Estimation of microbial biomass carbon and Nitrogen

1. fumigation-incubation method
2. the fumigation-extraction method
3. the substrate-induced respiration method
4. the ATP method 

The fumigation-incubation method is the basic technique which is also used for calibration of the three other methods. It is characterized by simple performance without the need of expensive equipment. Its application is limited to soils with a pH above 5 and to soils that do not contain easily degradable C sources. If these limitations are not considered, too low or even negative biomass values will be obtained. These restrictions are largely overcome by the fumigation-extraction method. 

The substrate-induced respiration requires expensive equipment for the hourly measurement of soil respiration. This method is also susceptible to amendment of soils with C sources, leading to an overestimate of biomass

The microbial biomass accounts for only 1-3 % of soil organic C but it is the eye of the needle through which all organic material that enters the soil must pass. During this process these materials are converted by microorganisms in order to generate energy and to produce new cellular metabolites to support their maintenance and growth. In the C-limited soil system available C in organic materials entering the soil is the driving force behind these processes but other essential nutrient elements (particularly N, P, K) are also involved. Under suitable environmental conditions the extent of the turnover will mainly be controlled by the size and activity of the microbial biomass. In order to elucidate the intricate interrelationships and controlling mechanisms of the input/output fluxes of nutrients and energy in the soil ecosystem a reliable quantification of the microbial biomass is required. Valuable information on biomass growth, turnover time, death rates, and the efficiency of C use can be derived from reliable biomass C data. The microbial biomass itself may represent a labile pool of C and nutrient elements. In agricultural soils 200-1000 ~tg biomass C g-~ soil is often found. This cell mass fixes 100-600 kg N and 50-300 kg P per hectare in the upper 30 cm of soil. These amounts often exceed the annual application of nutrients supplied as fertilizer to soils in agricultural practice. The liberation or fixation of these nutrients depends on the life dynamics of the microorganisms. Growth of biomass and fixation of nutrients is promoted by rhizodeposits and plant debris and the liberation of nutrients is the consequence of microbial death. These processes provide the inc centive for a reliable quantification of the microbial biomass as a whole and 88 for the inclusion of its life dynamics in considerations about nutrient cycling in soil. 

The fumigation-incubation method

As early as 1908, under the title "Uber die Wirkungen des Schwefelkohlenstoffs und tihnlicher Stoffe auf den Boden" (Effects of carbon disulfide and related compounds on soil) K. StOrmer described and interpreted the effects of biocidal fumigants on soils. He postulated that (1) the observed effect of improved plant growth after a transient treatment of soils with toxic fumigants is caused by a liberation of additional N; (2) this N originates from the bodies of the organisms killed by the toxicant; and (3) after treatment of the soils an increased proliferation of bacteria can be observed, which degrade the killed organisms and liberate the N fixed in the cell mass. This explanation for the observed phenomena, now accepted as correct, did not find the general acceptance it deserved and was overlain by other explanations. Jenkinson (1966) summarized the most important early theories. One hypothesis assumed that microbial activity and development is restrained in unsterilized soil by unknown toxic compounds, reduced microbial vigour, or by inhibiting, antagonistic effects between different sections of the microbial populations. Partial sterilization suspends these effects for a transient time. A second theory postulated a physical or chemical protection of otherwise unavailable substrates in unsterilized soil. This protecting barrier may consist of waxes which are dissolved by exposure to lipophilic solvents, such as CHC13, CS 2, or CC14. The third possible explanation was based on the observation that most ways of partial sterilization (heating, air drying, irradiation) increase the amount of water-soluble organic matter. This can be explained chemical alteration of the non-living parts of the soil organic matter but also by killing of microorganisms and the ensuing lysis. In order to examine these different theories, Jenkinson (1966) investigated the CO2 and 14CO2 liberation from soil samples subjected to different treatments. These

Effective Microorganism

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An effective microorganism (EM) refers to any of the predominantly aerobic organisms blended in commercial agricultural amendments, medicines and nutritional supplements These blends include:

·         Lactic acid bacteria: Lactobacillus casei

·         Photosynthetic bacteria: Rhodopseudomonas palustris

·         Yeast: Saccharomyces cerevisiae

·         Others: beneficial microorganisms that exist naturally in the environment may thrive in the mixture.

