Wastewater Report Reference
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Gram-Positive Phyla: Firmicutes, Actinobacteria
Acidogenic and Acetogenic Bacteria
Gram-Negative Bacteria Phyla
These are gram negative bacteria that include many environmental bacteria, especially freshwater and marine bacteria. This phyla includes anaerobic, aerobic and facultative bacteria (facultative means they can survive in aerobic or anaerobic environments). Many bacteria important in wastewater treatment fall into the Proteobacteria phyla including ammonia oxidizing bacteria, sulfate-reducing bacteria, and phosphorus accumulating bacteria.
Bacteroidetes is a very common phyla of gram negative environmental bacteria that includes both aerobic and anaerobic bacteria. One of the most well-studied genera in this phyla is Bacteroides a very common inhabitant of the human large intestine. The presence of Bacteroides in particular likely indicates the presence of fecal matter from warm-blooded animals and possibly humans.
Cyanobacteria are gram-negative bacteria that obtain most or all of their energy from sunlight and typically utilize inorganic carbon to build cell materials. These bacteria are commonly called blue-green algae, though they are actually bacteria, not eukaryotic algae. Most “algal blooms” are caused by Cyanobacteria. Some of these bacteria such as Microcystis contain neurotoxins. As a result, algal blooms can be dangerous to mammals. Algal blooms are typically triggered by warm, stagnant water and high nutrients (nitrogen and phosphorus). These blooms produce oxygen during daylight but consume oxygen during night-time and as cells die and decay. This decay can lead to oxygen-starved waters like those found in the Gulf of Mexico dead zone which is inhospitable to fish and most life forms.
Nitrospirae are a phyla of gram-negative bacteria that are important in the nitrogen cycle. These bacteria are very important in wastewater treatment and other freshwater and marine environments where they oxidize nitrite to nitrate and play a crucial role in removing ammonia from wastewater. These bacteria are able to compete well at low concentrations of nitrite and oxygen. In the past, it was thought that Nitrobacter was the primary nitrite-oxidizing bacteria found in wastewater treatment plants. This mistake was based upon culture studies that were able to grow Nitrobacter from wastewater samples. However, scientists have more recently learned (based on DNA methods) that Nitrobacter is not typically common in wastewater treatment plants and doesn’t compete well at low concentrations of nitrite even though it grows well in culture (which usually contains high concentrations of nitrite).
Synergistetes are a phyla of bacteria involved in methane production. These bacteria are frequently symbionts with methanogenic archaea.
Chloroflexi are a somewhat new and unstudied phyla. Some of these bacteria may be important filaments in wastewater treatment.
Verrucomicrobia are a somewhat new and unstudied phyla. These bacteria are frequently correlated with cyanobacterial blooms in freshwaters and likely consume simple carbohydrates released from cyanobacteria or other sources. They tend to be fast growing bacteria.
Planctomycetes are a unique phyla of bacteria that are ovoid and reproduce by budding. This phyla includes most or all of the bacteria capable of performing anammox (anaerobic ammonia oxidation) in wastewater treatment which is a novel method to remove ammonia with substantially lower energy consumption than conventional processes that rely on nitrifiers and denitrifiers.
Gram-Positive Bacteria Phyla
Firmicutes are gram-positive and are generally rod-like (bacilli) or round (cocci) cells. Many Firmicutes produce endospores that are resistant to drying and can survive extreme conditions. One example is Clostridia tetani, the bacteria that survives in soil and can cause tetanus. Firmicutes are important in beer, wine and cider spoilage. The primary subgroups of Firmicutes include the Clostridia class which are strictly anaerobic and the Bacilli which are obligate or facultative aerobes. In wastewater treatment, the presence of Firmicutes may indicate anaerobic conditions and could also indicate the presence of fermenting bacteria which are important for the production of volatile fatty acids necessary for biological phosphorus removal.
