Extremely acidophilic filamentous fungi are more prevalent in diverse ecosystems than previously documented

Abstract

The study of extremophiles can lead to the discovery of highly tolerant enzymes of value to biotechnology, and extreme environments are typically sampled to facilitate their discovery. Here, we show that extreme acidotolerant filamentous fungi, able to grow at pH < 1, can be found when sampling highly diverse environments, from industrial sites with low pH to typical non-acidic plant and soil samples. Over 100 fungal strains were isolated from over 2,000 samples taken from across Vietnam, and many of the strains were able to grow over wide pH ranges, displaying either acidotolerance or clear acidophilicity. ITS sequencing revealed that 63 isolates represent 12 previously undescribed species, with the majority from the Talaromyces or Penicillium genera. We furthermore report the rediscovery of the previously lost, historically significant acidophile Acontium velatum. Screening of selected fungal secretomes for polysaccharide-cleaving activity revealed that many show broad tolerance to harsh conditions (pH, temperature, organic solvents). Our work greatly expands on the diversity of identified extreme acidotolerant and acidophilic filamentous fungi, which can serve as sources of industrially relevant enzymes. For most species identified, acid tolerance or acidophilicity has not previously been reported, and our results showcase that acidophilicity is more widespread than previously appreciated.

Keywords: Acontium; Penicillium; Talaromyces; Acidophile; Acidotolerant; Ascomycota; Biodiversity; CAZymes; Extremophile; New fungal taxa.

Fig. 1

Origin of the acidotolerant and acidophilic fungal strains identified and screened. (A) Sampling locations. Green circles indicate coordinates of sampled locations where strains were subsequently isolated and determined to grow under acidic selection pressure. Yellow squares indicate the sampling sites where strains were selected for ITS sequencing. Red crosses indicate acidic environments where strains were successfully isolated under acidic conditions and ITS sequenced. Hanoi is shown in dark grey. Detailed sampling information, including coordinates, strain information and taxonomy, isolation location and environment are provided in Supplemental File 1. (B) Geographical heatmap of number of isolated strains (N) across Vietnam, sorted by province. Vector shapefiles were obtained from the Humanitarian Data Exchange (https://data.humdata.org/). The Python 3 code for this figure is provided in Supplemental File 2.

Fig. 2

Growth of selected strains of acid tolerant/acidophilic fungi on buffered malt agar at different pH values. The media were buffered and contained 1% sulfuric acid and 1% citric acid. Agar plate pH values were checked at the end of the growth period to ensure there was no change. Aspergillus oryzae CNTP 5138 was used as a control strain, as an industrial, non-acidotolerant species. The fungi were grown at 28 °C for one week, except for the slow-growing Acidomyces spp. and Acontium velatum (two weeks).

Fig. 3

Maximum likelihood phylogenetic tree depicting the diversity of extreme acid tolerant/acidophilic fungi isolated from Vietnam, and their relationship to closely related/known extreme acid tolerant/acidophilic fungi. The tree was constructed based on ITS sequences using the Maximum Likelihood method and Tamura-Nei model in MEGA11. An alignment of 870 positions was used for analysis. All ambiguous positions were removed for each sequence pair. GenBank accession numbers of ITS sequences are given after the taxon names. The scale bar represents 5% sequence divergence; genus groupings colored in alternating blue and orange (for clarity) represent strains obtained in this study; type strains are shown in black with no color bar; type strains are in bold and marked with an asterisk (*) are known extreme acid tolerant/acidophilic taxa.

Fig. 4

Plate cultures and conidiogenesis of selected acidophilic and acid tolerant fungi. By row: Av - Acontium velatum ASS 125.1 on malt agar containing 1% sulfuric acid, 3 weeks; As - Amplistroma sp1 ASS 88.1 on potato dextrose agar (PDA), 1 week; Pg - Penicillium griseolum ASS 45.1 on PDA, 1 week; At - Aspergillus turcosus ASS 350.1 on PDA, 1 week. Bars represent 10 μm (center) and 5 μm (right).

Fig. 5

Measurement of (A) xylanase and (B) CMCase activities of the secretomes of acidotolerant and acidophilic fungi grown on complex substrates (1.5 g/100 mL each of malt residue, sugarcane bagasse, and rice bran). All activities were measured at 50 °C. In each panel is shown the maximum specific activity (U/mg); the relative activity level at pH 1, 3, 5 and 7, scaled to the highest activity observed as 100%; and lastly the residual activity after heating at 70 °C for 20 min followed by overnight cooling at 4 °C before measuring activity at 50 °C. Each value is the average of duplicate measurements. Blanks containing no substrate were subtracted. On the left is shown an unscaled UPGMA (unweighted pair group method with arithmetic mean) tree.