EM Technology is purported to support sustainable practices in farming and to improve and support human health and hygiene, compost and waste management, disaster clean-up.

EM has been employed in many agricultural applications, but is also used in the production of several health products

The concept of "friendly microorganisms" was developed by Professor Teruo Higa, from the University of the Ryukyus in Okinawa, Japan. He reported in the 1980s that a combination of approximately 80 different microorganisms is capable of positively influencing decomposing organic matter such that it reverts into a "life promoting" process. Higa invoked a "dominance principle" to explain the effects of his "Effective Microorganisms". He claimed that three groups of microorganisms exist: "positive microorganisms" (regeneration), "negative microorganisms" (decomposition, degeneration), "opportunist microorganisms". In every medium (soil, water, air, the human intestine), the ratio of "positive" and "negative" microorganisms is critical, since the opportunist microorganisms follow the trend to regeneration or degeneration. Therefore, Higa claimed that it is possible to positively influence the given media by supplementing with "positive" microorganisms.

The concept has been challenged and no scientific studies support all of its claims. This was acknowledged by Higa in a 1994 paper co-authored by Higa and soil microbiologist James F Parr. They conclude "the main limitation...is the problem of reproducibility and lack of consistent results.".

Parr and Higa mention soil pH, shading, soil temperature and flooding as factors affecting the interaction of EM with local microorganisms and with each other. The approach that Higa and Parr recommend is maintaining pH and soil temperature within conditions known to be detrimental to negative microorganisms as well as the addition of EM to tip the balance of positive and negative microorganisms in favor of the former.

They dismiss inoculants that include only a single microorganism as generally ineffective due to the uncertainty about the conditions in which a single microorganism would be effective. They cite the acknowledgment by the scientific community that multiple microorganisms (as in the case of Bokashi, invented and marketed by Higa) in coordination with good soil management practices positively influence plant growth and yield.

Lwini and Ranamukhaarachchi published in 2006 a paper that discusses biological controls of bacterial wilt disease and showed that EM and EM Bokashi were most-effective as bio-control agents. Yamada and Xu examined the use of EM in making organic fertilizers. Hui-Lian Xu studied photosynthesis and yield of sweet corn, physiological characteristics in peanuts, and fruit yield and quality of tomato plants. Daiss, et al., looked at pre-harvest and post-harvest applications of EM.

Use in sanitation systems

Effective microorganisms have also been advocated for use in sanitation systems, in particular in pit latrines and septic tanks, where they are usually called "pit additives" or "septic tank additives". Most of these additives claim to be using some form of EM aspects, although some are simply used to improve odor or to reduce fat build-up. The products, consisting of packaged micro-organisms or enzymes or both, are marketed on their claimed ability to either reduce the pit or septic tank filling rate with faecal sludge, or to actually decrease the volume of material in the pit or septic tank.

Research studies in South Africa by the Water Research Commission during 2010-2012 as well as in the Netherlands in 2013-2014 have conclusively shown that it is very unlikely that any of the claims frequently made about the beneficial impacts of these additives are actually true. Such claims made by manufacturers include:

·         The products contain micro-organisms that can biologically break down the material in the pit to harmless compost products.

·         Nutrients present in the additive ensure optimal growth conditions for micro-organisms to break down pit contents.

·         Additives stimulate the micro-organisms in the pit to break down pit sludge faster.

·         Addition of aerobic micro-organisms create aerobic conditions in the pit that result in rapid degradation.

