Monday, December 19, 2022

Types of microbes in aquatic ecosystem

 An aquatic ecosystem includes freshwater habitats like lakes, ponds, rivers, oceans and streams, wetlands, swamp, etc. and marine habitats include oceans, intertidal zone, reefs, seabed. The aquatic ecosystem is the habitat for various animals, plants, and microbes.

 

Freshwater Microflora- Freshwater Microbial Diversity

  Freshwater environments provide excellent habitats for microorganisms. Large numbers of microorganisms in a body of water generally indicate high nutrient levels in the water. Water contaminated by inflows from sewage systems or from biodegradable industrial organic wastes is relatively high in bacterial numbers. Freshwater environments are highly variable in the resources and conditions available for microbial growth. Both oxygen producing and oxygen consuming organisms are present in aquatic environments, and the balance between photosynthesis and respiration controls the natural cycles of oxygen, carbon, and other nutrients (nitrogen, phosphorus, metals).

Neuston

Neuston is the uppermost layer of hydrosphere-it is the interface between hydrosphere and atmosphere and is occupied by phototrophic microorganisms. Organisms float on the top of the water -(Epineuston) or live right under the surface - (Hyponeuston) Primary producers are abundant here because of the availability of unrestricted light, carbon dioxide & mineral nutrients. Secondary producers also proliferate here. Microbial numbers in the surface layer are 10 to 100 fold higher than the underlying water column. Bubbles arising from the neuston layer burst out liberating bacteria and other microorganisms to air.  

Autochthonous (native) neuston microbiota include algae, bacteria, fungi and protozoa. Common bacteria are Pseudomonas, Caulobacter, Achromobacter, Flavobacterium, Alcaligenes etc. Gram positive and negative, pigmented and non-pigmented, motile and non-motile, rod and cocci, stalked and un-stalked forms seen. Common blue green algae or Cyanobacteria include Anabaena & Microcystis. Filamentous fungi like Cladosporium and various yeasts, algae like Nautococcus, Chromulina, and protozoa like Vorticella, Arcella etc. are present in neuston.

 

 Other Freshwater Microflora           

A variety of microorganisms live in fresh water. The region of a water body near the shoreline (the littoral zone) is well lighted, shallow, and warmer than other regions of the water. Photosynthetic algae and bacteria that use light as energy flourish in this zone. Further away from the shore is the limnetic zone. Areas of the limnetic zone with sufficient oxygen contain bacteria like Pseudomonads and species of Cytophaga, Caulobacter, and Hyphomicrobium. Photosynthetic algae are also located in the limnetic zone. 

Deeper waters of the profundal and benthic zones have low oxygen concentrations and less light. Algal growth near the surface often filters the light, and photosynthetic microbes in deeper zones  use different wavelengths of light from those used by surface-layer photosynthesizers. Purple and green sulfur bacteria are found in the profundal zone. These bacteria are anaerobic photosynthetic organisms that metabolize H2S to sulfur and sulfate in the bottom sediments of the benthic zone. Finally, at the bottom of fresh waters is the benthic zone containing the sediments, where few microbes survive. Bacteria that can survive in the absence of oxygen and sunlight, such as methane producing bacteria, thrive here. Clostridium species are common in bottom sediments and may include botulism organisms, particularly those causing outbreaks of botulism in waterfowl. 

Microbial photosynthesizers mainly include algae and cyanobacteria . Others feed on these organisms, forming the next link in the food chain . Plant material from the land also enters lakes and streams at their edges, providing an important nutrient source for many water bodies. Decomposers form an especially important part of fresh-water ecosystems because they consume dead bodies of plants, animals, and other microbes. These microbial agents of decay are an important part of the ecosystem because they convert detritus (dead and decaying matter) and organic materials into needed nutrients, such as nitrate, phosphate, and sulfate. Decomposers  are essential to the major biogeochemical cycles by which nutrients are exchanged between the various parts of the ecosystem, both living and nonliving.

 Aerobic decomposers in water need oxygen to survive and do their work which is ensured by the flowing water and waves. If there is not enough oxygen in the water, many parts of the system suffer-the aerobic decomposers cannot digest plant matter, insects cannot develop and mature, and the fish cannot grow properly. Eventually, the stream or pond will be changed, starting at the microbial level. Human interaction can jeopardize parts of this system in a variety of ways.

