Microbial communities from a lake and river flowing through a highly dense urbanized township in Malaysia were profiled by sequencing amplicons of the 16S V3-V4 and 18S V9 hypervariable rRNA gene regions via Illumina MiSeq. Results revealed that Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes were the dominant prokaryotic phyla; whereas, eukaryotic communities were predominantly of the SAR clade and Opisthokonta. The abundance of Pseudomonas and Flavobacterium in all sites suggested the possible presence of pathogens in the urban water systems, supported by the most probable number (MPN) values of more than 1600 per 100 mL. Urbanization could have impacted the microbial communities as transient communities (clinical, water-borne and opportunistic pathogens) coexisted with common indigenous aquatic communities (Cyanobacteria). It was concluded that in urban water systems, microbial communities vary in their abundance of microbial phyla detected along the water systems. The influences of urban land use and anthropogenic activities influenced the physicochemical properties and the microbial dynamics in the water systems.
Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-020-02617-3.
A rare fungal endophyte, identified as Buergenerula spartinae (C28), was isolated from the roots of Cymbidium orchids and was characterised and evaluated for its antimicrobial activities. Bio-guided fractionation revealed 4 fractions from B. spartinae (C28) having antibacterial activities against at least one bacterial pathogen tested (Bacillus cereus and Staphylococcus aureus). However, inhibitory activities were absent against pathogenic fungi (Ganoderma boninense, Pythium ultimum and Fusarium solani). Fraction 2 and fraction 4 of B. spartinae (C28) exhibited potent antibacterial activities against S. aureus (MIC: 0.078 mg/mL) and B. cereus (MIC: 0.313 mg/mL), respectively. LCMS analysis revealed the presence of antibacterial agents and antibiotics in fraction 2 (benoxinate, pyropheophorbide A, (-)-ormosanine and N-undecylbenzenesulfonic acid) and fraction 4 (kaempferol 3-p-coumarate, 6-methoxy naphthalene acetic acid, levofuraltadone, hinokitiol glucoside, 3-α(S)-strictosidine, pyropheophorbide A, 5'-hydroxystreptomycin, kanzonol N and 3-butylidene-7-hydroxyphthalide), which may be responsible for the antibacterial activities observed. Most of the bioactive compounds profiled from the antibacterial fractions were discovered for the first time from endophytic isolates (i.e. from B. spartinae (C28)). Buergenerula spartinae (C28) from Cymbidium sp. is therefore, an untapped resource of bioactive compounds for potential applications in healthcare and commercial industries.
The influence of light exposure on antioxidant and antimicrobial activities of nine fungal isolates [Pseudopestalotiopsis theae (EF13), Fusarium solani (EF5), Xylaria venustula (PH22), Fusarium proliferatum (CCH), Colletotrichum boninese (PL9), Colletotrichum boninese (PL1), Colletotrichum boninese (OL2), Colletotrichum gloeosporioides (OL3) and Colletotrichum siamense (PL3)] were determined. The isolates were incubated in blue, red, green, yellow and white fluorescent light (12 h photoperiod of alternating light/dark). It was observed that green light induced higher total phenolic content (TPC) (2.96 ± 0.16 mg-30.71 ± 1.03 mg GAE/g) and ferric reducing antioxidant power (FRAP) in most isolates (4.82 ± 0.04-53.55 ± 4.33 mg GAE/g), whereas red light induced higher total flavonoid content (TFC) levels (1.14 ± 0.08-18.40 ± 1.12 mg QE/g). The crude extracts from most fungal cultures exposed to green and red lights were also notably more potent against the tested pathogens, as larger zones of inhibition (ZOI) (9.00 ± 1.00-38.30 ± 2.90 mm) and lower minimum inhibitory concentration (MIC) (0.0196-1.25 mg/mL) were achieved for antimicrobial effect. This study showed that light treatments are effective strategies in enhancing production of more potent antimicrobial compounds and valuable antioxidants from fungal isolates.
