, it was determined that risk factors for developing such an infection include the insertion of catheters, intubation, and/or intravascular devices following a recent course in antibiotics. [2]

Another case of Pseudomonas putida infection was found in ten patients in and ear, nose, and throat outpatient clinic during the summer of 2000. All ten patients had chronicsinusitis, making them more susceptible to infection due to their challenged immune systems. Through investigation, it was discovered that all of the patients shared the same examination room. The source of the bacteria was from a contaminated bottle of StaKleer found in that room. StaKleer is an anti-fog solution used on mirrors and endoscopes to prevent condensation from occurring, allowing for the proper visualization of tissues. Other unopened bottles of the solution at the clinic were found to be contaminated withPseudomonas putida as well.[3]

Where and How used in water treatment

Pseudomonas putida play a huge role in bioremediation, or the removal or naturalization of soil or water contaminants. They can degrade toluene, xylene, and benzene, which are all toxic components of gasoline that leak into the soil by accidental spills. Other strains can convert styrene, better known as packing peanuts, which do not degrade naturally, into the biodegradable plastic polyhydroxyalkanoate (PHA). Methods used to get rid of styrene include incinerating it, spreading it on land, and injecting it underground, all of which release the toxins into the environment. Styrene can cause muscle weakness, lung irritation, and may even effect the brain and nervous system. Due to the fact that P. putida can use styrene as its only source of carbon and energy, it can completely remove this toxic chemical. P. putida can also turn Atrizine, an herbicide that is toxic to wildlife, into carbon dioxide and water

Nitrosomonas europa and eutropha - clean wastewater


Aerobic or Anaerobic,

Where and How used in water treatment


Microorganisms Used in Water Treatment

Dirty Water

Groundwater is below the soil surface and while this may seem to be of some distance the water is greatly affected by fertilizers and contaminants on the surface which leaches through to groundwater surface. However, there are microorganisms that have been used to treat contaminated ground water to make it less harmful as it makes its way to waterways. Arsenic is one such contaminant that the used to clean drinking water supplies and Ground water

Types of Organisms Used in remediation

Contaminants in Drinking Water (EPA)

Pathogenic Microorganisms

Disinfectants and their byproducts

Organic Chemicals

Inorganic Chemicals


Microorganisms used to biodegrade wastewater and sewage

Many types of bacteria and protozoa are commonly used in the biodegradation of organic material and the removal of contaminants in the secondary treatment of wastewater. Nitrosomonas and Nitrobacter are two types of bacteria commonly used to remove nitrates. Excess nitrates have the potential to cause serious harm to humans and to marine life as well.

Microorganisms used to clean up pollution

Eutrophication and Hypoxia- "Worldwide, the number of coastal areas impacted by eutrophication stands at over 500. In coastal areas, occurrences of dead zones, which are caused by eutrophic conditions, have increased from 10 documented cases in 1960 to 405 in 2008."(World Resource Institute)

Oil Spills

Types of Organisms Used to remediate

Microbes used to revitalize exhausted water by the introduction of previously inhabited microbes or to stimulate microbes to proliferate Conclusion

Bioremediation of Eutrophicated Water by Acinetobacter


Anammox stands for anaerobic ammonia oxidation and the organisms responsible were relatively recently discovered, in the late 1990s.[8] This form of metabolism occurs in members of thePlanctomycetes (e.g. Candidatus Brocadia anammoxidans) and involves the coupling of ammonia oxidation to nitrite reduction. As oxygen is not required for this process these organisms are strict anaerobes. Amazingly, hydrazine (N2H4 - rocket fuel) is produced as an intermediate during anammox metabolism. To deal with the high toxicity of hydrazine, anammox bacteria contain a hydrazine-containing intracellular organelle called the anammoxasome, surrounded by highly compact (and unusual) ladderane lipid membrane. These lipids are unique in nature, as is the use of hydrazine as a metabolic intermediate. Anammox organisms are autotrophs although the mechanism for carbon dioxide fixation is unclear. Because of this property, these organisms could be used in industry to remove nitrogen in wastewater treatment processes.[9] Anammox has also been shown have widespread occurrence in anaerobic aquatic systems and has been speculated to account for approximately 50% of nitrogen gas production in the ocean.[10]

