Fungal Monocultures Research Articles

Evaluation of cellulose degrading efficiency of some fungi and bacteria and their biofilms

In industrial applications like cellulosic biofuel production, efficient degradation of cellulose is one of the major concerns. But the efficiencies of cellulose degradation by different types of biofilms are not yet explored. This study was carried out to determine whether the community actions of fungal-bacterial biofilms may increase the rates of cellulose bio-degradation. Cellulolytic fungi and bacteria were isolated from soils, composts and leaf litter and screened for simple sugar production. To identify efficient sugar producing communities, combinations of these isolates were tested in batch cultures of cellulose broth prepared with commercial cellulose. Three different communities; fungal and bacterial monocultures, fungal-bacterial biofilms and fungal mixed cultures were tested. The monoculture of Acremonium sp. was the highest sugar yielder among all tested microorganisms. Among fungal mixed cultures, Acremonium sp. with Fusarium sp. was an effective sugar yielder. Acremonium sp. with a Bacillus sp. was effective among fungal-bacterial biofilms. These three cultures were also inoculated into broth media prepared with Eupatorium odoratum or Panicum maximum or Lantana camara or Mimosa pigra to assess their simple sugar production from plant materials . The highest sugar production was by Acremonium sp. monoculture and the lowest was by the Acremonium sp. with Bacillus sp. biofilm. This was true for all four weeds. Thus, all the fungal-bacterial biofilms tested were comparatively less effective sugar producers. DOI: http://dx.doi.org/10.4038/jnsfsr.v41i2.5710 J.Natn.Sci.Foundation Sri Lanka 2013 41 (2):155-163

Fungus-gardening ants prefer native fungal species: do ants control their crops?

Fundamental features of mutualisms are solutions to conflict. The mutualism between fungus-gardening ants and their fungi is characterized by pronounced evolutionary ant-fungus fidelity. Ant-fungus fidelity is thought to be controlled by the fungus, which secretes somatic incompatibility compounds that suppress incompatible fungal strains that may invade gardens. Accordingly, the ants are thought to perceive incompatibilities and then reject invading cultivars. During cultivar-switch experiments, we discovered that if switched colonies of the ant Trachymyrmex septentrionalis are successful in retaining a minute fragment of their original garden, the ants will gradually co-cultivate the novel and the original fungus strains in a chimeric (polycultural) garden; this chimeric garden will revert after 1–2 weeks completely to a monoculture of the original fungal strain. Experimental examination of worker preference suggests that symbiont rejection behavior seems to be an innate response toward foreign fungi rather than mediated solely by incompatibility compounds secreted by the fungus. These observations suggest that the fungal strain cultivated by a colony imprints the attending ants, and this imprinting modifies ant behavior so that the ants prefer their original cultivar even when experimentally forced to grow a foreign cultivar for several weeks. Fungal monocultures and ant-fungus fidelities in this symbiosis therefore seem to be reinforced by factors intrinsic to both the ants and the fungi. Key words: Attini, chimera, cultivar switch, somatic compatibility, symbiosis. [Behav Ecol]

C. elegans: An All in One Model for Antimicrobial Drug Discovery

One approach to identify new drugs with antimicrobial activities is to screen large libraries of molecules directly for their capacity to block the growth of bacterial or fungal monocultures. A more relevant way to assess both a product's efficacy and its potential cytotoxicity is undoubtedly to use an in vivo infection system. Testing banks containing thousands of natural or chemically synthesized molecules with rodents is generally neither desirable nor feasible. Therefore, invertebrate model organisms could represent a valuable alternative. In this review, we present the worm C. elegans as a suitable host model for the evaluation and characterization of drug effects in a pathogenesis context. This simple organism has been of great value in many fields of biology and is currently intensely used in studies of host-pathogen interactions. Infection of C. elegans induces a number of defense mechanisms, some of which are similar to those seen in mammalian innate immunity. Further, it has been demonstrated that several microbial virulence mechanisms required for full pathogenicity in mammals are also necessary for infection in nematodes. Based on these facts, a number of innovative antimicrobial drug screens have been carried out successfully and the development of new tools to monitor the interaction between worm and microbes in vivo opens promising perspectives.

Monoculture of leafcutter ant gardens.