Biodegradation is an eco-friendly measure to address plastic pollution. This study screened four bacterial isolates that were capable of degrading recalcitrant polymers, i.e., low-density polyethylene, polyethylene terephthalate, and polystyrene. The unique bacterial isolates were obtained from plastic polluted environment. Dermacoccus sp. MR5 (accession no. OP592184) and Corynebacterium sp. MR10 (accession no. OP536169) from Malaysian mangroves and Bacillus sp. BS5 (accession no. OP536168) and Priestia sp. TL1 (accession no. OP536170) from a sanitary landfill. The four isolates showed a gradual increase in the microbial count and the production of laccase and esterase enzymes after 4 weeks of incubation with the polymers (independent experiment set). Bacillus sp. BS5 produced the highest laccase 15.35 ± 0.19 U/mL and showed the highest weight loss i.e., 4.84 ± 0.6% for PS. Fourier transform infrared spectroscopy analysis confirmed the formation of carbonyl and hydroxyl groups as a result of oxidation reactions by enzymes. Liquid chromatography-mass spectrometry analysis showed the oxidation of the polymers to small molecules (alcohol, ethers, and acids) assimilated by the microbes during the degradation. Field emission scanning electron microscopy showed bacterial colonization, biofilm formation, and surface erosion on the polymer surface. The result provided significant insight into enzyme activities and the potential of isolates to target more than one type of polymer for degradation.
Basal stem rot (BSR), caused by Ganoderma boninense, is the most devastating oil palm disease in South East Asia, costing US$500 million annually. Various soil physicochemical parameters have been associated with an increase in BSR incidences. However, very little attention has been directed to understanding the relationship between soil microbiome and BSR incidence in oil palm fields. The prokaryotic and eukaryotic microbial diversities of two coastal soils, Blenheim soil (Typic Quartzipsamment-calcareous shell deposits, light texture) with low disease incidence (1.9%) and Bernam soil (Typic Endoaquept-non-acid sulfate) with high disease incidence (33.1%), were determined using the 16S (V3-V4 region) and 18S (V9 region) rRNA amplicon sequencing. Soil physicochemical properties (pH, electrical conductivity, soil organic matter, nitrogen, phosphorus, cation exchange capacity, exchangeable cations, micronutrients, and soil physical parameters) were also analyzed for the two coastal soils. Results revealed that Blenheim soil comprises higher prokaryotic and eukaryotic diversities, accompanied by higher pH and calcium content. Blenheim soil was observed to have a higher relative abundance of bacterial taxa associated with disease suppression such as Calditrichaeota, Zixibacteria, GAL15, Omnitrophicaeota, Rokubacteria, AKYG587 (Planctomycetes), JdFR-76 (Calditrichaeota), and Rubrobacter (Actinobacteria). In contrast, Bernam soil had a higher proportion of other bacterial taxa, Chloroflexi and Acidothermus (Actinobacteria). Cercomonas (Cercozoa) and Calcarisporiella (Ascomycota) were eukaryotes that are abundant in Blenheim soil, while Uronema (Ciliophora) and mammals were present in higher abundance in Bernam soil. Some of the bacterial taxa have been reported previously in disease-suppressive and -conducive soils as potential disease-suppressive or disease-inducible bacteria. Furthermore, Cercomonas was reported previously as potential bacterivorous flagellates involved in the selection of highly toxic biocontrol bacteria, which might contribute to disease suppression indirectly. The results from this study may provide valuable information related to soil microbial community structures and their association with soil characteristics and soil susceptibility to Ganoderma.
Three species of the lichen Usnea (U. baileyi (Stirt.) Zahlbr., U. bismolliuscula Zahlbr. and U. pectinata Stirt.) and nine associated endolichenic fungi (ELF) were evaluated using a metabolomics approach. All investigated lichen crude extracts afforded antibacterial activity against Staphylococcus aureus (minimum inhibitory concentration (MIC): 0.0625 mg/mL), but none was observed against Escherichia coli, while the ELF extract Xylaria venustula was found to be the most active against S. aureus (MIC: 2.5 mg/mL) and E. coli (MIC: 5 mg/mL). X. venustula was fractionated and tested for to determine its antibacterial activity. Fractions XvFr1 to 5 displayed bioactivities against both test bacteria. Selected crude extracts and fractions were subjected to metabolomics analyses using high-resolution LC-MS. Multivariate analyses showed the presence of five secondary metabolites unique to bioactive fractions XvFr1 to 3, which were identified as responsible for the antibacterial activity of X. venustula. The p-values of these metabolites were at the margin of significance level, with methyl xylariate C (P_60) being the most significant. However, their high variable importance of projection (VIP) scores (>5) suggest these metabolites are potential diagnostic metabolites for X. venustula for "dual" bioactivity against S. aureus and E. coli. The statistical models also showed the distinctiveness of metabolites produced by lichens and ELF, thus supporting our hypotheses of ELF functionality similar to plant endophytes.