Microorganisms used to clean drinking water supplies and ground water

The EPA estimates that the average annual baseline illnesses and deaths associated with viruses in ground water are about 185,000 and 3, respectively ("Federal Rule", 2006). The EPA currently monitors the contaminants often found in drinking water such as pathogenic microorganisms, disinfectants and their by-products, organic and inorganic chemicals and radionuclide. Waterborne pathogens can enter water sources through many entryways and accordingly the EPA suggests the threat of these contaminants may contribute to cancer, kidney failure,

Microorganisms Capable of Removing Toxic Heavy Metals (Srivastava, & Majumder, 2008)

Table 2

Bacterial species Metal

Rhodospirilium species

Cd Hg Pb Ni

Chatterjee [28]

Gallionella feruginea

As Mn Fe

Katsoyiannis and Zouboulis [29]

Leptothrix species

As Mn Fe

Katsoyiannis and Zouboulis [29]

Pseudomonous species

Cr As

Valls et al. [30]

Desulfovibrio species

Cu Zn Ni Fe

As Jong and Pany [31]

Thiomonous species

As Fe Casiot

et al. [32]

Escherichia coli

Hg Ni Deng

et al. [33]

Thauera selenatis

Zn Cd Co Cu Ni Pb Cr Hg

Mergray et al. [34]

Alcaligenes faecalis


Phillips and Taylor [35]

Fungal species

P. Chrysogenum

Zn Cu Ni As

Loukidou et al. [36]

Aspergillus niger

Ni Cu Pb Cr

Dursun et al. [37]

Coriolus hersutus


Miyata et al. [38]

Trametes versicolor

Cr Co

Blanquez et al. [39]

Mucor rouxi

Pb Cd Zn Ni

Yan and Viraraghavan [40]

Algal species

Brown algae

Cd Cu Zn Pb Cr Hg

Davis et al. [41]

Green algae

Cu Hg Fe Zn Pb Cd

Haritonidis and Malea [42]

Scenedesmus genus

Cu Ni Cd Cr Cu

Pena-Castro et al.

Works Cited

Environmental Protection Agency, Standards and Risk Management Division, Office of Ground Water and Drinking Water. (2006). National primary drinking water regulations: ground water rule. Federal Rule ( 40 CFR Parts 9, 141, and 142). Washington, DC, Retrieved from:

Srivastava, N.K., & Majumder, C.B. (2008). Novel biofiltration methods for the treatment of heavy metals. Journals of Hazardous Material, (151(1):1-8.), Retrieved from

Selmon, W. & Jones, C. (2010) Water Quality: Eutrophication and Hypoxia, World Resource Insitution Retrieved /17/2010 from:

Bioremediation of Eutrophicated Water by Acinetobacter


NOTES and thoughts

Genetically Modified Organisms

Heavy Metal Consuming Orgs

Carbon Consumers

Petroleum eating microorgs

Phospate reducers

Those helpful in nitrification Nitrosomanos and dentifrication are Nitrobacter

Wastewater treatment and disposal

The predominant method of wastewater disposal in large cities and towns is discharge into a body of surface water. Suburban and rural areas rely more on subsurface disposal. In either case, wastewater must be purified or treated to some degree in order to protect both public health and water quality. Suspended particulates and biodegradable organics must be removed to varying extents. Pathogenic bacteria must be destroyed. It may also be necessary to remove nitrates and phosphates (plant nutrients) and to neutralize or remove industrial wastes and toxic chemicals.

