Evaluation of co-culture of cellulolytic fungi for enhanced cellulase and xylanase activity and saccharification of untreated lignocellulosic material.

Bioethanol production from lignocellulosic materials is hindered by the high costs of pretreatment and the enzymes. The present study aimed to evaluate whether co-cultivation of four selected cellulolytic fungi yields higher cellulase and xylanase activities compared to the monocultures and to investigate whether the enzymes from the co-cultures yield higher saccharification on selected plant materials without thermo-chemical pretreatment. The fungal isolates, Trichoderma reesei F118, Penicillium javanicum FS7, Talaromyces sp. F113, and Talaromyces pinophilus FM9, were grown as monocultures and binary co-cultures under submerged conditions for 7 days. The cellulase and xylanase activities of the culture filtrates were measured, and the culture filtrates were employed for the saccharification of sugarcane leaves, Guinea grass leaves, and water hyacinth stems and leaves. Total reducing sugars and individual sugars released from each plant material were quantified. The co-culture of Talaromyces sp. F113 with Penicillium javanicum FS7 and of T. reesei F118 with T. pinophilus FM9 produced significantly higher cellulase activities compared to the corresponding monocultures whereas no effect was observed on xylanase activities. Overall, the highest amounts of total reducing sugars and individual sugars were obtained from Guinea grass leaves saccharified with the co-culture of T. reesei F118 with T. pinophilus FM9, yielding 63.5% saccharification. Guinea grass leaves were found to be the most susceptible to enzymatic saccharification without pre-treatment, while water hyacinth stems and leaves were the least. Accordingly, the study suggests that fungal co-cultivation could be a promising approach for the saccharification of lignocellulosic materials for bioethanol production.

High levels of microplastics in commercial salt and industrial salterns in Sri Lanka.

This study provides the first analysis and quantification of MPs in salt products in Sri Lanka. Commercial table salt brands, rock salt, lab-grade NaCl and raw salt from three salterns were analysed using microscopy and Fourier transform infrared spectroscopy. All salt samples were contaminated with MPs: in commercial salts products it ranged from 11 to 193 items/kg, rock salts had 64 items/kg and lab grade NaCl had 253 ± 8.9 items/kg. The MP levels in salterns varied significantly: Hambantota 3345.7 ± 311.4 items/kg, Puttalam 272.3 ± 10.6 items/kg, and Elephant Pass 36.3 ± 4.5 items/kg. Predominantly, MPs were presented as fibres, followed by fragments. Of the 23 polymer types identified; low-density polyethylene (LDPE; 17%), resin dispersion (15%) and high-density polyethylene (HDPE; 12%) were notable. This study provides the first comparison of MPs in raw salt and commercial salt. This information is important to trace the pollutant sources and then to take steps to eliminate MPs in food products consumed.

Antimicrobial potential of two traditional herbometallic drugs against certain pathogenic microbial species.