Metal removal potential of both alginate-immobilized and free-cells of Effective Microorganisms (EM-1™ Inoculant) was investigated in this study. Results revealed that removal of Cr(III), Cu(II) and Pb(II) followed a similar trend where alginate-immobilized EM were more efficient compared to free-cells of EM. For these metals, 0.940, 2.695 and 4.011 mg g(-1) of Cr(III), Cu(II) and Pb(II) were removed compared to only 0.160, 0.859 and 0.755 mg ml(-1) removed by free-cells, respectively. The higher efficiency of alginate-immobilized EM was primarily attributed to the alginate matrix. This was evident when both alginate-immobilized EM and plain alginate beads (without EM), were not significantly different in their removal efficacies. Presence of alginate also enhanced the use of the biosorbents as maximum metal sorption was achieved after 120 min as opposed to only 60 min for free-cells. EM per se in immobilized or free-cell forms did not enhance metal removal efficacy.
The influence of light regulation on the growth and enzyme production of three endolichenic fungal isolates, i.e. Pseudopestalotiopsis theae (EF13), Fusarium solani (EF5), and Xylaria venustula (PH22), was determined. The isolates were exposed to blue, red, green, yellow, white fluorescent light (12 h light-12 h dark photoperiod) (test), and 24 h dark (control) conditions. Results revealed that the alternating light-dark conditions resulted in the formation of dark rings in most fungal isolates but was absent in PH22. Red light induced sporulation while yellow light elicited higher biomass in all isolates (0.19 ± 0.01 g, 0.07 ± 0.00 g, and 0.11 ± 0.00 g, for EF13, PH22, and EF5, respectively) as compared to incubation in the dark. Results also showed that blue light induced higher amylase activity in PH22 (15.31 ± 0.45 U/mL) and L-asparaginase activity in all isolates (0.45 ± 0.01 U/mL, 0.55 ± 0.39 U/mL, and 0.38 ± 0.01 U/mL, for EF13, PH22, and EF5, respectively) compared to both control conditions. Green light enhanced the production of xylanase (6.57 ± 0.42 U/mL, 10.64 ± 0.12 U/mL, and 7.55 ± 0.56 U/mL for EF13, PH22, and EF5, respectively) and cellulase (6.49 ± 0.48 U/mL, 9.57 ± 0.25 U/mL, and 7.28 ± 0.63 U/mL, for EF13, PH22, and EF5, respectively). In contrast, red light was the least effective light treatment as production of enzymes was the least, with lower levels of amylase, cellulase, xylanase, and L-asparaginase detected. To conclude, all three endolichenic fungi are light-responsive, with fungal growth regulated with the use of red light and yellow light, and manipulation of enzyme production via blue and green light.
This study profiled the various endophytic fungi isolated from the orchid Cymbidium sp. and their L-asparaginase production and antioxidant potential. The L-asparaginase production was first screened through qualitative plate screening then quantified by the Nesslerization method. The antioxidant potential was quantified via the 2,2-diphenyl-1-picrylhydrazyl assay. A total of 30 endophytic fungi were isolated and all fungal isolates exhibited various degrees of radical scavenging activities (45.28% to 76.4%). Isolate Lasiodiplodia theobromae (C11) had the highest antioxidant capacity, represented by the lowest IC50 value (5.75 mg/mL) and highest ascorbic acid equivalent antioxidant capacity value (12.17 mg/g). Additionally, 16 isolates produced L-asparaginase (53.33%), which includes primarily species of Fusarium proliferatum, Fusarium fujikuroi, Fusarium incarnatum, and Fusarium oxysporum. A new isolate has also been discovered from Cymbidium orchid, Buergenerula spartinae (C28), which showed the highest L-asparaginase activity (1.736 unit/mL). These findings supported the postulation that medicinal species of Orchidaceae such as Cymbidium sp. harbor endophytes that are producers of L-asparaginase and antioxidants with various potential applications.
