Two papers about the process of mushroom feeding provide new information and confirm findings that were presented in THE FUNDAMENTALS OF MUSHRROM FEEDING AND FARMING TECHNOLOGY DEVELOPMENTS.

Carbohydrate composition of compost during composting and mycelium growth of Agaricus bisporus

Edita Jurak, Mirham A. Kabel, Harry Gruppen

Carbohydrate Polymers 101 (2014) 281-288  www.elsevier.com/locate/carbpol

I became interested in this paper because the presented data showed that water-insoluble xylans (for instance wheat flour, straw, stems, corn cobs and wheat oats) and glucans are degraded during mycelium growth into small particles soluble in water. During the mycelium growth, a process of degradation of these elements by mycelium was observed that resulted in increase of water-soluble products.  Xylans and glucans degraded by mushroom, according to literature, constitute the first source of carbohydrates for mushroom that later is utilized for primordia formation.

These results confirm that the established theory that the process of mushroom feeding might be improved by adding feeders that include corn meal after mycelium colonize the compost as well as by water-soluble carbohydrates, which are present in colonized composts.

Below I present a selection of the most practical information useful in the development of knowledge about the process of mushroom feeding that will also be an inspiration for further studies.

Carbohydrate utilization and metabolism is highly differentiated in Agaricus bisporus

Aleksandrina Patyshakuliyeva1†, Edita Jurak2†, Annegret Kohler3, Adam Baker4, Evy Battaglia1,5, Wouter de Bruijn2, Kerry S Burton6, Michael P Challen7, Pedro M Coutinho8, Daniel C Eastwood9, Birgit S Gruben1,5, Miia R Mäkelä10, Francis Martin3, Marina Nadal5, Joost van den Brink1, Ad Wiebenga1, Miaomiao Zhou1, Bernard Henrissat8, Mirjam Kabel2, Harry Gruppen2 and Ronald P de Vries1,5*

Patyshakuliyeva et al. BMC Genomics 2013, 14:663

http://www.biomedcentral.com/1471-2164/14/663

The most significant information that might be useful in educating about the process of mushroom feeding that was described in this article and that would become an inspiration for further research is presented below.

„Agaricus bisporus is commercially grown on compost, in which the available carbon sources consist

mainly of plant-derived polysaccharides that are built out of various different constituent monosaccharides. The major constituent monosaccharides of these polysaccharides are glucose, xylose, and arabinose, while smaller amounts of galactose, glucuronic acid, rhamnose and mannose are also present. We correlated the expression of genes encoding plant and fungal polysaccharide modifying enzymes identified in the A. bisporus genome to the soluble carbohydrates and the composition of mycelium grown compost, casing layer and fruiting bodies.

The compost grown vegetative mycelium of A. bisporus consumes a wide variety of monosaccharides. However, in fruiting bodies only hexose catabolism occurs, and no accumulation of other sugars was observed. This suggests that only hexoses or their conversion products are transported from the vegetative mycelium to the fruiting body, while the other sugars likely provide energy for growth and maintenance of the vegetative mycelium. Clear correlations were found between expression of the genes and composition of carbohydrates. Genes encoding plant cell wall polysaccharide degrading enzymes were mainly expressed in compost-grown mycelium, and largely absent in fruiting bodies. In contrast, genes encoding fungal cell wall polysaccharide modifying enzymes were expressed in both fruiting bodies and vegetative mycelium, but different gene sets were expressed in these samples.

