Role of Microbes in Ruminants

 Microbe-Animal Interaction - Role of Microbes in Ruminants 

Ruminants 

Ruminants, are mammals of the suborder Ruminantia (order Artiodactyla), which includes the Cow, sheep, giraffe, deer, antelope, moose, goat etc. Most ruminants have four-chambered stomach. The four chambers of ruminant stomach are named as Rumen, Reticulum, Omasum and Abomasum.  

Ruminant animals do not completely chew the grass or vegetation they eat. The partially chewed grass goes into the large rumen, which is the largest section and the main digestive center, where it is stored and broken down into balls of “cud”.

Cud is a portion of food that returns from a ruminant's stomach to the mouth to be chewed for the second time. It is a bolus of semi-degraded food regurgitated from the rumen. The rumen is filled with billions of tiny microorganisms that are able to break down grass and other coarse vegetation that animals with one stomach (including humans, chickens and pigs) cannot digest. 

e.g. Cellulose, which can be digested by the cellulase enzyme produced by the microbes. 

When the animal has eaten its fill, it will rest and “chew its cud”. That means, they later regurgitate the cud, and chew it again to further break down into smaller particle size and mix thoroughly with saliva. The cud is then swallowed once again where it will pass into the next three compartments—the reticulum, the omasum and the true stomach, the  abomasum. 

Digestive system of Ruminants 

The primary difference between ruminants and non-ruminants is that ruminants' stomachs have four compartments: Rumen, Reticulum, Omasum, Abomasum  

Rumen 

It is the largest compartment, it can hold as much as 50 gallons of food and other ingested substances at a time. It contains huge number of different microbes, including bacteria, fungi and protozoa. Its internal surface is covered with tiny projections, papillae, which increase the surface area and allow better absorption of digested nutrients. 

  Internal anatomy of Rumen showing papillae 

 

Reticulum / Honeycomb 

Reticulum is separated from the rumen by a ridge of tissue. Its lining has a raised honey comb like pattern, also covered with papillae. It traps hard, indigestible substances like rocks, nails, or wires that may be ingested by accident while the bovine is grazing. 

Internal anatomy of Reticulum showing honey comb projections and papillae

Omasum 

It is also known as “many-piles”, with leaf-like fold shaped compartment. Large plate like folds are known as laminae, which extend from the walls of the omasum. Omasum lies  between the reticulum and abomasum and act as a gateway to the abomasum. It sends back  large substances back to rumen and reticulum while allowing smaller, well-broken down  substances to pass through into abomasum. The materials entering omasum is made up of  90 to 95 % water. The primary function of omasum is to remove some of this water and to further grind and breakdown the feed. The laminae are covered in papillae which direct  the flow of food particles towards the next chamber, abomasum.

Internal anatomy of Omasum showing laminae and papillae 

Abomasum / true stomach 

It connects the omasum to the small intestine. It is much same as the human stomach. The acid and enzyme digestion takes place here. The lining of the abomasum is folded in to ridges, which produce gastric juices containing hydrochloric acids and enzymes (Pepsins). The pH of these gastric juices varies from 1 to 1.3 making the abomasum very acidic, with an average pH of about 2. The acidity in the abomasum kills the rumen microbes. The pepsins carry out the initial digestion of microbial and dietary proteins in the abdomen. 

Internal anatomy of Abomasum showing ridges

 

Process of Rumen digestion 

Once the food has been ingested by the animal, it is briefly chewed and mixed with saliva, swallowed and then moved down the oesophagus in to the rumen. The rumen is adapted for the digestion of fibre. The microbes breakdown the feed through the process of fermentation. The rumen and the reticulum, make up the fermentation vat, which is the major site of microbial activity.

Fermentation is crucial to digestion because it breaks down complex carbohydrates, such as cellulose, and enables the animal to utilize them. Microbes function best in a warm, moist, anaerobic environment with a temperature range of 37.7 to 42.2 °C (100 to 108 °F) and a pH between 6.0 and 6.4. Without the help of microbes, ruminants would not be able to utilize nutrients from forages. 