BACKGROUND: Mineral based preparations are widely used for centuries as antimicrobial agents. However, the efficacy and the mode of action of mineral based preparations are uncertain due to the insufficient antimicrobial studies. Arogyawardhana Vati (AV) and Manikya Rasa (MR) are such two Rasashastra herbo-minerallic drugs commonly in India and other countries in South Asia. Despite of their well known traditional use of skin diseases, reported antimicrobial and mineralogical studies are limited. Therefore, in this study antimicrobial activities of the drugs and their organic, inorganic fractions were evaluated against Pseudomonas aeruginosa, Escherischia coli, Staphylococcus aureus, Methecilline Resistance Staphylococcus aureus - MRSA and Candida albicans. METHODS: Antimicrobial activity of the drugs, their inorganic residues and organic extracts were determined using four assay techniques viz agar well diffusion, modified well diffusion, Miles and Misra viable cell counting and broth turbidity measurements. Mineralogical constituents of the drugs were determined using X-ray diffraction, while total cation constituents and water soluble cation constituents were determined using inductively coupled plasma-mass spectrometer and the atomic absorption spectrophotometer respectively. Thermogravimetric analysis was used to determine the weight percentages of organic and inorganic fraction of the drugs. Particle sizes of the drugs were determined using the particle size analyzer. RESULTS: AV and MR drugs showed antibacterial activity against both gram positive and gram negative bacterial species when analyzed separately. Inorganic residues of the drugs and organic extracts showed activity at least against two or more bacterial species tested. All tested components were inactive against C. albicans. Common mineral constituents of drugs are cinnabar, biotite and Fe-rich phases. Drugs were rich in essential elements such as Na, K, Ca, Mg and Fe and toxic elements such as Zn, Cu and As. However, the water soluble concentrations of the toxic elements were below the detection limits. Both drugs have significantly higher percentages of organic constituents and volatile minerals and particle sizes of drugs are in the nanometer range. CONCLUSIONS: AV and MR Rasashastra preparations could provide alternatives to synthetic antibiotics against human bacterial infections. Improved solubility and reduced particle sizes are influential physicochemical properties used to enhance the antimicrobial efficacy of the drugs. Therefore, traditional knowledge on the use of antimicrobial mineral sources could provide a novel path for the producing of effective antimicrobial drugs. However, further chemical and toxicological studies are urgently needed for a greater understanding of their toxicity to humans.

First Report of Mango Malformation Disease Caused by Fusarium mangiferae in Sri Lanka.

Mango malformation disease (MMD) is one of the most devastating diseases causing severe economic losses to this crop worldwide. MMD has not been reported in Sri Lanka although the disease was reported in neighboring India over a century ago. Abnormal, thick, and fleshy mango panicles (40%) and proliferating stunted shoots (<1%) showing characteristic malformation symptoms were observed in Peradeniya-Kandy area (7°17'4.15" N, 80°38'14.08" E). Malformed inflorescences and vegetative shoots were collected during January to March and September to November, in 2008 through 2012. Pieces of malformed tissues were surface sterilized in 1% sodium hypochlorite and transferred to potato dextrose agar (PDA). The plates were incubated at 26 ± 2°C for 7 days. Monoconidial cultures of 41 isolates that resembled Fusarium spp. were obtained. Colonies showed white sparse aerial mycelium and magenta-dark purple pigmentation on the underside. Growth rate of the isolates averaged 3.67 mm/day in the dark at 25°C on PDA. To stimulate conidia development, Fusarium isolates were transferred to carnation leaf agar (CLA). Sympodially branched conidiophores bearing mono- and polyphialides with 2 to 3 conidiogenus openings originated erect and prostrate on aerial mycelium. Oval to allontoid, abundant microconidia were produced in false heads on mono- and polyphialides. Dimensions of aseptate conidia were 2.5 to 12.5 (6.47) × 1.25 to 3.8 (2.29) µm. Macroconidia were long and slender, 3 to 5 celled and 27.5 to 47.5 (38.59) × 2.5 to 5 (2.94) µm. Chlamydospores were absent. These characters are consistent for F. mangiferae. DNA was extracted from 30 monoconidial Fusarium isolates (1) and amplified with species-specific PCR primers 1-3F/R (forward: 5'-TGCAGATAATGAGGGTCTGC-3'; reverse: 5'-GGAACATTGGGCAAAACTAC-3') (3). Eight isolates from malformed inflorescences (I6, I13, I15, and I16) and malformed vegetative tissues (V1, V2, V3, and V4), were identified as F. mangiferae based on a 608-bp species-specific amplified DNA fragment. Pathogenicity of F. mangiferae isolates, I15 and V2, was tested on 1-year-old seedlings cv. Willard planted in 10-liter plastic pots. Conidia suspensions (107 conidia/ml of 0.1% water agar) were obtained from 10-day-old monoconidial cultures. Each isolate was inoculated onto 15 apical buds by placing drops (20 µl) of conidia (2). Both F. mangiferae isolates, I15 and V2, on artificial inoculation produced typical floral malformation symptoms in 40% of the buds, up to 10 weeks after inoculation. The Fusarium isolates recovered were identical in colony and mycelia morphology and conidia dimensions to the original F. mangiferae isolates. No Fusarium species were recovered from control flower buds. To our knowledge, this is the first report of MMD in the inflorescence and the vegetative shoots caused by F. mangiferae in Sri Lanka. Isolation of other Fusarium spp. that were not identified as F. mangiferae in this study suggests that additional Fusarium spp. may be associated with the MMD in Sri Lanka. Further studies are needed to confirm the identity of these Fusarium isolates, their role in MMD, and the distribution over the island. Since the disease is likely to drastically reduce productivity, measures will be required to protect 12,160 ha of mango cultivation from this devastating disease. References: (1) S. Freeman et al. Exp. Mycol. 17:309, 1993. (2) S. Freeman et al. Phytopathology 89:456, 1999. (3) Q. I. Zheng and R. C. Ploetz. Plant Pathol. 51:208, 2002.

