Effects of lithium compounds on the growth of white-rot fungi

Identifying the most suitable lithium compounds for fungal growth is important for the lithium (Li) enrichment of mushrooms. However, enrichment thus far has been carried out using LiCl. In an effort to identify an alternative source of lithium, we investigated the effect of five lithium compounds on lag phase, growth rate and biomass of ten species of white-rot fungi. The objective of this work was to make a screening of promising lithium compounds for further studies of Li enrichment of mushrooms. The fungal growth was affected depending on the species and lithium form tested. Lithium sulfate and lithium chloride were found to be the most promising candidate compounds for the Li enrichment of mushrooms.


INTRODUCTION
Lithium is not an essential mineral for humans (Schrauzer, 2002), but studies have suggested that its uptake can influence human behavior (Dawson et al., 1970;Schrauzer and Shrestha, 1990;Severus et al., 2009).Indeed, lithium salts are commonly used to treat bipolar disorder, but the side effects caused by ingestion of these salts are numerous and severe (Kjølholt et al., 2003;Aral and Vecchio-Sadus, 2008;Ghaemi, 2010), so much so that their sale in the USA was prohibited in 1949 by the Food and Drug Administration and not resumed until 1970.The main sources of lithium for human consumption are vegetables and grains (Schrauzer, 2002).Lithium level in mushrooms varies depending lithium availability and ability of fungi to accumulate lithium.In the environmental, this element has been found at average concentration of 0.189 ppm in mushrooms (Vetter, 2005).According to Vetter (2005) due to the low levels of lithium, the mushrooms are not suitable source of lithium for humans.However, mushrooms can be enriched with lithium (de Assunção et al., 2012).
Li-enriched mushrooms have been shown by de Assunção et al. (2012) to be a promising alternative source of lithium due to the higher solubility in water of the lithium found in the mushrooms than lithium carbonate, which is interesting as solubility in water is one of the factors that affect the absorption of compounds in the intestine.Therefore, the lithium found in the mushrooms can be more bioavailable to humans than lithium carbonate.As the control of lithium consumed by direct consumption of Li-enriched mushrooms is difficult, development of medicines based on the lithium found in the mushrooms seems to be a good approach to provide new lithium compounds for humans and perhaps, decrease the side-effects currently observed.
Only the mushroom Pleurotus ostreatus was shown to be capable of Li enrichment.Nunes et al. ( 2014) screened 12 white-rot fungi with the goal of identifying other mushrooms suitable for Li-enrichment and found that Pleurotus djamor and Pholiota nameko were more resistant to LiCl than P. ostreatus, making these species promising candidates.
However, both studies use LiCl for enrichment (de Assunção et al., 2012;Nunes et al., 2014).Chlorine is known to have antimicrobial properties (Wilson et al., 2005).Therefore, other lithium compounds may be more suitable for use in Li enrichment.Furthermore, the accessibility and price of different lithium compounds vary between regions.Therefore, knowing which lithium compounds can be used effectively is useful information.
To make a screening of promising lithium compounds for further studies of Li enrichment of mushrooms, we tested the effect of five lithium compounds on development of nine white-rot species that produce commercially available edible mushrooms.

Culture media and cultivation conditions
Fungi were grown on PDA containing one of the following lithium compounds: lithium acetate, lithium chloride, lithium hydroxide, lithium sulfate, or lithium carbonate.The pH of all media was adjusted to 5.5.The concentrations chosen for enrichment of the PDA medium were determined by the lithium content of each compound (Table 1) and the concentration of LiCl that allowed mycelial growth for each fungus in previous experiments (Nunes et al., 2014).It should be noted that lithium concentrations used were different for each fungus, but lithium molar concentrations were equal among different compounds.The culture medium was then autoclaved at 121C for 20 min.PDA plugs of inoculum 5 mm in diameter containing active mycelium were cut from the board of the colony.Inoculum plugs were firmly placed with the mycelium side down in the centers of Petri dishes.Four replicate plates were prepared for each lithium salt and fungus and were incubated at 22 ± 1ºC with room moisture.

Lag phase and growth rate
After incubation, colonies were observed daily to determine the start of mycelial growth.The fungal growth rate was determined by measuring each colony's diameter in two orthogonal directions.Measurements were made for 45 d or until maximum Petri dish colonization.Measurements were taken every 48 h.