The breakdown of food starts in the mouth itself due to the mechanical action of chewing. The chemical breakdown starts in the rumen by the action of microbial enzymes. The walls of the rumen and reticulum moves continuously, churning and mixing the ingested feed with the rumen fluid and microbes. The feed is returned to the mouth for cud chewing, which further breaks the feed in to smaller pieces.

Cud chewing increase the rate of microbial digestion in the rumen. The contraction of the rumen and reticulum help the flow of finer food particles in to the next chamber, the omasum. Omasum controls what is able to pass into the abomasum. It keeps the particle size as small as possible in order to pass into the abomasum. Abomasum is the gastric compartment of the ruminant stomach. This compartment releases acids and enzymes that further digest the material passing through.  This is also where the ruminant digests the microbes which may reach from rumen. 

 

Microbes of Rumen and their role in Digestion 

 

The microbes in the rumen include, Bacteria, Protozoa and Fungi. Rumen is estimated to contain 10–50 billion bacteria and 1 million protozoa, as well as several yeasts and fungi. Since the environment inside a rumen is anaerobic, most of these microbial species are obligate or facultative anaerobes.

Bacteria: 

Rumen bacteria account for 10¹⁰ organism/mL of rumen fluid and several hundred  species have been characterized to date. By volume, they comprise up to 50% of the total microbial biomass. Bacteria species are an important source of microbial protein, which supply the ruminant with 75-80% of its metabolizable protein. Bacteria are also important for producing enzymes that digest fiber (cellulose, hemicellulose), starch and sugars.

Examples: Ruminococcus flavefacians, Ruminococcus albus,  Bacteriodes   succinogenes,  Butyrivibrio fibrisolvens,  Bacteriodes ruminocola,  Bacteriodes amylophilus,  Methanomicrobium sp., Methanobacterium sp.,  Methanosarcina sp, Selenomonas ruminantium,  Streptococcus bovis,  Succinomonas amylolytica, Methanobrevibacter ruminatium, Methanosphaera stadtmanae, Butyrivibrio sp., Eubacterium sp.,  Lactobacillus sp. 

 

Protozoa: 

Ciliate protozoa are organisms larger than bacteria and account for 10⁶organisms / mL of  rumen fluid, however they still make up to 50% of the total microbial biomass.  They have various activities: 

✓ Cellulolytic and hemicellulolytic protozoa can digest plant particles. 

✓ Different protozoa have a positive role digesting starch (more slowly than bacteria) ✓ Other protozoa can consume lactic acid, thereby limiting the risk of acidosis. 

           ✓ Some types of protozoa are able to remove oxygen so they have a stabilizing effect upon anaerobiosis. 

          ✓ Most of them degrade proteins very efficiently and release ammonia, so they can waste dietary protein.  These proteins represent around 25% of the microbial protein available for the animal.  

          ✓ Ciliate protozoa produce large amounts of hydrogen, which is a substrate for     methanogens.  

✓ The ciliate species are predators of other rumen microbes.  A single protozoal cell can swallow up to several thousand bacteria in an hour so they play a very important role in rumen microbial population stability. 

 

Examples -Ophryoscolex monoacanthus, Entodinium exiguum, Eudiplodinium maggii, Isotricha intestinalis, Epidinium sp., Entodinium sp., Diplodinium sp., Sarcodina sp. 

 

Fungi: 

Rumen fungi comprise up to 8-10% of microbial biomass and are strictly anaerobic. They  play an essential role in fiber digestion due to the production of  filamentous rhizoids which invade plant tissues, and their efficient enzymatic activities.  This physical action to plant cell walls, can facilitate access to more digestible tissues and  help release polysaccharides, which are linked to lignin increasing the pool of digestible  energy for the other rumen microflora. 

Examples - Neocallimastix sp., Caecomyces sp., Piromyces sp., Anaeromyces sp., Orpinomyces sp., Cyllamyces sp 

Microbial digestion 

 

Rumen microbes can decompose complex plant material, such as cellulose, starch, hemicellulose, and proteins.  

The major end products of microbial fermentation are; 

1. Volatile fatty acids, including acetate, propionate and butyrate, which are the major energy source of cow. 

2. Ammonia, which is used to manufacture microbial proteins. Bacteria are made up of 60 % protein. These bacteria are digested in the abomasum and become the major source of protein for the cow. 