The degree to which wastewater must be treated varies, depending on local environmental conditions and governmental standards. Two pertinent types of standards are stream standards and effluent standards. Stream standards, designed to prevent the deterioration of existing water quality, set limits on the amounts of specific pollutants allowed in streams, rivers, and lakes. The limits depend on a classification of the "maximum beneficial use" of the water. Water quality parameters that are regulated by stream standards include dissolved oxygen, coliforms, turbidity, acidity, and toxic substances. Effluent standards, on the other hand, pertain directly to the quality of the treated wastewater discharged from a sewage treatment plant. The factors controlled under these standards usually include biochemical oxygen demand (BOD), suspended solids, acidity, andcoliforms.

There are three levels of wastewater treatment: primary, secondary, and tertiary (or advanced). Primary treatment removes about 60 percent of total suspended solids and about 35 percent of BOD; dissolved impurities are not removed. It is usually used as a first step before secondary treatment. Secondary treatment removes more than 85 percent of both suspended solids and BOD. A minimum level of secondary treatment is usually required in the United States and other developed countries. When more than 85 percent of total solids and BOD must be removed, or when dissolved nitrate and phosphate levels must be reduced, tertiary treatment methods are used. Tertiary processes can remove more than 99 percent of all the impurities from sewage, producing an effluent of almost drinking-water quality. Tertiary treatment can be very expensive, often doubling the cost of secondary treatment. It is used only under special circumstances.

Sludge treatment and disposal

The residue that accumulates in sewage treatment plants is called sludge (or biosolids). Treatment and disposal of sewage sludge are major factors in the design and operation of all wastewater treatment plants. Two basic goals of treating sludge before final disposal are to reduce its volume and to stabilize the organic materials. Stabilized sludge does not have an offensive odour and can be handled without causing a nuisance or health hazard. Smaller sludge volume reduces the costs of pumping and storage.

Treatment methods

Treatment of sewage sludge may include a combination of thickening, digestion, and dewatering processes.


Thickening is usually the first step in sludge treatment because it is impractical to handle thin sludge, a slurry of solids suspended in water. Thickening is usually accomplished in a tank called a gravity thickener. A thickener can reduce the total volume of sludge to less than half the original volume. An alternative to gravity thickening is dissolved-air flotation. In this method, air bubbles carry the solids to the surface, where a layer of thickened sludge forms.


Sludge digestion is a biological process in which organic solids are decomposed into stable substances. Digestion reduces the total mass of solids, destroys pathogens, and makes it easier to dewater or dry the sludge. Digested sludge is inoffensive, having the appearance and characteristics of a rich potting soil.

Most large sewage treatment plants use a two-stage digestion system in which organics are metabolized by bacteria anaerobically (in the absence of oxygen). In the first stage the sludge is heated and mixed in a closed tank for about 15 days, while digestion takes place. The sludge then flows into a second tank, which serves primarily for storage and settling. As the organic solids are broken down by anaerobic bacteria, carbon dioxideand methane gas are formed. Methane is combustible and is used as a fuel to heat the first digestion tank as well as to generate electricity for the plant. Anaerobic digestion is very sensitive to temperature, acidity, and other factors. It requires careful monitoring and control.

Sludge digestion may also take place aerobically-that is, in the presence of oxygen. The sludge is vigorously aerated in an open tank for about 20 days. Methane gas is not formed in this process. Although aerobic systems are easier to operate than anaerobic systems, they usually cost more to operate because of the power needed for aeration. Aerobic digestion is often combined with small extended aeration or contact stabilization systems.


Candidatus Brocadia anammoxidans (Ca. B. anammoxidans) are aquatic autotrophs best known for their unique ability to anarobically oxidize ammonia to dinitrogen gas, a reaction that has been patented and is otherwise known as the "anammox" reaction (5). The bacteria were first discovered in a wastewater treatment plant in the Netherlands where it was observed that ammonia concentrations dropped while dinitrogen gas concentrations rose in airtight effluent reactors (5). Ca. B. anammoxidans were subsequently held responsible for this process and were deemed to be the first bacteria to demonstrate anaerobic ammonia oxidation abilities (5). The bacteria were isolated from enrichment cultures by density centrifugation and have received ample attention from ecologists who suspect the bacteria's participation in consuming substantial amounts of nitrogen in the ocean, and from researchers who see the bacteria's metabolism as a potential in revolutionizing wastewater treatment (3, 8). These microbes are no bigger than one micron in diameter and grow optimally in a pH range of 6.4-8.3 and in a temperature range of 20-43 oC (6, 9). The bacteria is named as such, as Candidatusindicates an uncultureable yet well-characterized organism, Brocadia refers to the Gist-Brocades, the place of its discovery, and anammoxidans describes the process of anaerobic ammonium oxidation (3, 10).