Fresh water is host to numerous microorganisms that affect human health directly.  Polluted drinking water is a major source of illness and death throughout the world, particularly in developing countries. Some common microbes in lakes and streams that are responsible for disease include:

· The protozoa Giardia lambliafound in fresh-water bodies throughout the world. Giardiasis is a common waterborne illness.

· The bacterium Vibrio choleraeremains a significant source of disease and death .

· The bacterium Escherichia coli, is a very common waterborne pollutant. Humans have a large and harmless population of E. coli in their large intestines, and bacteria make up a large fraction of the volume of human feces. When released into drinking water or recreational water sources, E. coli can be ingested causing diarrhea. 

Thus, many microorganisms are found naturally in fresh water including bacteria, cyanobacteria, protozoa, algae and tiny animals such as rotifers. These can be important in the food chain that forms the basis of life in the water.

 


 Biodiversity of the marine environment

On Earth, formed 4.6 billion years ago, life appeared in the oceans about 3.8 billion years ago. And it was only very recently, about 400 million years before our era, that it conquered the land. As a result, extremely diverse lifestyles have developed in the oceans, where light only penetrates the upper layers, gradually adapting to the specific conditions of this marine environment. Most of the biodiversity on our planet is found in the marine environment but most of these species, especially those living in deep water, are still unknown.

 Marine Microflora

Neuston and pleuston are organisms that live near the surface of a water body. The small aquatic organisms inhabiting the surface layer or moving on the surface film, are neuston and the organisms that live at the air-water interface is the pleuston. These organisms are exposed to harsh environmental conditions such as high-temperature variations, high-light variations, including UV irradiation, marine, and aerial predators, etc. . 

Marine bacteria

Pelagibacter ubique and its relatives may be the most abundant organisms in the ocean, and they are possibly the most abundant bacteria in the world. They make up about 25% of all microbial plankton cells, and in the summer they may account for approximately half the cells present in temperate ocean surface water.

The largest known bacterium, the marine Thiomargarita namibiensis, can be visible to the naked eye and sometimes attains 0.75 mm (750 μm).

Marine bacteria perform all kinds of chemical processes in the open ocean, including most of the steps in nitrogen cycling. Cyanobacteria are a large group of photosynthetic bacteria, they “fix” nitrogen, converting nitrogen gas into more biologically useful compounds.

Trichodesimium is one of the most important and well-studied nitrogen-fixing cyanobacteria found in open-ocean areas such as Station ALOHA. It is one of the few organisms involved in the oceanic nitrogen cycle that is visible to the naked eye. Other cyanobacteria in open-ocean areas is Richelia, which is found living inside diatoms, a type of marine algae. Uncultivated cyanobacteria group A (UCYN-A) is a group of nitrogen-fixing cyanobacteria that cannot perform photosynthesis and possibly form “partnerships” (symbioses) with other photosynthetic organisms.

Proteobacteria are an extremely diverse group of bacteria. Some alphaproteobacteria and gammaproteobacteria fix nitrogen. Other important ones are the ammonium oxidizing bacteria (AOB) such as betaproteobacteria and gammaproteobacteria involved in nitrification and nitrite oxidizing bacteria including Nitrobacter, Nitrospira, and Nitrospina etc. Photosynthetic cyanobacteria which do not fix nitrogen include Prochlorococcus, Synechococcus.

Marine archaea are also common in oceans, including ammonium oxidizing archaea (AOA) in the group Crenarchaeota. These archaea are common in the deeper parts of the open ocean, where there are little light and oxygen concentrations are relatively low.

 Archaea are extremophiles living in harsh environments, such as the yellow archaea in a hot spring, but they are also found in a much broader range of habitats. Archaea are particularly numerous in the oceans and may play roles in both the carbon cycle and the nitrogen cycle. Thermoproteota (also known as eocytes or Crenarchaeota) are a phylum of archaea thought to be very abundant in marine environments and one of the main contributors to the fixation of carbon. Eocytes may be the most abundant of marine archaea. Halobacteria, found in water near saturated with salt, are now recognised as archaea. Methanosarcina barkeri, is a marine archaea that produces methane. Marine thermophiles, such as Pyrolobus fumarii, survive well over 100 °C.

 Redtide

Red tide is a colloquial term used to refer the natural phenomena harmful algal blooms (HABs), (or excessive algae growth) that causes negative impacts to other organisms by production of toxins, mechanical damage to other organisms, or by other means. After the bloom dies, the microbes that decompose the dead algae use up more of the oxygen, generating a "dead zone" which can cause fish death. When these zones cover a large area for an extended period of time, neither fish nor plants are able to survive. HABs are induced by eutrophication, which is an overabundance of nutrients (nitrogen and phosphate) in the water. The excess nutrients are emitted by agriculture, industrial pollution, excessive fertilizer use in urban/suburban areas, and associated urban runoff.