·         Addition of non-pathogenic bacteria in the sludge out-compete and in fact eat disease-causing pathogenic micro-organisms in the pit sludge, rendering it safe.

·         Odours are reduced as a result of accelerated sludge breakdown.

The main reason why pit additives do not change the pit or septic tank filling rate is that the quantity of bacteria introduced to the pit or septic tank by dosing additives is insignificant compared to the number already present in the faecal sludge.

As the costs and health risks associated with manual pit emptying are huge, if a product was ever developed which significantly impacted the filling rate of pits, e.g. based on EM, this would be of enormous significance.

 source:wiki

Trichoderma

Trichoderma

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Trichoderma is a genus of fungi that is present in all soils, where they are the most prevalent culturable fungi. Many species in this genus can be characterized as opportunistic avirulent plant symbionts. This refers to the ability of several Trichodermaspecies to form mutualistic endophytic relationships with several plant species. The genomes of severalTrichoderma species have been sequenced and are publicly available from the JGI.

Taxonomy

The genus was described by Christiaan Hendrik Persoon in 1794, but the taxonomy has remained difficult to resolve. For a long time it was considered to consist of only one species, Trichoderma viride, named for producing green mold.

Subdivision

The genus was divided into five sections in 1991 by Bissett, partly based on the aggregate species described by Rifai:

·         Pachybasium (20 species)

·         Longibrachiatum (10 species)

·         Trichoderma

·         Saturnisporum (2 species)

·         Hypocreanum

With the advent of molecular markers from 1995 onwards, Bissett's scheme was largely confirmed but Saturnisporum was merged withLongibrachiatum. While Longibrachiatum and Hypocreanum appearedmonophyletic, Pachybasium was determined to be paraphyletic, many of its species clustering with Trichoderma. Druzhina and Kubicek (2005) confirmed the genus as circumscribed was holomorphic. They identified 88 species which they demonstrated could be assigned to two major clades. Consequently the formal description of sections has been largely replaced by informal descriptions of clades, such as the Aureoviride clade or the Gelatinosum clade.

Species

The belief that Trichoderma was monotypic persisted until the work of Rifai in 1969, who recognised nine species. Currently there are 89 accepted species in the Trichoderma genus. Hypocrea areteleomorphs of Trichoderma which themselves have Hypocrea asanamorphs.

Characteristics

Cultures are typically fast growing at 25–30°C, but some species ofTrichoderma will grow at 45°C. Colonies are transparent at first on media such as cornmeal dextrose agar (CMD) or white on richer media such as potato dextrose agar (PDA). Mycelium are not typically obvious on CMD, conidia typically form within one week in compact or loose tufts in shades of green or yellow or less frequently white. A yellow pigment may be secreted into the agar, especially on PDA. Some species produce a characteristic sweet or 'coconut' odor.

Conidiophores are highly branched and thus difficult to define or measure, loosely or compactly tufted, often formed in distinct concentric rings or borne along the scant aerial hyphae. Main branches of the conidiophores produce lateral side branches that may be paired or not, the longest branches distant from the tip and often phialides arising directly from the main axis near the tip. The branches may rebranch, with the secondary branches often paired and longest secondary branches being closest to the main axis. All primary and secondary branches arise at or near 90° with respect to the main axis. The typical Trichoderma conidiophore, with paired branches assumes a pyramidal aspect. Typically the conidiophore terminates in one or a few phialides. In some species (e.g. T. polysporum) the main branches are terminated by long, simple or branched, hooked, straight or sinuous, septate, thin-walled, sterile or terminally fertile elongations. The main axis may be the same width as the base of the phialide or it may be much wider.

Phialides are typically enlarged in the middle but may be cylindrical or nearly subglobose. Phialides may be held in whorls, at an angle of 90° with respect to other members of the whorl, or they may be variouslypenicillate (gliocladium-like). Phialides may be densely clustered on wide main axis (e.g. T. polysporum, T. hamatum) or they may be solitary (e.g. T. longibrachiatum).