This study characterized and identified the antimicrobial compounds from an endophytic fungus (Fusarium incarnatum (C4)) isolated from the orchid, Cymbidium sp. Chromatographic techniques were employed to separate the bioactive compounds from the crude extracts of F. incarnatum (C4). Following bio-guided fractionation, two fractionated extracts (fractions 1 and 2) of F. incarnatum (C4) exhibited antibacterial and antifungal activities against Bacillus cereus (MIC: 0.156 mg/mL) and Ganoderma boninense (MIC: 0.3125 mg/mL), respectively. The active fractions were discovered to comprise of a variety of bioactive compounds with pharmacological importance (alkaloids, flavonoids, phenolic compounds, terpenoids, peptides and fatty acids). Liquid chromatography mass-spectrometry (LCMS) analysis detected the presence of antibacterial (kanzonol N, rifaximin, linoleic acid (d4), cannabisativine, docosanedioic acid, and stearamide) and antifungal components (3-methyl-quinolin-2-ol, prothiocarb, kanzonol N, peganine, 5Z-tridecene, and tetronasin) in fractions 1 and 2, respectively, which may have contributed to the antimicrobial effects. Findings from this study highlighted the important potential of fungal endophytes from medicinal hosts as producers of antimicrobials and antibiotics.
The influence of light regulation on fungal growth and enzyme production was tested on endophytic isolates of Fusarium proliferatum (CCH), Colletotrichum boninense (PL1, PL9, OL2), Colletotrichum gloeosporiodes (OL3) and Colletotrichum siamense (PL3). The isolates were treated with blue, red, green, and yellow light, while white fluorescent light (12 h light/12 h dark photoperiod) and 24 h dark conditions were applied as control. Results revealed that coloured light treatments induced formation of circadian rings, while exposure to white light and dark conditions showed less pronounced circadian rings. Growth and sporulation of endophytes were not significantly influenced by light. By contrast, enzyme production was affected by coloured light treatments, notably with red (amylase), blue (cellulase) and yellow (cellulase, xylanase, L-asparaginase) light, resulting in lower enzyme levels for certain isolates. Under control conditions, enzyme production was relatively higher for amylase, cellulase, xylanase (for cultures incubated in the dark), and for L-asparaginase (for cultures incubated in white fluorescent light). Among the endophytic isolates, F. proliferatum (CCH) showed better response to coloured light treatment as higher sporulation and enzyme production was detected, although growth was significantly suppressed. On the contrary, C. gloeosporiodes (OL3) showed better growth but significantly lower enzyme production and sporulation when treated with the various coloured light. This study revealed that coloured light may have the potential to manipulate growth, sporulation and enzyme production in certain fungal species as strategies for fungal control or for harnessing of valuable enzymes.
Trichoderma asperellum (Ta) was first cultured in synthetic medium (Potato Dextrose Broth, PDB) of various concentrations (100, 75, 50, 25%). The biomass was harvested and inoculated into dye solutions (crystal violet, CV; methyl violet, MV; malachite green, MG; and cotton blue, CB). Reduced concentrations (20, 50, 75%) affected growth rate but their decolourization efficacies remained unaffected. This was attributed to similar numbers and types of functional groups (hydroxyl, amine, ester-lipid, alkane groups) found on the surface of fungal biomass, as revealed by the Fourier transformed infrared spectroscopy (FTIR) analysis. Their production of NADH-reductase for degradation, and their biosorption activities were also unaffected. In general, Ta cultured in reduced concentrations (20, 50, 75%) retained the ability to perform biosorption and biodegradation, similar to cultures from control (100% PDB). This suggested that reduced nutrient levels (as a cost-feasible strategy) could be used to cultivate biomass of Ta for dye removal activities.