Actinobacteria are gram-positive and are frequently soil-dwelling bacteria, though a major portion of freshwater bacteria also fall into this phyla. Actinobacteria are known for producing secondary metabolites (compounds not directly necessary for the cell’s survival) such as antibiotics and they are also known for degrading complex organics as part of the natural decay process. Streptomyces is one example which is famous for producing the majority of antibiotics used in medicine. In wastewater treatment, these bacteria are mostly known for causing problems such as filamentous bulking and foaming. Microthrix and Gordonia (aka Nocardia) are the two most-well known problematic Actinobacteria. The Actinobacteria are known for creating filamentous forms that can cause settling issues in wastewater treatment plants.
Fermenting bacteria produce volatile fatty acids (such as acetic acid) necessary for biological phosphorus removal. Fermentation can also occur upstream in sewer systems that have extended residence times. Fermenting bacteria are generally anaerobic bacteria. Figure 1 demonstrates the steps involved in fermentation in wastewater. The first step may be hydrolysis of large insoluble organics with extracellular microbial enzyme to break down the organics and produce soluble compounds that can be taken into a cell. The next step is production of volatile fatty acids in anaerobic conditions.
Phosphorus accumulating organisms (PAOs) are Proteobacteria with a unique metabolic ability to take up and store volatile fatty acids (VFAs) such as acetic acid during anaerobic conditions. The PAOs consume polyphosphate for energy during anaerobic conditions and take up organics and store them as PHA (polyhydroxyacetate). They then consume PHA for energy during aerobic conditions and take up phosphate to store energy as a polyphosphate granule.
The Microbe Detectives table includes several groups of bacteria important in nitrogen processes. The first group labelled SND includes bacteria important in simultaneous nitrification and denitrification. This is a unique process which is usually operated at low dissolved oxygen concentrations (<0.5 mg/L) which results in aerobic conditions in the outside of the floc but anoxic conditions inside, allowing these bacteria to both oxidize ammonia aerobically and perform nitrate reduction in anoxic conditions. This process can save energy over conventional nitrification-denitrification if the bacteria utilize the nitrite shunt, in which they oxidize ammonia to nitrite then reduce nitrite to nitrogen gas without ever producing nitrate. It’s essentially a shortcut.
The second group are AOBs (ammonia oxidizing bacteria). These bacteria are autotrophs (i.e. fix inorganic carbon into organic carbon) that obtain energy by oxidizing ammonia to nitrite. Nitrosomonas is the most common of these bacteria.
The third group are NOBs (nitrite oxidizing bacteria). These bacteria are autotrophs (i.e. fix inorganic carbon into organic carbon) that obtain energy by oxidizing nitrite to nitrate. Nitrospira is the most common of these bacteria in wastewater treatment plants.
The next group includes nitrogen fixing bacteria such as Bradyrhizobium. The presence of these bacteria may indicate nitrogen-limiting conditions (i.e. plenty COD and P but not enough N to produce proteins for growth).
Next are the denitrifiers. Thauera is the most well-known denitrifier in wastewater treatment systems. However, denitrification is not a truly unique function. Many aerobic bacteria are capable of this metabolic function.
The final group are anammox bacteria. These are very unique bacteria capable of oxidizing ammonia and reducing nitrite in anaerobic conditions. This is the most energy efficient method of removing ammonia in wastewater treatment. These bacteria are very slow growing and are typically present only if retention times are in excess of 50 days. As a result, biofilm retention is one method of keeping these bacteria in a system.
Foaming bacteria are typically caused by the presence of FOG (fats, oils and grease). Most of these bacteria are mycolic-acid producing bacteria in the Acidomicrobiales or Actinomycetales orders of Actinobacteria. They are hydrophobic bacteria (water repellant) and thus naturally cling to air bubbles present in wastewater. As a result they float to the surface where they entrap air into bubbles and create a foamy broth that can become several feet thick. This foam layer can overflow basins, creating high effluent TSS, permit violation and a difficult mess to clean up.
Table 1 indicates the potential cause of each possible filament.