In nature plant biomass is the main carbon source for many fungal species. A. bisporus (the white button mushroom) is commercially cultivated on a composted mixture of lignocellulose containing materials (mainly wheat straw and horse manure), which is highly selective for this fungus [2,3]. The major constituents of the lignocellulose fraction of compost are cellulose and the hemicellulose xylan (70% of the biomass) [4] and lignin [5-7]. Due to their diverse and complex polymeric nature, degradation of plant cell wall polysaccharides to their monomeric constituent requires a large range of enzymes [8,9]. Most of these enzymes have been divided into families in a classification system for Carbohydrate Active enZymes (CAZy, www.cazy.org) [10]. It has been shown that during mycelial growth and fruiting A. bisporus produces a range of extracellular enzymes, which are involved in the degradation of the lignocellulosic fraction in compost [11-14]. A shift in fungal metabolism takes place during development of the fruiting body of A. bisporus that is closely linked to an increased rate of cellulose and hemicellulose degradation [15]. The production of laccase and cellulase was suggested to be connected to the high rate and flow of carbon metabolism during fruiting body development [16,17]. Lignin degradation by A. bisporus decreases towards the end of the mushroom production cycle [18-20].

Mannitol functions as an osmolyte, which accumulates to high levels during fruiting body growth while after sporulation the level of mannitol decreases rapidly [35]. It might also serve as a post-harvest reserve carbohydrate [31,33,36]. Trehalose also serves as a reserve carbohydrate, which is present at lower levels than mannitol that decline during fruiting body development. It has been suggested that trehalose is synthesized in the mycelium and translocated to the fruiting body [16,32,34].

Expression of most PPP genes is similar in casing, compost and fruiting bodies compared to plate grown mycelium, while only some genes are slightly up- (in compost and casing layer) or down-regulated (in fruiting bodies) (Figure 1, Additional files 3 and 4). There Oxalic acid and citric acid are among the two most commonly produced organic acids by fungi [38]. No specific upregulation for oxalic acid metabolic genes was observed in any of the samples. In contrast, several of the citric acid metabolic genes were expressed at higher levels in fruiting bodies than in compost or the casing layer. These genes are expressed at significantly higher levels in compost than in the other samples. For xylan and cellulose related genes, 90% and 64%, respectively, were expressed in compost while in casing layer and fruiting bodies less than 15% of these genes were expressed. In compost, expression of genes encoding enzymesn targeting other polysaccharides (e.g. starch, pectin and xyloglucan) was also observed.

Fungal cell wall degrading and modifying enzymes have received less attention than plant cell wall degrading enzymes, resulting in a less well defined assignment of function. During growth A. bisporus needs to synthesize and modify its cell wall. As growth occurs in compost, casing layer and fruiting bodies, genes encoding fungal cell wall modifying enzymes need to be expressed in all growth stages. However, as the morphology of these stages is not identical, different genes may be expressed in compost and fruiting bodies. (czy rzeczywiście potrzebne są obumarłe komórki grzybni? Jeżeli tak to dodatek przykładowo drożdży powinien być przydatny) When the A. bisporus mushrooms have matured, compost consists of lignin (41% w/w) and ash (36% w/w), carbohydrates (17% w/w) and proteins (13%).

The casing layer is a mixture of calcium and peat that consists mainly of lignin (52% w/w) and ash (29% w/w). There are few carbohydrates present (14% w/w) and the main monosaccharides released after acid hydrolysis were xylose (1.4% w/w), mannose (0.6% w/w) and glucose (7.5% w/w) [41]. As mentioned above, the actual lignin amount is likely to be lower than measured due to calcium and sandy particles that remain on the filter after acid hydrolysis. Aqueous extraction of compost, casing layer and fruiting bodies revealed that more than 95% of carbohydrates are insoluble.

Changes in free soluble monosaccharides were observed in these samples. Concentrations of arabinose, galactose and xylose were high in compost, while only traces of these monosaccharides were found in casing layer and fruiting bodies (Table 3). High levels of glucose were observed in all samples. Mannitol and trehalose levels were significantly higher in fruiting bodies than in compost and casing layer (Table 3), as were the levels of citric acid (data not shown), while no oxalic acid was detected in the samples. The very high level of sorbitol in the compost samples could suggest a role as a transportable carbon compound from the vegetative mycelium to the fruiting body (Table 3).