Phyllosticta musarum Infection-Induced Defences Suppress Anthracnose Disease Caused by Colletotrichum musae in Banana Fruits cv 'Embul'.

Anthracnose development by Colletotrichum musae was observed to be significantly less in the fruits of the banana cultivar 'Embul' (Mysore, AAB) infected with Phyllosticta musarum than in fruits without such infections. Anthracnose disease originates from quiescent C. musae infections in the immature fruit. P. musarum incites minute, scattered spots, referred to as freckles, in the superficial tissues of immature banana peel which do not expand during maturation or ripening. P. musarum does not appear to have a direct suppressive effect on C. musae as conidia of C. musae germinate on both freckled and non-freckled fruit forming quiescent infections. Our investigations have shown that P. musarum infection induced several defence responses in fruit including the accumulation of five phytoalexins, upregulation of chitinase and ß-1,3-glucanase, phenylalanine ammonia lyase (PAL) activity and cell wall lignification. (1)H and (13)C NMR spectral data of one purified phytoalexin compared closely with 4'-hydroxyanigorufone. Some of the P. musarum-induced defences that retained during ripening, restrict C. musae development at the ripe stage. This paper examines the potential of P. musarum-induced defences, in the control of anthracnose, the most destructive postharvest disease in banana.

Temporal variation of microbiological and chemical quality of noncarbonated bottled drinking water sold in Sri Lanka.

Use of bottled water in Sri Lanka has increased over the last decade, while new brands of bottled water are often introduced to the market. However, the manufacturers' adherence to bottled water regulations is questionable, raising concerns regarding the quality of bottled water. The objective of the current study was to investigate the microbiological and chemical quality of bottled water in Sri Lanka. Thirty bottled water brands were sampled and their chemical and microbiological parameters were analyzed. Microbiological analysis was carried out within 1 to 3, 3 to 6, 6 to 9, and 9 to 12 mo after the date of manufacture. The results indicated that 63% of brands tested exceeded the levels permitted by the Sri Lanka Standards Institution (SLSI) for presumptive total coliforms (TC) (<10 cfu per 100 mL) whereas 97% brands exceeded the World Health Organization (WHO) permitted level. Thirty percent of brands exceeded the limit for presumptive fecal coliforms (FC) (0 cfu per 100 mL in accordance with WHO permitted levels, SLSI and the Sri Lanka Health Ministry requirement). Eighty percent of brands showed higher heterotrophic plate counts (HPC) which exceeded the WHO guidelines for bottled drinking water. Throughout their shelf life, the counts of TC, FC, and HPC bacteria decreased. Bacteria identified were Klebsiella pneumoniae ssp. pneumoniae, Enterobacter cloacae, Pseudomonas aeruginosa, and Pasteurella haemolytica, the most frequently being P. aeruginosa. The dominant fungi identified were Aspergillus sp. and Penicillium sp. Inorganic chemical parameters were within permitted levels for all brands except for initial content of ammonia. The results of this study show the need for the bottling industry to be monitored closely by relevant authorities, in order to provide safe bottled drinking water to consumers in Sri Lanka.