Genome structure

The genome of Ca. B. anammoxidans has yet to be sequenced. The unculturable nature of the bacteria (and of its closest relatives) has made it difficult to ascertain its genomic particulars. Researchers have found through PCR, however, the complete sequences of the 16S ribosomal RNA gene, tRNA-Ile gene, and tRNA-Ala genes, and a partial sequence of the 23S RNA gene. The known sequences of these genes total 4032 contiguous base pairs. The size of the genome, the shape and number of chromosomes, and the presence or absence of plasmids are not yet known (7).

Cell structure and metabolism

Ca. B. anammoxidans is a spherical bacterium that lacks peptidoglycan, a common compound found in most microbial cell walls, and displays small cavities known as 'crateriform structures' on its surface. The organism also exhibits a compartmentalized cytoplasm -- a rare find in bacteria. The anammoxosome, one of the cellular compartments, comprises 30-60% of the cell volume and is arguably the most integral structure Ca. B. anammoxidans possesses, as it plays the chief role in the bacteria's unique metabolic process (3).

Nitrogen tracer studies have shown that Ca. B. anammoxidans obtain their energy by anaerobically combining ammonia and nitrite to produce dinitrogen gas (3, 6):

NH4+ + NO2- → N2 + 2H2O

The reaction, which takes place inside the ammoxosome, yields and requires two toxic intermediates, hydroxylamine (NH2OH) and hydrazine (N2H4, otherwise known as rocket-fuel). These intermediates serve as electron generators for the initial step of the anammox reaction, the reduction of nitrite (2, 3). The safe containment of the noxious intermediates would be impossible were it not for the unparalleled molecular structure of the anammoxosome membrane. Lipids composed of five linearly cis-linked cyclobutane rings make the membrane unusually dense, limiting the diffusion of hydroxylamine and hydrazine. The diffusion limiting aspect of the membrane not only protects the rest of the cell from the toxic intermediates, but also prevents a substantial decrease in the bacteria's metabolism. From a bioenergetics perspective, if one molecule of hydrazine diffuses through the anammoxosome, a 50% decrease in the catabolic activity will ensue. The bacteria have low growth rates to begin with; in an optimal environment, they double once every eleven days at best (9).


Ca. B. anammoxidans plays a substantial role perpetuating the nitrogen cycle in the ocean. Nitrogen tracer studies and calculations have demonstrated that Ca. B. anammoxidans consume 20-40% of inorganic nitrogen that drops into the suboxic zones of the ocean (4). The bacterium use nitrite as the electron acceptor to anaerobically oxidize ammonia to dinitrogen gas, which promotes the growth and productivity of aquatic organisms by limiting the amount of inorganic nitrogen found in the ocean. It is impossible, however, for Ca. B. anammoxidans to carry this reaction out in oxic ocean areas. The anammox bacteria are extremely sensitive to concentrations of oxygen (as low as 2 μM) and will terminate metabolic processes upon oxygen sensation (4). Albeit this limitation, Ca. B. anammoxidanscan cooperate with aerobic ammonium oxidizing bacteria to carry out the anammox reaction if it finds itself in slightly oxygenated marine areas. For example, at the oxic/anoxic ocean interface, Ca. B. anammoxidans cooperates with members of the genus Nitrosomonas. The Nitrosomonas bacteria aerobically oxidize ammonia to nitrite and suppress oxygen concentrations, while Ca. B. anammoxidansaccepts the nitrite and combines it with ammonia to produce dinitrogen gas anaerobically (3, 4).