Biomass
To determine the mycelial dry mass, the entire contents of the Petri dish (mycelium + culture media) were placed in a bottle with approximately 200 mL of distilled water and heated in a water bath for 1-5 min to dissolve all culture medium (da Silva et al., 2013).The solution was then filtered, and retained mycelium was dried in an oven at 80 ºC until a constant weight was reached.

Statistical analysis
Experiments used a randomized design.The data were subjected to analysis of variance (ANOVA), and the averages were compared by Tukey's test (P < 0.05) using Minitab statistical software (Version 16.0).

RESULTS AND DISCUSSION
The lag phase of five fungi increased due to the addition of lithium compounds to the culture medium (Figure 1).One-way ANOVA revealed significant effects of lithium shimeji showed the highest increase in lag phase (Figure 1).Moreover, we observed that different lithium compounds had a similar negative influence on the lag phase (Figure 1), which suggest that the lithium is the main factor influencing this fungal growth phase.
We observed a significant effect (P < 0.05) of lithium compounds on the growth rate and biomass of all fungi tested (Figures 2 and 3).Richter et al. (2008) noted that the radial growth rate may not represent fungal biomass reduction.Indeed, biomass evaluation has been shown to be a more sensitive method (Nunes et al., 2014).The profiles of biomass and growth rate data observed clearly show that lithium acetate and lithium carbonate were the most toxic compounds (Figures 2 and 3).Acetate is an organic acid that is able to cross the plasmatic membrane and affect cytoplasmic pH, negatively affecting many metabolic pathways (Cheung et al., 2010).Carbonate has been shown to strongly inhibit Nce103, an enzyme that participates in many physiological processes in eukaryotes (Innocenti et al., 2008).The fungal growth inhibition observed (Figure 2) suggests that these phenomena may be occurring in the fungal cell.Thus, these compounds are not recommended for use in fungal Li enrichment.
The effect of lithium hydroxide on growth rate and biomass varies among the fungal strains tested (Figures 2 and 3).For some fungi, lithium hydroxide results were similar (P > 0.05) to those obtained for lithium sulfate and lithium chloride (Figures 1 and 3).In contrast, the addition of lithium hydroxide decreased (P < 0.05) the growth rate and biomass of P. nameko (PHOLI), G. subamboinense var.laevisporum (GR 117), P. eryngii (PLE 04) and L. edodes (UFV 73), clearly showing that this lithium compound was more toxic for these fungi.Furthermore, lithium hydroxide increased (P < 0.05) the lag phase of P. nameko (PHOLI).In addition, Xu (1997) showed that the activity of laccase, an important factor for white-rot fungus growth, was affected by hydroxide.Furthermore, it is not known how the effects of lithium hydroxide may vary with variation among fungal strains.Therefore, we do not recommend using lithium hydroxide for the Li enrichment of fungi.
The similar profile for lag phase, growth rate and dry mass observed in this study shows that lithium sulfate and lithium chloride were the less toxic compounds to fungi (Figures 1 and 3 ).As observed by de Assunção et al. (2012), Li is incorporated in the fungus when LiCl is added to the substrate used for mycelial growth.Thus, we can assume that lithium sulfate and chloride are the most promising for the Li enrichment of mushrooms.However, the effect of these lithium compounds on important parameters of mushroom cultivation and bioaccumulation of Li should be investigated.

Figure 1 .
Figure 1.Lag phase of white rot fungi in culture media supplemented with different lithium compounds.Mean growth of 4 replicates.Missing bars indicates fungal growth inhibition.Letters above columns compare individual fungi by lithium compounds level (Tukey´s test; p ≤ 0.05).

Figure 2 .
Figure 2. Mycelial growth rate of white rot fungi in culture media supplemented with different lithium compounds.Mean growth of 4 replicates Missing bars indicates fungal growth inhibition.Letters above columns compare individual fungi by lithium compounds level (Tukey´s test; p ≤ 0.05).

Figure 3 .
Figure 3. Mycelial dry mass of white rot fungi in culture media supplemented with different lithium compounds.Mean growth of 4 replicates.Missing bars indicates fungal growth inhibition.Letters above columns compare individual fungi by lithium compounds level (Tukey´s test; p ≤ 0.05).

Table 1 .
Concentration of lithium in culture medium according to fungi specie.