The harmful effects from such blooms is due to the toxins they produce or from using up oxygen in the water which can lead to fish die-offs. Some only discolor water, producing a smelly odor, or adding a bad taste to the water. There are three main types of phytoplankton which can form into harmful algal blooms: cyanobacteria, dinoflagellates and diatoms.  Some cyanobacteria, such as Microsystis, can produce hazardous cyanotoxins such as microcystins, which are hepatotoxins that harm the liver of mammals. Other types of cyanobacteria can also produce hepatoxins, as well as neurotoxins, cytotoxins, and endotoxins. Diatoms and dinoflagellates (in marine coastal areas) also cause HAB. Diatoms produce domoic acid, another neurotoxin, which can cause seizures in higher vertebrates and birds as it concentrates up the food chain. Domoic acid  accumulates in the bodies of shellfish, sardines, and affect the nervous system of the consumers such as sea lions, otters, cetaceans, birds or people causing serious injury or death. Blooms of harmful algae can have large and varied impacts on marine ecosystems, depending on the species involved, the environment where they are found, and the mechanism by which they exert negative effects.

Redfield ratio

 In 1934, Alfred Redfield discovered that the ratio of carbon to nitrogen to phosphorus is a nearly constant 106:16:1 throughout the world's oceans, in both phytoplankton biomass and in dissolved nutrient pools. Redfield noticed that the ratio between the quantities of Carbon, Nitrogen and Phosphorus constituting the healthy oceanic phytoplankton, as well as the Nitrogen and Phosphorus in the waters of healthy seas remained close to this value. Thus, "Redfield Ratio" is maintained as the optimal ratio between Carbon, Nitrogen and Phosphorus in natural aquatic ecosystems.

 Marine viruses

Marine phages parasite marine bacteria and archaea, such as cyanobacteria. They are the most abundant biological entity in marine environments. Tailed phages of the order Caudovirales, non-tailed viruses, Phages belonging to the families Corticoviridae, Inoviridae, Microviridae are also known to infect diverse marine bacteria. There are also archaean viruses which replicate within archaea and giant viruses such as the giant mimivirus and the largest known virus, Tupanvirus, as marine microflora.

Microorganisms make up about 70% of the marine biomass. There are 15 times as many viruses in the oceans as there are bacteria and archaea. The viruses kill 20% of microbial biomass and harmful algal blooms. Viruses are an important natural means of transferring genes between different species, which increases genetic diversity and drives evolution.

 Marine protists

Protists are eukaryotes that cannot be classified as plants, fungi or animals. They are usually single-celled and microscopic. Common examples are red and brown algae, diatoms, some dinoflagellates, foraminiferans, radiolarian, some marine amoebae, ciliates and flagellates, slime moulds and slime nets. Diatoms have glass like cell walls made of silica. Diatoms generate about 20% of world oxygen production. Coccolithophores are minute unicellular photosynthetic protists with two flagella for locomotion. Most of them are protected by a shell covered with  circular plates or scales called coccoliths, made from calcium carbonate.

 Single-celled alga, Gephyrocapsa oceanica,and groups of small-sized algae- Zoochlorellae or zooxanthellae -that live inside freshwater protozoans and invertebrate hosts like coral are common in marine environments. They have a symbiotic association with the host and use carbon dioxide, nitrogenous, phosphorous waste, and provide oxygen and essential nutrients to the host. Most protists are single-celled and microscopic. Some single-celled marine protists are macroscopic. Spiculosiphon oceana, a unicellular foraminiferan has an appearance and lifestyle that mimics a sponge. Xenophyophore, another single-celled foraminiferan, lives in abyssal zones. Giant kelp, a brown algae, is not a true plant, but is multicellular and can grow to 50m. 

Marine fungi

Over 1500 species of fungi are known from marine environments. These are parasitic on marine algae or animals, or are saprobes feeding on dead organic matter from algae, corals, protozoan cysts, sea grasses, and other substrata. Marine fungi can also be found in sea foam and around hydrothermal areas of the ocean. Many unusual secondary metabolites is produced by marine fungi. The aquatic fungi play a significant role in heterotrophic mineralization and nutrient cycling. Lower fungi  adapted to marine habitats include mastigomycetes: oomycetes and chytridiomycetes. Higher fungi are filamentous hyphomycetes, ascomycetes, basidiomycetes.