Soluble oligosaccharides were detected in the compost, while none were detected in the casing layer or fruiting bodies (Figure 3).

Discussion

In this study, genes encoding carbon metabolic genes  were identified in the genome of A. bisporus and their expression in different growth stages was compared to the available carbohydrates and the expression of genes encoding carbohydrate modifying enzymes.

Analysis of the expression of genes encoding plant and fungal polysaccharide modifying enzymes identified in the A. bisporus genome [37] revealed correlation between these genes and composition of carbohydrates.

A large decrease of carbohydrate content and, therefore, polysaccharides was revealed in the compost after growth of A. bisporus and fruiting body production.

Expression of genes encoding other plant polysaccharide degrading enzymes that are not normally associated with compost, e.g. starch, pectin and xyloglucan related genes, was also detected. In nature A. bisporus can grow on various substrates ranging from leaf litter and soil under cypress in coastal California to manured soil, composts of plant debris, and other horticultural and agricultural situations reported in Europe [43]. Growth on these different substrates is likely due to the ability of A. bisporus to produce a wide range of plant polysaccharide degrading enzymes and it may co-express genes aimed at different polysaccharides.

The casing layer serves as an intermediate phase In the casing layer, which is a mixture of peat and lime, it is likely that the detected glucose and mannose at least partially drive from the mycelial cell wall, in the form of glucans and mannoproteins, respectively. While some genes encoding putative plant cell wall degrading enzymes were expressed in the casing layer, the level of up-regulation compared to plate-grown mycelium is much smaller than that in compost. In addition, expression

of some chitinase encoding genes was detected. The casing layer seems to be an intermediate phase in which some genes related to plant biomass degradation are expressed, but also modification of the A. bisporus cell wall is an important process for the conversion to fruiting body morphology. The lack of soluble polysaccharides indicates that the role of the mycelium in the casing layer is mainly to supply carbohydrates to the fruiting body.

This suggests that A. bisporus has specific genes for mycelium development and growth and others for fruiting body formation and modification.

These results support the compositional and morphological differences found between mycelium and fruiting bodies [35]. Expression of different sets of genes encoding fungal cell wall modifying enzymes has also been described for other fungi. Enzymes from families GH5 and CE4 have several described activities, some of which are related to plant cell wall polysaccharides, while others are related to fungal cell wall polysaccharides (www.cazy.org). For some of the enzymes from these families upregulation in compost was observed, while others were upregulated in fruiting bodies.

Expression analysis demonstrated that the pentose catabolic pathway and galacturonic acid pathway were strongly upregulated in compost and moderately upregulated in the casing layer, while they were down regulated in fruiting bodies.

The concentration of mannitol in fruiting bodies was six times higher than in compost. However, expression of mannitol pathway genes was significantly lower in fruiting bodies than in compost, suggesting that Mannitol is synthesized in the vegetative mycelium and transported to the fruiting body. Earlier studies observed that mannitol functions as an osmoregulatory compound and facilitates a continuous influx of water from compost to the fruiting body to support turgor and fruiting body development [58,59]. This would suggest that mannitol is unlikely to be transported by diffusion from the mycelium. Therefore, it should either be transferred by active transport or alternatively, be synthesized in the fruiting body. If the latter is the case, a possible explanation for the observed expression of the genes could be that the encoded enzymes are transported into the fruiting body.

As citric acid is known to have preservative properties against bacteria in food [60], it is tempting to speculate that the accumulation of citric acid in fruiting bodies may also be involved in the defence mechanism of the mushroom against bacteria.

A. bisporus is highly dependent on obtaining carbon from its surroundings. In contrast, the mycorrhizae L. bicolor obtains carbon from its symbiotic partner in the form of sucrose, placing a much lower demand on a versatile carbon metabolism.

Conclusions

The data from our study demonstrates that overall there is a clear correlation between expression of genes related to plant and fungal polysaccharides and the ability of A. bisporus to degrade these polysaccharides.