Filamentous bacteria are necessary for bridging between flocs to create a strong floc and blanket necessary for clear effluent. However, an abundance of filaments can prevent flocs from coalescing, resulting in a condition called bulking in which the sludge blanket does not thicken well. This condition can limit plant hydraulic capacity and cause high effluent TSS if the blanket goes over the clarifier weir.
Table 1. Causes of Filaments
|Bacterial Genus||Description/Potential Cause|
|Zoogloea||Slime, typically caused by low nutrients|
|Thiothrix||Type 021N, Sulfur|
|Acinetobacter||Type 1863, FOG (fats, oils, grease)|
|Microthrix||FOG (fats, oils, grease); can also produce foam|
|Runella||Type 0411; septicity/organic acids|
|Gordonia||Nocardia; FOG (fats, oils, grease)|
|Haliscomenobacter||Low DO, Low P|
|Trichococcus||Nostocoida Limnicola II, septicity/organic acids|
|Tetrasphaera||Nostocoida Limnicola I; Septicity/organic acids|
|Isosphaera||Nostocoida Limnicola III; Septicity/organic acids|
|Caldilinea||Eikelboom Type 803; Low F:M|
|Skermania piniformis||PTLO – Pine tree like organisms, can cause foam; possibly FOG (fats, oils, grease)|
Iron oxidizing bacteria consume reduced iron for energy in aerobic conditions, typically producing a rust color in the water. These bacteria can also play a role in corrosion. Gallionella is one of the most well-known iron oxidizing which is frequently found in drinking water wells. The presence of iron oxidizing bacteria indicates the presence of dissolved iron.
The presence of sulfate reducing or sulfur oxidizing bacteria indicates the presence of sulfur. Sulfur can hinder ammonia removal and can also cause filamentous issues such as Thiothrix (aka 021N). Sulfate reduction can also produce hydrogen sulfide gas which may cause aesthetic issues due to the rotten egg odor. The presence of sulfur-associated bacteria could increase corrosion in certain environments such as sewers as these bacteria oxidizing hydrogen sulfide in the head space and produce corrosive sulfuric acid
Hydrolytic bacteria convert raw feedstocks into smaller organic molecules that can be used by other microbial groups. The majority of hydrolytic bacteria are in the two phyla Firmicutes and Bacteroidetes, which were well represented in this study. Hydrolytic bacteria are more robust in response to environmental change than methanogens (Venkiteshwaran 2016). Other studies (Sundberg, 2016, Carballa 2015, Vanwonterghem 2014) found Clostridia, a different family of Firmicutes, were abundant contributors to hydrolysis. In this study, no single Clostridia group contributed more than 1% to the total, but when combined, Clostridia accounted for 7% of all sequences observed. Clostridia were especially abundant (>15%) in municipal samples from Fond du Lac, Region of Waterloo and industrial samples from Paper Mill 1.
Acidogenic and Acetogenic Bacteria
Acidogenic and acetogenic bacteria form acetate and other organic acids from the products of hydrolysis.
Firmicutes, including Clostridia, and Bacteroidetes along with Proteobacteria include acidogens and acetogens. Because the ability to perform hydrolysis, acidogenesis, and acetogenesis are widespread among bacteria and many groups can perform different functions depending on existing conditions, it is not feasible to identify which bacterial types are performing these preliminary metabolic steps.
Methanogens produce methane from acetate or hydrogen. Methanogenesis can only be accomplished by archaea; no known bacteria can produce methane. Three primary methanogen groups were observed in this study: Methanobacterium spp., Methanothermobacter spp., and Methanosaeta spp. The first two are hydrogenotrophic, meaning that they get their energy from hydrogen produced by other microbes in the digester. Methanosaeta spp. is acetotrophic, meaning that it gets energy from organic acids, and typically dominates the methanogenic community at low acetate concentrations. Methanothermacter spp. prefers high temperatures and was primarily found in thermophilic reactors in this study. Generally, thermophilic reactors also have a higher ratio of Methanobacterium spp. to Methanosaeta spp., indicating that syntrophic hydrogen metabolism is more favorable at high temperatures.