 Lichens are mutualistic associations between a fungus, usually an ascomycete, and an alga or a cyanobacterium. Several lichens are found on rocks in marine environments or covering sea snails. Fossil marine lichens 600 million years old have been discovered in China

 Estuaries

An estuary is a partly enclosed coastal body of water with one or more rivers or streams flowing into it, and opening to the sea.

 Microbial communities

Bacteria

Bacteria are the most numerous organisms in the estuary. Sediments and salt marsh soil generally harbor more bacteria per unit volume than does the water column. Aerobic and facultative anaerobic bacteria are most common, and Pseudomonads and Vibrio are the most often isolated species. Higher bacteria densities have been found in most estuaries than in nearby coastal seawater and river water 

Fungi

The number of fungi living in estuaries is extremely large. Some of fungi are unique in estuaries, while others have a broader range of habitats. Aquatic fungi and yeast dominate species in aquatic environment, few of fungi associate with particles or solid matters in the water. In sediments, the active species of fungi primarily are found in surface aerobic zones. The densities of fungi decrease rapidly with soil depth, but the spores of fungi are found throughout sediments 

Most of the bacterioplankton are closely related to surrounding freshwater or marine bacterial groups and belong to the phyla ProteobacteriaBacteroidetes, and Actinobacteria. Cyanobacteria play an important role as primary producers,  Oscillatoriales, chroococcoid colonies and  Synechococcus-like Cyanobacteria are prevalent. Methanogenic Archaea are important for the mineralization of organic matter in anoxic estuarine environments. Sulfate-reducing bacteria often outcompete methanogens for hydrogen and acetate in estuarine sediments.

 

Carbon & Nitrogen cycling

Bacteria show a variety of metabolic pathways related to carbon flow and cycling. Photosynthesis is mainly carried out by algae and phytoplankton in estuarine. As many of the sediment and water-logged soils of estuaries are anoxic, anaerobic decomposition is important. Complex organic matter is used by the fermenters and dissimilatory nitrogenous oxide reducers.

Nitrogen is a major limiting nutrient for primary production in estuaries. The N-cycling processes that are dominated by microbial activity include nitrification, dissimilatory nitrous oxide reduction, and nitrogen fixation. Nitrogen cycling in estuaries is related to the water mixing and microbial community dynamics.

Water movement is the dominant controlling factor in estuarine ecosystem. Circulation stimulates fluxes of dissolved constituents and particulate materials such as sediments, detritus, bacteria, and plankton. In Estuaries, salt water mixes with water derived from land drainage. The estuarine circulation movements are the primary mechanism of mixing. 

Much of the organic matter carried to an estuary by rivers, produced by phytoplankton, or derived from marshes, is deposited on the sediment surface. Oxygen is the most important electron acceptor in organic matter respiration, but at the water column of anerobic estuarine or saturated sediment sulfate become more significant electron acceptors. The major product of sulfate reduction is hydrogen sulfide, which gives the habitat a pungent smell. 

Autotrophic nutrients are important for the functional estuarine ecosystems, because they are the raw materials for the primary producers. The concentrations of these nutrients change in estuaries due to the mixing of river and ocean water. Microbial heterotrophic activity and primary production play very important roles in the formation and turnover of organic matter in eutrophic estuaries

Friday, December 16, 2022

DNA Sequencing- Maxam–Gilbert Method


DNA sequencing is the process of determining the sequence of nucleotide bases (As, Ts, Cs, and Gs) in a piece of DNA. 

Sequencing an entire genome (all of an organism’s DNA) is complex. It requires breaking the DNA of the genome into many smaller pieces, sequencing the pieces, and assembling the sequences. New methods have been developed over the past two decades, that make genome sequencing much faster and less expensive

History

Watson and Crick discovered the structure of DNA in the year 1953. In 1964, Richard Holley performed the sequencing of the tRNA as the first attempt to sequence the nucleic acid.

Using the technique of Holley and Walter Fieser, they sequenced the genome of bacteriophage MS2 (RNA sequencing). The sequenced molecules were RNA, yet DNA sequencing was not performed.

In the year 1977, Fredrick Sanger postulated the first method for sequencing the DNA, named a chain termination method.