BackgroundLeafcutter ants depend on the cultivation of symbiotic Attamyces fungi for food, which are thought to be grown by the ants in single-strain, clonal monoculture throughout the hundreds to thousands of gardens within a leafcutter nest. Monoculture eliminates cultivar-cultivar competition that would select for competitive fungal traits that are detrimental to the ants, whereas polyculture of several fungi could increase nutritional diversity and disease resistance of genetically variable gardens.Methodology/Principal FindingsUsing three experimental approaches, we assessed cultivar diversity within nests of Atta leafcutter ants, which are most likely among all fungus-growing ants to cultivate distinct cultivar genotypes per nest because of the nests' enormous sizes (up to 5000 gardens) and extended lifespans (10–20 years). In Atta texana and in A. cephalotes, we resampled nests over a 5-year period to test for persistence of resident cultivar genotypes within each nest, and we tested for genetic differences between fungi from different nest sectors accessed through excavation. In A. texana, we also determined the number of Attamyces cells carried as a starter inoculum by a dispersing queens (minimally several thousand Attamyces cells), and we tested for genetic differences between Attamyces carried by sister queens dispersing from the same nest. Except for mutational variation arising during clonal Attamyces propagation, DNA fingerprinting revealed no evidence for fungal polyculture and no genotype turnover during the 5-year surveys.Conclusions/Significance Atta leafcutter ants can achieve stable, fungal monoculture over many years. Mutational variation emerging within an Attamyces monoculture could provide genetic diversity for symbiont choice (gardening biases of the ants favoring specific mutational variants), an analog of artificial selection.

Caste-specific symbiont policing by workers of Acromyrmex fungus-growing ants

The interaction between leaf-cutting ants and their fungus garden mutualists is ideal for studying the evolutionary stability of interspecific cooperation. Although the mutualism has a long history of diffuse coevolution, there is ample potential for conflicts between the partners over the mixing and transmission of symbionts. Symbiont transmission is vertical by default, and both the ants and resident fungus actively protect the fungal monoculture growing in their nest against secondary introductions of genetically dissimilar symbionts from other colonies. An earlier study showed that mixtures of major and minor Acromyrmex workers eliminate alien fungus fragments even in subcolonies where their resident symbiont is not present. We hypothesize that the different tasks and behaviors performed by majors and minors are likely to select for differential responses to alien fungi. Major workers forage and cut new leaves and masticate them after delivery in the upper parts of the fungus garden and so are likely to more frequently encounter alien fungus than minor workers maintaining the established fungus garden and caring for the brood. We show that major workers of Acromyrmex echinatior indeed express stronger incompatibility reactions toward alien fungus garden fragments than minor workers. This implies that only the major workers, through recognition and exclusion of foreign fungus clones at their point of entry to the nest, have a realistic possibility to eliminate alien fungal tissue before it gets incorporated in the fungus garden and starts competing with the resident fungal symbiont. Key words: evolutionary stability, leaf-cutting ants, mutualism, symbiont policing. [Behav Ecol]

Polycyclic Aromatic Hydrocarbons (PAHs) Biodegradation by Basidiomycetes Fungi, Pseudomonas Isolate, and Their Cocultures: Comparative In Vivo and In Silico Approach

The polycyclic aromatic hydrocarbons (PAHs) biodegradation potential of the five basidiomycetes' fungal monocultures and their cocultures was compared with that of a Pseudomonas isolate recovered from oil-spilled soil. As utilization of hydrocarbons by the microorganisms is associated with biosurfactant production, the level of biosurfactant production and its composition by the selected microorganisms was also investigated. The Pseudomonas isolate showed higher ability to degrade three of the five PAHs but the isolate did not produce biosurfactant higher than C. versicolor and P. ostreatus. Among the PAHs, the most effective biodegradation of PAH--pyrene (42%)--was obtained with the fungus C. versicolor. Cocultures involving the fungi and Pseudomonas could not significantly degrade the selected PAHs compounds above that degraded by the most efficient monoculture. A slight increase in pyrene degradation was observed in cocultures of C. versicolor and F. palustris (93.7% pyrene). The crude biosurfactant was biochemically characterized as a multicomponent surfactant consisting of protein and polysaccharides. The PAH biodegradation potential of the basidiomycetes fungi positively correlated with their potential to express ligninolytic enzymes such as lignin peroxidase (Lip), manganese peroxidase (Mnp), and laccase. The present study utilized in silico method such as protein-ligand docking using the FRED in Open Eye software as a tool to assess the level of ligninolytic enzymes and PAHs interactions. The in silico analysis using FRED revealed that of the five PAHs, maximum interaction occurred between pyrene and all the three ligninolytic enzymes. The results of the in silico analysis corroborated with our experimental results showing that pyrene was degraded to the maximum extent by species such as C. versicolor and P. ostreatus.