Ca. B. anammoxidans is not known to cause any disease.

Application to Biotechnology

The performance of the anammox reaction by Ca. B. anammoxidans has revolutionized wastewater treatment. Before the discovery of Ca. B. anammoxidans, effluent treatments were carried out by aerobic bacteria that were obligated to perform nitrification in conjunction with denitrification in order to free the wastewater of ammonia (5). Furthermore, the long aerobic process necessitated an expensive supply of methanol (5). The use of the anammox reaction of Ca. B. anammoxidans in wastewater treatment eliminates these substantial inconveniences. The anaerobic removal of ammonia from wastewater by Ca. B. anammoxidans leads to a faster treatment and a 90% reduction in operational costs, as the anammox process bypasses the denitrification step of the nitrogen cycle completely and does not require expensive methanol as fuel (3). Effluent cure is arguably the most pragmatic application of Ca. B. anammoxidans, and is the frequent study of modern anammox research (11, 12).

Current Research

1) A recent phylogenic study at the University of Queensland was conducted to investigate the similarities and differences of the primary and secondary genetic sequences of Candidatus Brocadia anammoxidans and Candidatus Kuenenia stuttgartiensis. The two planctomycetes' ribonuclease P RNA gene sequences and secondary structures were compared and found to be identical in helix number. In phylogenetic studies, the two bacteria were repeatedly found to be closely related to Gemmata obscuriglobus, a sister planctomycete. It was verified that P13, a unique bacterial helix insert was found in only these three planctomycetes. This study has been deemed significant as it shows that since the helix insert is exclusively shared between the three planctomycetes, it can be deduced that there is a shared common ancestor for ribonuclease P RNA molecules among these three species (1).

2) Recent and significant improvements on the quality of wastewater treatment with Ca. B. anammoxidans have been made through new anammox reactor developments. The newly developed anammox reactors yield a nitrogen removal rate of 25 kg-N m-3 per day, a number that triples the current nitrogen removal rate, 8.7 kg-N m-3 per day. The bacteria were also able to double in an unprecedented 3.6-5.4 days -- a period that is less than half the doubling time of the bacteria mentioned in current reports. According to the study, the increased rate of nitrogen removal and bacterial doubling is attributed to special inter-reactor fabric sheets and a high total nitrogen loading rate. These two improvements allow for a higher density of anammox bacteria (70% of total bacteria) to be used in the new effluent reactors. The newly developed non-woven fabric sheets are used as biofilm carriers and effectively keep the bacteria active inside the reactor, and the high total nitrogen loading rate encourages internal substrate transport, which plays a part in encouraging anammox bacteria division. These improvements made in effluent reactors promise a more effective means of anaerobic wastewater treatment (12).

3) A study in Hokkaido University, Japan, is innovating current quantification methods of Ca. B. anammoxidans by quantifying the bacteria using a real-time polymerase chain reaction (PCR). The quantification of the bacteria has traditionally been carried out using fluorescence in situ hybridization (FISH) but the method has proved to be impractical. Anammox bacteria have low counts of rRNA molecules per cell and tend to form heavy clusters. These obstacles make FISH difficult to perform. The use of real-time PCR for quantification is shown in the study to be a more convenient and advantageous method for anammox bacteria enumeration. The real-time method is better adapted to quantify dense microbial clusters and is sensitive enough to quantify the slow-growing Ca. B. anammoxidans in an uncultured environment, as it is based on continual fluorescent monitoring. Real-time PCR was used to quantify the 16S rRNA gene of anammox bacteria subsequent to the development of specific PCR primers for an enrichment culture of anammox bacteria from a rotating disk reactor biofilm (11). The autotrophic and obligate anaerobic nature of Ca. B. anammoxidans makes culturing the bacteria very difficult, but the quantification of the bacteria's 16S rRNA gene using real-time PCR sheds light on the intricate details of the bacteria's physiology and kinetics, which is an important step in isolating Ca. B. anammoxidans in pure culture (11).

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