The isolate Coriolopsis sp. (1c3) was cultured on muslin cloth to induce formation of filamentous biofilm. The biofilm and the free-mycelium forms (control) were then used to treat two triphenylmethane dyes; Cotton Blue (CB) and Crystal Violet (CV). The biofilm comprised primarily of a compact mass of mycelium while sparse mycelium network was detected in free-mycelium forms. Results revealed significant decolourization activities by filamentous biofilm of 1c3 for CB (79.6%) and CV (85.1%), compared to free-mycelium forms (72.6 and 58.3%, for CB and CV, respectively). Biodegradation occurred in both biofilm and free-mycelium forms. FTIR spectra revealed that biofilm formation (stacking of mycelium), did not have severe implications to the number and types of functional groups available for dye biosorption. The findings here suggested that formation of biofilm in 1c3 was induced effectively on muslin cloth, leading to enhanced decolourization activities. This technology is simple, feasible and can be adopted and further improved to obtain biofilm to enhance their dye removal efficiency in aqueous solutions.
The increasing consumer demand for higher quality fruit juices has encouraged the use of non-thermal processing to extend the shelf life of perishable juice, watermelon juice. Ozone with its high oxidizing effect serve as an effective non-thermal processing treatment. The aim of this study was to investigate the impact of ozone treatment on the physico-chemical, bioactive compounds, pectin methylesterase (PME) activity and microbiological properties of unclarified and clarified watermelon juice. The ozone gas was pumped into watermelon juice for up to 25 min in a closed chamber. The microorganism inactivation in unclarified and clarified watermelon juices improved across the increasing processing time. Among these juices, the microorganism inactivation efficiency of ozone was found higher on clarified juice (3.466 log) than unclarified juice (3.150 log). It was found that °Brix value and PME activity were not altered by ozone treatment. The other physico-chemical properties (titratable acidity, pH, total colour difference, non-enzymatic browning, cloudiness) and bioactive compounds reduced across processing time. This study demonstrated that ozone treatment is an effective non-thermal processing technique to reduce the microorganism in watermelon juice. Further study is required to optimise the processing parameters of ozone treatment to maintain the overall quality of the watermelon juice.
Fresh food products are highly prone to oxidation and microbial attack, rendering them unsuitable for consumption. Thus, active food packaging was developed to protect and prolong food shelf-life. Zein/gellan gum (GG) based active film is developed by incorporating rosemary oleoresin extract (ROE) (0-20%). The films were characterized by their barrier and antioxidant properties. The release behavior of ROE in fatty and hydrophilic food stimulants was investigated via mathematical modeling. The active films incorporated with 20% ROE have significantly higher oxygen barrier and oxygen transfer is reduced by 20% compared to the control. A tortuous path is created with ROE, which impedes oxygen movement across the film. ROE addition improved water resistance performance by reducing the active film swelling ratio by 31%. This improvement is attributed to the hydrophobic nature of ROE. FTIR shows that the interaction between ROE and the active film is primarily hydrogen bonding and electrostatic interactions. Active film exhibits excellent antioxidant activity, with high TPC, DPPH scavenging activity, and FRAP. Mathematical modeling revealed a higher diffusivity (D) of ROE in fatty food stimulants at 24 °C, attributed to high polarity and solubility in fatty food stimulants. Overall, this active film has an excellent antioxidant effect and could potentially be used as food packaging for high-fat food products to prevent oxidation.
Volatile organic compounds (VOCs) are important to determine the aroma and sensory perception of cocoa. Starter cultures can modulate the volatile profile of cocoa beans during fermentation. This study aimed to determine the VOCs and sensory of chocolates produced using cocoa beans fermented with yeast starters (Pichia kudriavzevii (MH979681), Hanseniaspora thailandica (MH979675) and the mixture of the two yeasts (Mix)). The VOCs of chocolates were determined by Head-Space Solid Phase Microextraction followed by Gas Chromatography-Mass Spectrophotometry. Sensory analysis was determined by using trained panels. VOCs profiles of chocolates produced using beans fermented with HT, PK or Mix were noticeably different from Ghana and control chocolates (no starter). The addition of yeast starters during cocoa fermentation produced chocolates that were preferred by trained panels. Bitterness and astringency were the more intense flavour attributes in chocolates produced using cocoa beans added with yeast starters. The chocolate produced using cocoa beans fermented with PK was the most acidic; whereas chocolate produced using beans fermented with Mix had the sweetest taste. The addition of PK or HT is helpful in producing chocolate with a distinct flavour.
