Qualitative and Molecular Screening of Potential Ligninolytic Microbes from Termite (Coptotermes curvignathus) Gut

Ligninolytic microbes have great potential in converting high lignin by-products to more utilisable products by decomposing the lignin-rich agricultural and industrial wastes. Thus, the aim of this study are to screen and identify the potential ligninolytic microbes from the termite (Coptotermes curvignathus) gut. The study was conducted at Universiti Putra Malaysia Bintulu Sarawak Campus, Malaysia. Twenty-seven microbes isolated from termite gut obtained from the Microbiology Laboratory, Faculty of Agricultural Science and Forestry, were used for the ligninolytic activity screening. Media with four different ligninolytic indicator dyes (Azure B, phenol red, methylene blue, and Remazol Brilliant Blue) were streaked with microbial isolates and incubated at 37 °C for 48 h. Out of twenty-seven microbe isolates, only three (CH2, CH5, and CH9) isolates showed decolourisation zone indicating the positive presence of ligninolytic activity. The 16S rRNA gene sequence data indicated the isolates are highly homologous to Bacillus spp.

Ligninolytic activity of the Penicillium chrysogenum and Pleurotus ostreatus fungi involved in the biotransformation of synthetic multi-walled carbon nanotubes modify its toxicity

Multi-walled carbon nanotubes (MWCNTs) are of multidisciplinary scientific interest due to their exceptional physicochemical properties and a broad range of applications. However, they are considered potentially toxic nanoparticles when they accumulate in the environment. Given their ability to oxidize resistant polymers, mycorremediation with lignocellulolytic fungi are suggested as biological alternatives to the mineralization of MWCNTs. Hence, this study involves the ability of two fungi specie to MWCNTs biotransformation by laccase and peroxidases induction and evaluation in vivo of its toxicity using Caenorhabditis elegans worms as a model. Results showed that the fungi Penicillium chrysogenum and Pleurotus ostreatus were capable to grow on media with MWCNTs supplemented with glucose or lignin. Activities of lignin-peroxidase, manganese-peroxidase, and laccase in cultures of both fungi were induced by MWCNTs. Raman, FTIR spectroscopy, HR-TEM, and TGA analyses of the residue from the cultures of both fungi revealed structural modifications on the surface of MWCNTs and its amount diminished, correlating the MWCNTs structural modifications with the laccase-peroxidase activities in the fungal cultures. Results indicate that the degree of toxicity of MWCNTs on the C. elegans model was enhanced by the structure modification associated with the fungal ligninolytic activity. The toxic effect of MWCNTs on the in vivo model of worms reveals the increment of reactive oxygen species as a mechanism of toxicity. Findings indicate that the MWCNTs can be subject in nature to biotransformation processes such as the fungal metabolism, which contribute to modify their toxicity properties on susceptible organisms and contributing to environmental elimination.

Pretreatment of lignocellulosic biomass with autochthonous fungi from Serbia

This research examined the potential use of isolated Serbian autochthonous fungi in lignocellulosic biomass pretreatment. Among 12 isolated fungi, the isolates identified as Trametes hirsuta F13 and Stereum gausapatum F28 stood out as ligninolytic enzyme producers and were selected for potential use in the pretreatment of a waste lignocellulosic biomass. An isolate identified as Myrmaecium fulvopruinatum F14 showed high hydrolytic activity, but negligible ligninolytic activity, and it was selected as a potential producer of important industrial hydrolytic enzymes. Further, the breakdown of lignocellulosic waste, beechwood sawdust, by T. hirsuta F13 and S. gausapatum F28 was examined. Both isolates efficiently degraded biomass, but T. hirsuta F13 exhibited greater selectivity (selectivity coefficient of 1.7) than S. gausapatum F28 (1.1). The isolate F13 was considered a better candidate for the pretreatment, and it was selected for further analysis which involved the use of molasses stillage as a supplement to improve the pretreatment.

Biotechnological potential of fungi and bacteria with ligninolytic activity (mini-review)

The presented mini-review gives a general idea of oxidative enzymes of fungi and bacteria. Possible directions of their practical application are shown.

Determination of Pleurotus abieticola ligninolytic activity on Norway spruce wood