Screening for bacterial–fungal associations in a south-eastern US salt marsh using pre-established fungal monocultures

Both bacteria and fungi play critical roles in decomposition processes in many natural environments, yet only rarely have they been studied as an integrated community. We examined whether physical associations exist between individual bacterial and fungal species that co-occur on decaying smooth cordgrass, Spartina alterniflora, in a south-eastern US salt marsh. Fungal-pervaded decaying Spartina was used as “bait” for potential bacterial associates. The bundles (infiltrated with one of three dominant fungal members of the decomposer assemblage, or an autoclaved control) were placed in a salt marsh and collected biweekly for 6 weeks during the first experiment (late summer 2002), and weekly for 3 weeks during the second experiment (early summer 2003). Terminal-restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes was used to track colonization by bacterial taxa in association with the established fungal species. T-RFLP analysis of 18S-to-28S internal transcribed spacer (ITS) regions was used to monitor changes in fungal communities once bundles had been placed in the field. Results from both years were nearly identical, and showed that invasion by fungi other than the bait species was slow, resulting in a virtual fungal monoculture for several weeks into the experiments. Surprisingly, bacterial communities were unaffected by the identity of the fungal bait. Regardless of the fungal species, and even in the absence of prior fungal colonization, bacterial 16S rRNA profiles were remarkably similar. These results suggest that few species-specific associations, either positive or negative, exist between bacterial and fungal members of the Spartina decomposer community during initial colonization.

Biodegradation of gossypol by the white oyster mushroom, Pleurotus florida, during culturing on rice straw growth substrate, supplemented with cottonseed powder

The capacity of the white oyster mushroom, Pleurotus florida to biodegrade gossypol was studied, when grown on rice straw supplemented with cottonseed powder. The mushroom fruiting bodies did not contain any residues of gossypol at concentrations of cottonseed powder ≡0.15–0.60% nitrogen contents of rice straw at the end of mycelial ramification. However, the cottonseed supplementation (at 0.30% N level itself) caused a doubling in the mushroom yield and its protein content, per unit weight straw substrate. The mushroom mycelium when grown on synthetic medium in liquid cultures was able to biodegrade gossypol. A pre-incubation period of 5 days before the addition of gossypol into the culture medium, an inoculum load ≡10 mg and an incubation period of 10 days at 25 °C caused the biodegradation of 100 μg gossypol. Increased concentrations of gossypol required increased duration and increased inoculum levels to effect biodegradation. However, the effect was more pronounced with an increase in inoculum density. The fungal monoculture when grown in rice straw (powder) (5%) + glucose (1%) liquid culture medium, showed an increase in hexosamine content and laccase activity that produced an increased degradation of gossypol over an incubation period from 5 to 25 days. Enzymic extracts of the mycelial monoculture raised on the chopped rice straw substrate when incubated with 100 μg of gossypol demonstrated its biodegradability; the increase in enzyme concentration showed enhanced gossypol degradation. This study adds to the world list of organic compounds that Pleurotus is able to biodegrade, and explains the cause of non-yellowing of the white oyster mushroom (P. florida) fruiting bodies, during culture on rice straw with supplementation of cottonseed powder for enhancing the mushroom yields.

Fungus-growing ants use antibiotic-producing bacteria to control garden parasites

The well-studied, ancient and highly evolved mutualism between fungus-growing ants and their fungi has become a model system in the study of symbiosis1,5. Although it is thought at present to involve only two symbionts, associated with each other in near isolation from other organisms1,5, the fungal gardens of attine ants are in fact host to a specialized and virulent parasitic fungus of the genus Escovopsis (Ascomycotina)6. Because the ants and their fungi are mutually dependent, the maintenance of stable fungal monocultures in the presence of weeds or parasites is critical to the survival of both organisms. Here we describe a new, third mutualist in this symbiosis, a filamentous bacterium (actinomycete) of the genus Streptomyces that produces antibiotics specifically targeted to suppress the growth of the specialized garden-parasite Escovopsis. This third mutualist is associated with all species of fungus-growing ants studied, is carried upon regions of the ants' cuticle that are genus specific, is transmitted vertically (from parent to offspring colonies), and has the capacity to promote the growth of the fungal mutualist, indicating that the association of Streptomyces with attine ants is both highly evolved and of ancient origin.