Conidia typically appear dry but in some species they may be held in drops of clear green or yellow liquid (e.g. T. virens, T. flavofuscum). Conidia of most species are ellipsoidal, 3–5 x 2–4 µm (L/W = > 1.3); globose conidia (L/W < 1.3) are rare. Conidia are typically smooth but tuberculate to finely warted conidia are known in a few species.

Synanamorphs are formed by some species that also have typicalTrichoderma pustules. Synanamorphs are recognized by their solitaryconidiophores that are verticillately branched and that bear conidia in a drop of clear green liquid at the tip of each phialide.

Chlamydospores may be produced by all species, but not all species produce chlamydospores on CMD at 20°C within 10 days. Chlamydospores are typically unicellular subglobose and terminate short hyphae; they may also be formed within hyphal cells. Chlamydospores of some species are multicellular (e.g. T. stromaticum).

Trichoderma genomes appear to be in the 30–40 Mb range, with approximately 12,000 genes being identifiable.

Teleomorph

Teleomorphs of Trichoderma are species of the ascomycete genusHypocrea. These are characterized by the formation of fleshy, stromata in shades of light or dark brown, yellow or orange. Typically the stroma is discoidal to pulvinate and limited in extent but stromata of some species are effused, sometimes covering extensive areas. Stromata of some species (Podostroma) are clavate or turbinate. Perithecia are completely immersed. Ascospores are bicellular but disarticulate at the septum early in development into 16 part-ascospores so that the ascus appears to contain 16 ascospores. Ascospores are hyaline or green and typically spinulose. More than 200 species of Hypocrea have been described but few have been grown in pure culture and even fewer have been described in modern terms.

Occurrence

Trichoderma species are frequently isolated from forest or agricultural soils at alllatitudes. Hypocrea species are most frequently found on bark or on decorticated wood but many species grow on bracket fungi (e.g. H. pulvinata), Exidia (H. sulphurea) or bird's nest fungi (H. latizonata) or agarics (H. avellanea).

Biocontrol agent

Several strains of Trichoderma have been developed as biocontrol agents against fungal diseases of plants. The various mechanisms include antibiosis, parasitism, inducing host-plant resistance, and competition. Most biocontrol agents are from the species T. harzianum, T. viride and T. hamatum. The biocontrol agent generally grows in its natural habitat on the root surface, and so affects root disease in particular, but can also be effective against foliar diseases.

Causal agent of disease

T. aggressivum (formerly T. harzianum biotype 4) is the causal agent of green mold, a disease of cultivated button mushrooms. Trichoderma viride is the causal agent of green mold rot of onion.

Toxic house mold

The common house mold, Trichoderma longibrachiatum, produces small toxic peptides containing amino acids not found in common proteins, like alpha-aminoisobutyric acid, called trilongins (up to 10% w/w). Their toxicity is due to absorption into cells and production of nano-channels that obstruct vital ion channels that ferry potassium and sodium ions across the cell membrane. This affects in the cellsaction potential profile, as seen in cardiomyocytes, pneumocytes andneurons leading to conduction defects. Trilongins are highly resistant to heat and antimicrobials making primary prevention the only management option.

Medical uses

Cyclosporine A (CsA), a calcineurin inhibitor produced by the fungiTolypocladium inflatum and Cylindrocarpon lucidum, is an immunosuppressant prescribed in organ transplants to prevent rejection.

Industrial use

Trichoderma, being a saprophyte adapted to thrive in diverse situations, produces a wide array of enzymes. By selecting strains that produce a particular kind of enzyme, and culturing these in suspension, industrial quantities of enzyme can be produced.

·         Trichoderma reesei is used to produce cellulase and hemicellulase

·         Trichoderma longibrachiatum is used to produce xylanase 

·         Trichoderma harzianum is used to produce chitinase.

source:wiki