The effect of Phlebiopsis gigantea treatment in control of Heterobasidion parviporum in Norway spruce is less effective than that in control of Heterobasidion annosum in pine. It is necessary to apply other fungi, for example, Pleurotus abieticola in Norway spruce stands. Thus, it is necessary to assess the activity of major ligninolytic enzymes, that is, laccase, lignin peroxidase (LiP), manganese peroxidase (MnP) and versatile peroxidase (VP) produced by P. abieticola, which may be effective in the fast degradation of Norway spruce wood. Three strains of P. abieticola (Pa1-3) were grown on pieces of Norway spruce sapwood and heartwood for 50 days in laboratory conditions. Enzymatic activity was determined using spectrophotometry. Pleurotus abieticola produced laccase, LiP, MnP and VP. The activity of laccase was low, ranging 0–3.696 and 0–0.806 mU/μg of protein in sapwood and heartwood, respectively. The highest activity in Pa1 = 3.696 mU/μg of protein in sapwood and in Pa3 = 0.806 mU/μg of protein in heartwood was observed after 30 and 50 days of incubation, respectively. The activity of LiP was also low, ranging 0–0.188 and 0–0.271 mU/μg of protein in sapwood and heartwood, respectively. The highest activity in Pa1 = 0.271 mU/μg of protein in sapwood and in Pa2 = 0.188 mU/μg of protein in heartwood was observed after 40 and 20 days of incubation, respectively. The activity of MnP ranged 0–17.618 and 0–12.203 mU/μg of protein in sapwood and heartwood, respectively. This enzymatic activity peaked at the 50th day of culture on sapwood for the Pa3 strain (17.618 mU/μg of protein) and at the 20th day of culture on heartwood for the Pa1 strain (12.203 mU/μg of protein). The activity of VP with manganese-oxidising properties was found to be high in all strains of P. abieticola, ranging 0–39.19 and 0–59.153 mU/μg of protein in sapwood and heartwood, respectively, whereas the activity of VP with guaiacol-oxidising properties was very low for all P. abieticola strains, ranging 0–0.248 and 0–0.225 mU/μg of protein in sapwood and heartwood, respectively. The values of released hydroxyphenols in P. abieticola strains ranged 24.915–139.766 and 25.19–84.562 µg of protocatechuic acid/ml in sapwood and heartwood, respectively. The values of released methoxyphenols for the evaluated strains of P. abieticola ranged 7.225–23.789 and 1.953–20.651 µg of vanillic acid/ml in sapwood and heartwood, respectively. Further studies with a higher number of strains of this species as well as an optimisation of conditions for the measurement of ligninolytic activity are needed.

SKRINING DAN IDENTIFIKASI MIKROBA LIGNINOLITIK PADA PENGOMPOSAN ALAMI TANDAN KOSONG KELAPA SAWIT

Screening and Identification of Ligninolytic Microbes in the Natural Decomposition of Oil Palm Empty Fruit Bunch ABSTRACTOPEFB (oil palm empty fruit bunch)could potentially be utilized as organic fertilizer or animal feed through composting. Information on microorganisms that play important roles in the natural decomposition of OPEFB is to date not much known yet. This research was aimed to obtain and, subsequently, to molecularly identify lignin-degrading microbial isolates responsible for naturally decomposing OPEFB in the Oil Plant Plantation and Palm Oil Refinery Plant, PTPN VIII Cikasungka, Bogor. Screening for active lignin-degrading isolates was carried out on 17 naturally decomposing OPEFB samples. A total of 19 isolates of fungi and 80 isolates of bacteria were obtained. Ligninolytic activity was measured by Sundman and Nase testing methods. Ligninolytic activity was found on 13 fungal isolates and 15 bacterial isolates. The active isolates were subsequently identified molecularly based on ITS sequence in the ribosome DNA area for fungi and in 16S rRNA genes for bacteria. The results showed that the lignin-degrading microorganisms obtained consisted of 5 bacterial isolates from the genus Bacillus and 3 fungal isolates from the genus Rhizopus and Aspergillus. Keywords: composting, lignin, microbes, OPEFB, 16S rRNA ABSTRAKTKKS (tandan kosong kelapa sawit) berpotensi dimanfaatkan sebagai pupuk organik atau pakan ternak dengan cara pengomposan. Informasi mikroba yang berperan dalam pengomposan alami TKKS hingga saat ini belum banyak diketahui. Penelitian ini bertujuan mendapatkan isolat mikroba pendegradasi lignin dalam pengomposan alami TKKS asal Perkebunan dan Pabrik Pemerasan Kelapa Sawit, PTPN VIII Cikasungka, Bogor, serta mengidentifikasi mikroba tersebut secara molekuler. Skrining mikroba aktif pendegradasi lignin dilakukan terhadap 17 sampel TKKS yang sudah lapuk secara alami. Sebanyak 19 isolat jamur dan 80 isolat bakteri telah dihasilkan. Aktivitas ligninolitik diukur dengan metode pengujian Sundman dan Nase. Isolat jamur yang memiliki aktivitas ligninolitik sebanyak 13 isolat, sedangkan bakteri sebanyak 15 isolat. Isolat-isolat aktif tersebut selanjutnya diidentifikasi secara molekuler berdasarkan pada sekuen ITS di daerah DNA ribosom untuk jamur dan menggunakan gen 16S rRNA untuk bakteri. Hasil menunjukkan bahwa 5 isolat bakteri yang memiliki kemampuan mendegradasi lignin berasal dari genus Bacillus, sedangkan 3 isolat jamur pendegradasi lignin berasal dari genus Rhizopus dan Aspergillus Kata Kunci: lignin, mikroba, pengomposan, TKKS, 16S rRNA