Effects of ruminal protozoa on cellulose degradation and the growth of an anaerobic ruminal fungus, Piromyces sp. strain OTS1, in vitro

An anaerobic rumen fungus, Piromyces sp. strain OTS1, was incubated in the presence or absence of a mixed, A-type, protozoal population obtained from a goat, in a medium containing filter paper cellulose as energy source and antibiotics to suppress bacterial growth. Fermentation end products, cellulose degradation, and chitin as an indicator of fungal biomass were examined. In the presence of protozoa, total volatile fatty acids, notably propionate and butyrate, increased, and lactate decreased. In fungus-protozoan coincubations, formate was not detected at the end of the experiment and the amount of reducing sugars remained low throughout the incubation period. The fungal growth in the coincubations was negatively affected. While protozoal predation on zoospores was one mechanism of inhibition, mature fungal cells were also affected. Total cellulose degradation was greater in fungal monocultures, but the amount of cellulose degraded per unit of fungal biomass was 25% larger in the coincubations. The negative effects that the protozoal predatory activity had on the fungal growth and subsequently on the amount of cellulose degraded by Piromyces sp. strain OTS1 were partially attenuated by the protozoal fibrolytic activity or by an enhanced fungal activity due to a more favorable environment.

Degradation of wheat straw and maize stem by a monocentric and a polycentric rumen fungi, alone or in association with rumen cellulolytic bacteria

Two rumen anaerobic fungi, Neocallimastix frontalis MCH3 and Orpinomyces (Neocallimastix) joyonii TP 90-9 were cultured on wheat straw or maize stem alone or in association with one of two rumen cellulolytic bacteria, Ruminococcus flavefaciens 007 and Fibrobacter succinogenes S85. Observations by scanning electron microscopy showed that the behaviour of the different microorganisms was qualitatively similar. The phloem and inner parenchyma of the wheat straw and maize stem were completely degraded after 48 h in both the monocultures and cocultures. Quantitatively, the two fungal species degraded comparable amounts of wheat straw and maize stem as the two cellulolytic bacteria. In the coculture of N. frontalis or O. joyonii with F. succinogenes, wheat straw or maize stem were digested to the same extent as in the fungal monocultures. The associations of N. frontalis with R. flavefaciens on the two substrates and of O. joyonii with R. flavefaciens on wheat straw were less efficient than the fungal monocultures. R. flavefaciens exerted an inhibitory effect on the cellulolytic activity of N. frontalis and O. joyonii while F. succinogenes did not interact with the two fungal species.

Effect of Eubacterium limosum, a ruminal hydrogenotrophic bacterium, on the degradation and fermentation of cellulose by 3 species of rumen anaerobic fungi

The degradation and fermentation of cellulose filter paper were studied in axenic cultures of 3 species of rumen anaerobic fungi, Neocallimastix frontalis, Piromyces communis and Caecomyces communis, and in cocultures containing 1 of these fungal strains and Eubacterium limosum, a hydrogenotrophic rumen bacterial species. When E limosum was introduced into fungal cultures a slight decrease in fungal cellulolytic activity was observed. The end products of the fermentation of cellulose found in the cocultures were different from those found in the fungal monocultures. E limosum used formate and part of the hydrogen produced by the fungi and probably created a shift in the metabolism of the fungi resulting in a reduction of lactate and ethanol production.

Degradation and fermentation of cellulose by the rumen anaerobic fungi in axenic cultures or in association with cellulolytic bacteria

Three rumen anaerobic fungi—Neocallinastix frontalis MCH3,Piromyces (Piromonas) communis FL, andCaecomyces (Sphaeromonas) communis FG10—were cultured on cellulose filter paper alone or in association with one of two rumen cellulolytic bacteria,Ruminococcus flavefaciens 007 andFibrobacter succinogenes S85. Cocultures ofN. frontalis orP. communis andR. flavefaciens were markedly less effective than the fungal monocultures in degrading cellulose but more effective than the bacterial monocultures.R. flavefaciens had an antagonistic effect against both of the fungal species. In contrast, no interaction was observed between the two fungal species andF. succinogenes. Cellulose was more effectively degraded by the cocultureC. communis-R. flavefaciens than by the corresponding fungal and bacterial monocultures. The effectiveness of degradation of the cocultureC. communis-F. succinogenes was comparable to that of the bacterial strains but greater than that of the fungi; no interaction was observed between these two microorganisms.