3. Gases, like carbon dioxide and methane, which are wasted energy, as they are belched  out regularly.  

 

Freshwater ecosystems - threats and management

 Freshwater ecosystems account for less than 0.01% of the planet’s total surface area but they support more than 100,000 species, including fish, worms, mollusks, crayfish and insect larvae. But these freshwater systems are now among the most endangered habitats in the world, due to human development, pollution, and climate change.

Fewer than 70 of the world’s 177 longest rivers remain free of man-made obstructions. Also, more than half of the world’s wetlands have disappeared since 1900, a particular concern because these places serve as nature’s water treatment facilities—cleaning water of chemicals and other pollutants. Development, logging, pollution, agriculture and poor management put freshwater systems—and the water they produce for people—at risk.

Freshwater biodiversity loss

Freshwaters cover only about 0.5% of the earth’s surface, but are home to nearly 10% of all known species, including a third of all vertebrates.

 Despite this abundance of biodiversity, freshwater ecosystems are among the most threatened on Earth. According to the World Wildlife Fund’s Living Planet Index, freshwater fish, birds, mammals and reptiles and amphibians have declined by three-quarters over the last 40 years, which is significantly more than the declines in ocean and land wildlife.

The International Union for Conservation of Nature Red List reveals that 35% of freshwater amphibians are threatened or extinct, 46% of mammals and 38% of turtles.

Freshwater threats

Of all the Earth’s ecosystems, freshwater ecosystems have been hit hardest by human activities. Key threats include dams, farming and industry, water extraction, pollution, flow change, invasive species, over-harvesting of species, and climate change.To make the management of freshwater ecosystems even more challenging, these threats often interact in ways that are difficult to predict. These complex and interacting threats are often ignored, leading to the loss of species. 

Habitat loss and degradation are primarily due to deforestation, farming activities and dams. When these activities occur in an upper-catchment area, sediment is carried into rivers and lakes, causing significant negative impacts on freshwater species.

Unsustainable water extraction – for irrigation, industry and urban consumption – is a major threat to freshwater species. Over-harvesting of freshwater species (particularly fish) is, in turn, a threat to these ecosystems.

Infrastructure development – including dams  – also modifies water flow. There are perhaps one million dams globally, fragmenting rivers into isolated sections. Freshwater species – including fish, molluscs and reptiles – often can’t adapt to these changes and are at increased risk of extinction.

 Pollution is another significant threat to these habitats. Fertiliser runoff from farming and the dumping of industrial pollutants directly into rivers and lakes have resulted in areas so poisoned that they can no longer support their normal range of species.

Invasive species have played a major role in disrupting freshwater ecosystems. For example, The European carp (Cyprinus carpio), is a pest that out-competes native fish. It was first introduced to Australian waterways more than 100 years ago and has spread to every state and territory and now the government has plans to introduce a herpes virus to control carp.

Climate change presents another threat to freshwater habitats, particularly to those species that can’t migrate or compensate for higher temperatures. In Australia, extreme weather fluctuations and natural disasters such as floods and droughts are projected to become more common, placing freshwater biodiversity under further stress.

As threats intensify, the risk to freshwater wildlife will increase. Vulnerable freshwater ecosystems will be particularly susceptible to further loss of species.

The best way to help freshwater species is to restore rivers. This might include fencing out livestock, stabilising river banks, removing weeds, replanting native vegetation and expanding floodplain areas. Farm and land management such as rotating pasture, reducing erosion through smart burning practices, and better management of pesticides and nutr

 

Plasmids

 

Plasmids are small, circular molecules of DNA that are capable of replicating independently. They do not depend on chromosomal DNA of the organism for replication so are also referred to as extra-chromosomal DNA

Plasmids are made up of circular double chains of DNA and measure between a few kilobases and several hundred kilobases. Some plasmids have a linear structure and do not form a circular shape.

 

Plasmids provides certain properties to bacteria and can get transferred from one bacteria to another through a process known as conjugation (contact between cells that is followed by transfer of DNA content). There are different types of plasmids like,

Resistance Plasmids-Also referred to as antimicrobial resistance plasmids, resistance plasmids are a type of plasmids that carry genes that play an important role in antibiotic resistance. They are also involved in bacterial conjugation by producing conjugation pili which transfer the R plasmid from one bacterium to another.