In the same year, the chemical method of DNA sequencing was explained by Allan Maxam and Walter Gilbert. The genome of bacteriophage X174 was sequenced in the same year using the chemical degradation method.

Because of the lack of automation, both the methods (chemical degradation and chain termination) were tedious and time-consuming.

The first semi-automated DNA method was developed by Lorey and Smith in the year 1986. In 1987, the Applied Biosystem had developed a fully automated machine-controlled DNA sequencing method. After the development of fully automated machines, the era of the 2000s become a golden period for sequencing platforms.

Furthermore, in 1996, Applied Biosystem developed another innovative sequencing platform known as capillary DNA sequencing. After that, the human genome project was completed by using the combination of these methods in the year 2003.

A fast, accurate, reliable, and highly efficient next-generation sequencing platform was developed in the year 2005 by Solexa/Illumina.



Steps in DNA sequencing

       Sample preparation (DNA extraction)

       PCR amplification of target sequence

       Amplicons purification

       DNA Sequencing

       Data analysis

Different methods of DNA sequencing:

Various methods of DNA sequencing

       Maxam and Gilbert method

       Chain termination method

       Automated method

       Pyrosequencing

       Whole-genome shotgun sequencing method

       Next-generation sequencing method

 

Two main methods are widely known to be used to sequence DNA:

  1. The Chemical Method (also called the Maxam–Gilbert method).
  2. The Chain Termination Method (also known as the Sanger dideoxy method).

Maxam–Gilbert technique is chemical cleavage method (depends on the relative chemical liability of different nucleotide bonds) whereas the Sanger method is the chain termination method (interruption of elongation of DNA sequences by incorporating dideoxynucleotides into the sequences)

The chain termination method is the method more usually used because of its speed and simplicity.


Chemical Cleavage Method (Maxam–Gilbert Method)

Maxam–Gilbert Method

Maxam and Gilbert method was developed in 1977. It is also referred to as a chemical cleavage method. The single-stranded DNA is cleaved at the specific location with the help of the chemicals and the fragments of DNA is then run on polyacrylamide gel.

       DNA extraction is the first step. After that, the DNA is denatured using the heat denaturation method and single-stranded DNA is generated.

       The phosphate (5’ P) end of the DNA is removed (by alkaline phosphatase enzyme) and radiolabeled with P32 (by polynucleotide kinase).

       4 different chemicals are used to cleave DNA at four different positions; hydrazine and hydrazine NaCl are selectively attack pyrimidine nucleotides while dimethyl sulfate and formic acid attack purine nucleotides. The modified DNAs may then be cleaved by hot piperidine

      Hydrazine: T + C

      Hydrazine NaCl: C

      Formic acid: A + G

      Dimethyl sulfate: G

         An equal volume of 4 different ssDNA samples is taken into 4 different tubes each containing these 4 different chemicals. The samples are incubated for some time and electrophoresed in polyacrylamide gel electrophoresis.

      A series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule

      Fragments electrophoresed in polyacrylamide gel electrophoresis for size separation.

      To visualize the fragments, the gel is exposed to X-ray film for autoradiography (due to radiolabelled 32P end of the DNA-a series of dark bands each showing the location of identical radiolabeled DNA molecules.

      From presence and absence of certain fragments the sequence may be inferred




Important features

       Base-specific cleavage of DNA by certain chemicals

       Four different chemicals, one for each base

       A set of DNA fragments of different sizes

       DNA fragments contain up to 500 nucleotides

Advantages

       Purified DNA can be read directly

       Homopolymeric DNA runs are sequenced as efficiently as heterogeneous DNA sequences

       Can be used to analyze DNA protein interactions (i.e. footprinting)

       Can be used to analyze nucleic acid structure and epigenetic modifications to DNA

Disadvantages

       It requires extensive use of hazardous chemicals.

       It has a relatively complex set up / technical complexity.

       It is difficult to analyze more than 500 base pairs.

       The read length decreases from incomplete cleavage reactions.

       It is difficult to make Maxam-Gilbert sequencing based DNA kits.

However, the method is more accurate than Sanger sequencing. It is more advantageous over the Sanger method because the purified DNA is directly used for sequencing.

It’s best suitable for DNA footprinting and DNA structural studies.  It is used in automated techniques for DNA fingerprinting and genetic engineering studies.


Anoxygenic photosynthesis with reference to photosynthesis in green bacteria and purple bacteria

Phototrophs use light energy to generate a proton motive force (PMF), which is then used to synthesize ATP –this process is called photophos...