Degradation of maize stem by two rumen fungal species, Piromyces communis and Caecomyces communis, in pure cultures or in association with cellulolytic bacteria.

Two species of rumen fungi, Piromyces (Piromonas) communis FL and Caecomyces (Sphaeromonas) communis FG10, were cultured alone or in association with the cellulolytic bacteria Ruminococcus flavefaciens or Fibrobacter succinogenes on maize stem. A kinetic study of the degradation of the substrate was then made. After 48 h of culture, all non-lignified tissues observed by scanning electron microscopy disappeared with P communis and degradation was as complete as that observed in the rumen. In contrast, C communis degraded little of the plant cell walls. The ability of P communis to more rapidly degrade maize stem was probably due to the presence of filamentous rhizoids. The extent of dry matter loss after 8 days of incubation was practically the same in all the monocultures and in the 4 cocultures. However, the rate of degradation was faster in the bacterial than in the fungal monocultures and the co-cultures. No metabolic interaction was observed.

Improved cellulolytic activity of actinomycetes in axenic mixed fermentations

AbstractFermentations with defined mixed cultures of cellulolytic actinomycetes or fungi and non‐cellulolytic microorganisms were performed on insoluble cellulose as well as on soluble cellulose derivatives. Degradation of insoluble Whatman‐cellulose with co‐cultures of cellulolytic actinomycetes and fungi occurred much faster and more completely than with the corresponding monocultures. Fungi appeared mainly responsible for initial cellulose degradation, while the actinomycetes subsequently metabolised the formed soluble oligosaccharides. Both fungi and actinomycetes produced active cellulases in cocultures, resulting in an increased cellulase activity of the culture filtrates as compared with the monocultures. With carboxymethylcellulose (CM‐cellulose) as a carbon source, similar results were obtained, but here the actinomycetes were mainly responsible for CM‐cellulose degradation rather than the fungi. Hydrolysis of insoluble cellulose by cocultures of cellulolytic actinomycetes or fungi with cellobiose‐utilising bacteria or yeasts was similar to that with the corresponding actinomycete or fungal monocultures. Apparently the soluble oligosaccharides, formed from cellulose by the actinomycetes or fungi, could not be utilised as a carbon source by these bacteria or yeasts, while the reducing sugar level was probably too low to allow detectable growth. With CM‐cellulose as a carbon source, the reducing sugars formed by the actinomycetes or fungi are actively metabolised by the bacteria or yeasts. Thus by keeping the readily utilisable sugar concentration very low, carbon catabolite repression of cellulase synthesis is lowered, resulting in an increased cellulase formation and improved cellulose hydrolysis.

Production of Citrate by Anaerobic Fungi in the Presence of Co-culture Methanogens as Revealed by (1)H NMR Spectrometry.

The metabolomic profile of the anaerobic fungus Piromyces sp. F1, isolated from the rumen of goats, and how this is affected by the presence of naturally associated methanogens, was analyzed by nuclear magnetic resonance spectroscopy. The major metabolites in the fungal monoculture were formate, lactate, ethanol, acetate, succinate, sugars/amino acids and α-ketoglutarate, whereas the co-cultures of anaerobic fungi and associated methanogens produced citrate. This is the first report of citrate as a major metabolite of anaerobic fungi. Univariate analysis showed that the mean values of formate, lactate, ethanol, citrate, succinate and acetate in co-cultures were significantly higher than those in the fungal monoculture, while the mean values of glucose and α-ketoglutarate were significantly reduced in co-cultures. Unsupervised principal components analysis revealed separation of metabolite profiles of the fungal mono-culture and co-cultures. In conclusion, the novel finding of citrate as one of the major metabolites of anaerobic fungi associated with methanogens may suggest a new yet to be identified pathway exists in co-culture. Anaerobic fungal metabolism was shifted by associated methanogens, indicating that anaerobic fungi are important providers of substrates for methanogens in the rumen and thus play a key role in ruminal methanogenesis.