Degradative Plasmids-These enable the host organism to degrade/break down xenobiotic compounds. Also referred to as recalcitrant substances, xenobiotic compounds include a range of compounds released into the environment as a result of human actions and are therefore not naturally occurring or common in nature. Degradative plasmids are found in Burkholderia spp, Escherichia coli, Pseudomonas fluorescens etc

Fertility Plasmids- Fertility plasmids (F plasmid) plays an important role in reproduction and they contain genes that code for the production of sex pilus as well as enzymes required for conjugation. F plasmid also contains genes that are involved in their own transfer.

Col Plasmids-Col plasmids confer to bacteria the ability to produce toxic proteins known as colicins. Bacteria as E. coli, Shigella and Salmonella use these toxins to kill other bacteria.

Virulence Plasmids -Pathogenic bacteria carry genes for virulence factors that allow them to invade and infect their respective hosts. The virulence factors are the result of the organisms' own genetic material or due to extra-chromosomal DNA such as transposons, plasmids etc

Most of these plasmids can be transmitted from one bacterium to another.

Some of the other types of plasmids include:

• Recombinant plasmids - Plasmids that have been altered in the laboratory and introduced into the bacteria for the purposes of studies
• Cryptic plasmids - No known functions
• Metabolic plasmids - Enhance metabolism of the host
• Conjugative plasmids - Promote self-transfer
• Suicide plasmids - Fail to replicate when transferred from one cell to another

Inclusion Bodies

A variety of inclusion bodies, granules of organic or inorganic material; for storage (carbon compounds, inorganic substances, and energy)

•      Reduce osmotic pressure by tying up molecules in particulate form

•      Some inclusion bodies lie free in the cytoplasm, not bounded by a membrane- polyphosphate granules, cyanophycin granules, and some glycogen granules

•      Other inclusion bodies are enclosed by a single-layered membrane (protein or lipid) - poly-β-hydroxybutyrate granules, some glycogen and sulfur granules, carboxysomes, and gas vacuoles

•      Quantity will vary with the nutritional status of the cell. For example, polyphosphate granules will be depleted in freshwater habitats that are phosphate limited.

 

Organic inclusion bodies 

• usually contain either glycogen or poly—hydroxybutyrate

•       Glycogen is a polymer of glucose units (long chains formed by (1→4) glycosidic bonds and branching chains connected to them by (1→6) glycosidic bonds)

•      PHB contains -hydroxybutyrate molecules joined by ester bonds 

 

•       Usually only one of these found in a species, purple photosynthetic bacteria have both

 

•      Poly--hydroxybutyrate readily stained with Sudan black for light microscopy and are clearly visible in the electron microscope

•      Glycogen can be seen only with the electron microscope. Stained with iodine 

•      Glycogen and PHB inclusion bodies are carbon storage reservoirs providing material for energy and biosynthesis. Many bacteria also store carbon as lipid droplets.

•      Cyanobacteria (blue green algae) - has two distinctive organic inclusion bodies

•       Cyanophycin granules -large polypeptides containing equal amounts of  arginine and aspartic acid. The granules are large enough to be visible in the light microscope and store extra nitrogen for the bacteria.

•       Carboxysomes are present in many cyanobacteria, nitrifying bacteria, and Thiobacilli. They serve as a reserve of the enzyme ribulose-1,5-bisphosphate carboxylase - may be a site of CO₂ fixation.

Gas vacuoles

•      In many cyanobacteria, purple and green photosynthetic bacteria, and a few other aquatic forms such as Halobacterium and Thiothrix

•      Help them to float at or near the surface; buoyancy

•      Gas vacuoles are aggregates of enormous numbers of small, hollow, cylindrical structures called gas vesicles ; composed entirely of a single small protein, to form a rigid enclosed cylinder that is hollow and impermeable to water but freely permeable to atmospheric gases

•       Bacteria with gas vacuoles can regulate their buoyancy to float at the depth necessary for proper light intensity, oxygen concentration, and nutrient levels

•       They descend by simply collapsing vesicles and float upward when new ones are constructed

Inorganic inclusion bodies

•      Polyphosphate granules or volutin granules – Polyphosphate is a linear polymer of orthophosphates joined by ester bonds.