We see a clear difference in genes expressed within mycelium grown compost and fruiting bodies supporting the hypothesis that different genes are expressed in A. bisporus mycelium and fruiting bodies. This supports previous results that this fungus produces different enzymes during its life cycle [64]. However, it should also be recognized that gene expression is likely to be dynamic and here we have examined it at the time point when first flush was harvested (approximately 34 days after compost was inoculated with spawn.

Moreover, our study demonstrates a clear correlation between the expression of genes encoding plant and fungal cell wall polysaccharides with the composition of carbohydrates in compost, casing layer and fruiting bodies. Genes encoding plant cell wall polysaccharide degrading enzymes were mainly expressed in compost grown mycelium, and largely absent in fruiting bodies. In contrast, genes encoding fungal cell wall polysaccharide modifying enzymes were expressed in both fruiting bodies and vegetative mycelium in the compost, but different gene sets were expressed in these samples. In the present study an in silico metabolic reconstruction of the central carbon metabolism in A. bisporus was performed and combined with expression analysis of the relevant genes in different growth stages of A. bisporus. The analysis of metabolic pathways in A. bisporus may provide information about the requirements of carbon source and energy metabolism during commercial growth of A. bisporus. We showed that during growth in compost and casing a much larger variety of carbon sources was used by A. bisporus than during growth on synthetic medium. In contrast, carbon metabolism in fruiting bodies appears to be mainly aimed at hexoses. This could indicate that only these sugars are transported towards the fruiting body from the vegetative mycelium, which implies that carbon transport to the fruiting bodies is a highly regulated and selective process. “

My comments:

1. The casing layer can participate in the process of mushroom feeding in addition to its basic role, which is providing moisture essential for high yields and environment transporting nutrients by rhizomorphic mycelium. The possibility of digestion of polysaccharides existing in a casing layer during its colonization signifies necessity of performing tests with double casing layer and the first conducted test confirms that approach. Forming nutritional sources within casing materials seem to be particularly interesting because of the short transport of nutrients to fruiting bodies and primordia. It creates opportunity of decreasing the amount of substrate in mushroom production and thus might become essential element of cultivation without compost.
2. Information provided in this paper confirms that the main role in mushroom feeding is degradation of polysaccharides into monosaccharides – digestible nutritional elements. The studies concentrate on function of Carbohydrate Active enzymes. The paper is missing information regarding the role of proteins in the feeding process, and their utilization when they are applied within a supplement based on soybean. This justifies the use of corn meal.
3. Attention is also drawn to the possibility of employing starch in the feeding process, the main component of corn kernel.
4. There is interesting data, which says that mushrooms contain enzymes degrading cell walls of fungi, which are present in compost. This fact makes usage of waste products in the feeding process possible, for instance from alcohol fermentation. The performed tests confirm that.
5. The paper provides explanation regarding a yield decrease in the third and fourth flush that is caused by a different set of enzymes being formed after harvesting first flush. It requires reevaluation of the mushroom feeding process from this phase, particularly in the cultivation model without compost and possibilities of increasing yields in the third and following flushes.
6. Mushroom mycelium has the capability of producing a range of enzymes depending on a carbon source. Therefore, the usage of components other than compost, soybeans and corn in the feeding process might have a much broader application.
7. Does introduction of mannitol into a diet make sense? If yes, it raises a question how much and when.
8. The presence of citric acid in the feeding process can be questioned as far as its usefulness in mushroom cultivation is considered. Its addition to the irrigation system might be improving mushroom appearance and also protect against bacterial diseases.
9. Both soluble carbohydrates adsorbed by mushroom and insoluble that require enzyme degradation are present in the compost. In the tested cultivation model without a compost, introduction of monosaccharides into a subtract significantly accelerated colonization of supplement and structural part that supports spawn structure and spawn construction of mycelium

The presented conclusions do not illustrate all benefits of the conducted experiments and it might be valuable to discuss this data once again.