Four fungal isolates: Simplicillium chinense (iso 9, accession no. KX425621), Penicillium simplicissimum (iso 10, KP713758), Trichoderma asperellum (iso 11, KP792512), and Coriolopsis sp. (1c3, KM403574) were subjected to a series of induced-tolerance training under high metal concentrations to determine if greater tolerance could be achieved from constant exposure to such conditions. Adaptive tolerance assay (Tolerance Index, TI) and Field-Emission Scanning Electron Microscopy with Energy Dispersive X-ray (SEM-EDX) characterized their metal tolerance. "Untrained" S. chinense, P. simplicissimum and T. asperellum showed tolerance towards 4000-4500ppm Al(III) (TI: 0.64-0.71), 1000ppm Cr(III) (0.52-0.83) and Pb(II) (0.32-0.88). With tolerance training, tolerance towards 2000-6000ppm Al(III), 500-3000ppm Pb(II) and 2000-3000ppm Cr(III) were achieved (TI: 0.01-0.82) compared to untrained cultures (0.00-0.59). In contrast, tolerance training for Coriolopsis sp. and P. simplicissimum was less successful, with TI values similar or lower than untrained cultures. SEM-EDX analysis proposed biosorption and bioaccumulation as mechanisms for metal removal. The latter was demonstrated with the removal of Cr(III) and Pb(II) by S. chinense (12.37 and 11.52mgg-1, respectively) and T. asperellum (10.44 and 7.50mgg-1). Induced-tolerance training may render benefit in the long run, but this delicate approach is suggestively species and metal dependent.
Penicillium simplicissimum (isolate 10), a metal tolerant fungus, tolerated 1000 mg/L Cu and 500 mg/L Zn, but were inhibited by Cd (100 mg/L), evident by the Tolerance Index (TI) of 0.88, 0.83, and 0.08, respectively. Live cells of P. simplicissimum were more effective in removing Cr (88.6%), Pb (73.7%), Cu (63.8%), Cd (33.1%), and Zn (28.3%) than dead cells (5.3-61.7%). Microscopy approach via SEM-EDX and TEM-EDX suggested that metal removal involved biosorption and bioaccumulation, with metal precipitates detected on the cell wall, and in the cytoplasm and vacuoles. FTIR analysis revealed metals interacted with amino, carbonyl, hydroxyl, phosphoryl (except Cd) and nitro groups in the cell wall. Biosorption and bioaccumulation of metals by live cells reduced Cu and Pb toxicity, observed from good root and (4.00-4.28 cm) and shoot (8.07-8.36 cm) growth of Vigna radiata in the phytotoxicity assay.
L-asparaginase from endophytic Fusarium proliferatum (isolate CCH, GenBank accession no. MK685139) isolated from the medicinal plant Cymbopogon citratus (Lemon grass), was optimized for its L-asparaginase production and its subsequent cytotoxicity towards Jurkat E6 cell line. The following factors were optimized; carbon source and concentration, nitrogen source and concentration, incubation period, temperature, pH and agitation rate. Optimization of L-asparaginase production was performed using One-Factor-At-A-Time (OFAT) and Response surface methodology (RSM) model. The cytotoxicity of the crude enzyme from isolate CCH was tested on leukemic Jurkat E6 cell line. The optimization exercise revealed that glucose concentration, nitrogen source, L-asparagine concentration and temperature influenced the L-asparaginase production of CCH. The optimum condition suggested using OFAT and RSM results were consistent. As such, the recommended conditions were 0.20% of glucose, 0.99% of L-asparagine and 5.34 days incubation at 30.50 °C. The L-asparaginase production of CCH increased from 16.75 ± 0.76 IU/mL to 22.42 ± 0.20 IU/mL after optimization. The cytotoxicity of the crude enzyme on leukemic Jurkat cell line recorded IC50 value at 33.89 ± 2.63% v/v. To conclude, the enzyme extract produced from Fusarium proliferatum under optimized conditions is a potential alternative resource for L-asparaginase.