•      Storage reservoirs for phosphate, an important component of cell constituents such as nucleic acids.

•      In some cells they act as an energy reserve, and energy source in reactions.

•      Also called metachromatic granules -show the metachromatic effect;  appear red or a different shade of blue when stained with the blue dyes methylene blue or toluidine blue

•      Sulfur granules - store sulfur temporarily

•      Eg., purple photosynthetic bacteria

•      Inorganic inclusion bodies can be used for purposes other than storage

Magnetosome (contain iron in the form of magnetite)-      Used by some bacteria (eg., Aquaspirillum magnetotacticum) to respond to the earth’s magnetic field

Bacterial Cell structure- Plasma Membrane & Cytoplasm

  The Plasma Membrane

•        Most widely accepted model for membrane structure is the fluid mosaic model of S. Jonathan Singer and Garth Nicholson

 

•        Membranes contain lipid bilayers with floating proteins- both proteins and lipids; higher proportion of protein

 

•        Membrane-associated lipids (mostly phospholipids) with polar and nonpolar ends (amphipathic); organized in two layers, or sheets

•        The outer surfaces are hydrophilic, whereas hydrophobic ends are buried in the interior away from the surrounding water

 

•        Bacterial membranes lack sterols such as cholesterol ; contain pentacyclic sterol-like molecules called hopanoids ; synthesized from the same precursors as steroids; probably stabilize the bacterial membrane.

 

•        Two types of membrane proteins.

•        Peripheral proteins are loosely connected to the membrane, easily removable, soluble in aqueous solutions -about 20 to 30% of total membrane protein.

•        Integral proteins- About 70 to 80% of membrane proteins; not easily extracted from membranes and are insoluble in aqueous solutions when freed of lipids.

•        The plasma membrane retains the cytoplasm, and separates it from the surroundings

•        Selectively permeable barrier: it allows particular ions and molecules to pass, either into or out of the cell, while preventing the movement of others.

•        Prevents the loss of essential components through leakage

•        Many substances cannot cross the plasma membrane without assistance; Transport systems for nutrient uptake, waste excretion, and protein secretion

•        Location of a variety of crucial metabolic processes: respiration, photosynthesis, the synthesis of lipids and cell wall constituents, and chromosome segregation

•         Membrane contains special receptor molecules that help procaryotes detect and respond to chemicals in their surroundings

 

Cytoplasm

•        The plasma membrane and everything within is called the protoplast; thus the cytoplasmic matrix is a major part of the protoplast

•        Procaryotes have few well defined internal structures; so main component is semifluid cytoplasm

•        4/5th water and 1/5th substances dissolved/suspended in water (Enzymes and other proteins, carbohydrates, lipids and a variety of inorganic ions)

•        Site of Anabolic and catabolic chemical reactions

Ribosomes

•         Site of protein synthesis

•        Made of both protein and ribonucleic acid (RNA)

•        Large and small subunits

•        Abundant in cytoplasm; as long chains (Polyribosomes)

•        Size expressed in Swedberg Units (Sedimentation rates –rate at which they sediment when centrifuged); vary with molecular weight

•        Bacterial ribosomes – 70S (30S + 50S)

•        Streptomycin & Erythromycin bind to 70S ribosomes- inhibit protein synthesis

Nucleoid

•        No  well-defined nucleus & nuclear membrane

•        Central nuclear region/nucleoid- mainly DNA, some RNA and protein

•        DNA arranged in one or two large circular chromosomes (Rhodobacter)/one circular and one linear chromosome (Agrobacterium)/One large circular and one small circular (Vibrio)

•        Some bacteria have plasmids -extrachromosomal, genetic elements

No mitochondria/chloroplasts

 

Internal Membrane Systems

•        Photosynthetic bacteria & Cyanobacteria – internal membrane systems called Chromatophores; Membranes derived from cell membrane- house pigments to capture light for synthesis of sugars

•        Nitrifying bacteria- internal membranes to house enzymes for oxidation of nitrogen compounds

•        Mesosomes -invaginations of the plasma membrane in the shape of vesicles, tubules, or lamellae- involved in cell wall formation during division or play a role in chromosome replication and distribution to daughter cells (Artefacts!?)