Mechanisms of lignocellulosic conversion by the brown rot fungus serpula lacrymans

Cost effective processing of wheat straw using solid state fermentation (SSF) would provide a source for value added chemicals from agricultural waste biomass. Fungi natural breakdown lignocellulosic biomass hence could have received a lot of attention. In this study the ability of S. lacrymans to convert straw waste was compared with other Basidiomycetes (Postia placenta, Phanerochaete chrysosporiom, and Schizophyllum commune). S. lacrymans out performed the other Basidiomycetes both in its growth (as measured by ergosterol and fatty acid production (linoleic acid);18:2n6c) , and in the comounds released which included; total soluble phenols, total reducing sugars, and low molecular organic chemicals (MW<400). Non-enzymatic breakdown requiring the presence of Fe2+ was also demonstrtated and influenced by the production of quinone and low molecular organic acid. The amount of the fungal extract used and the concentration of chelator/reducing agents also affected the production of Fe2+. Changes in the lignocellulose structure was also detected as key functional group, such as the pyranose ring and aromatic skeletal vibration were significantly reduced following culture with S. lacrymans and a significant reduction in mass was measured. Iron reductase genes IR1 and IR2 suspected to be involved in the lignocellulose degradation were cloned. It seems that these genes are more closely related to the cellulose binding module (CBM) family instead of cellobiose dehydrogenase (CDH) genes as first suspected. IR1 has an open reading frame of 774 bp which encoding 258 amino acid (55 kDa), whilst IR2 642 bp encoding 214 amino acid ( 49 kDa). The IR1 gene contains a CBM1 domain which is lacking in IR2. Gene expression analysis using qRT-PCR showed that in the early stage of fungal growth, the level of IR2 genes expression was higher than IR1 while IR1 became more dominant in the latter stages of culture. The time at which these genes are highly expressed correlated with the release of soluble and aromatic phenolic compounds. The functionality of the recombinant IR1 and IR2 on the decomposition of lignocellulose was shown using several assays including; iron reductases, nitrated lignin and the reduction of electron acceptor (DCPIP). In addition, using both synthetic and nature sources of cellulose or lignocellulose (Avicel and wheat straw powder) the recombinant IR proteins were shown to break down cellulose. This suggested that these enzymes represent a significant addition to those currently used within biomass based biorefinerie

Optimization of Vanillin Extraction from Biodegradation of Oil Palm Empty Fruit Bunches by Serpula lacrymans

Abstract This research aims to determine the combination of the ethyl acetate solvent volume and the extraction time that resulted in the optimum response of vanillin content and vanillin yield from the degradation of lignocellulose components from oil palm empty fruit bunches (OPEFB). First, OPEFB degraded using Serpula lacrymans to break down lignocellulosic components. The research design used a centralized composite design with two factors, the volume of ethyl acetate solvent (ml) and the extraction time (minutes). The responses of the experiment are vanillin content and vanillin yields. The optimization analysis results showed that the volume of ethyl acetate solvent and extraction time have a quadratic effect on the vanillin content and vanillin yields. The optimal solution was obtained by treatment with ethyl acetate volume 101.1 ml and extraction time 123.5 minutes. The optimal solution prediction results obtained vanillin content 0.014% and vanillin yield 7.302 μg/g with desirability of 92.8%. Validation based on the optimal solution’s prediction brought response vanillin content 0.013% and vanillin yield 6.950 μg/g. The vanillin content and yield validation results differed respectively by 4.081% and 4.826% lower when compared to predictions on the optimal solution.Keywords: ethyl acetate, vanillin content, vanillin yield AbstrakPenelitian ini bertujuan untuk mengetahui kombinasi dari volume pelarut etil asetat dan lama waktu ektraksi yang menghasilkan respon kadar vanillin dan yield vanillin optimum dari hasil degradasi komponen lignoselulosa tandan kosong kelapa sawit. Tandan kosong kelapa sawit terlebih dahulu didegradasi menggunakan jamur pelapuk Serpula lacrymans untuk memecah komponen lignoselulosa. Rancangan penelitian menggunakan rancangan komposit terpusat dengan dua faktor, yaitu volume pelarut etil asetat (ml) dan lama waktu ekstraksi (menit). Respon dari percobaan tersebut adalah kadar vanillin dan yield vanillin. Hasil penelitian untuk analisis optimasi menunjukkan bahwa faktor volume pelarut etil asetat dan lama waktu ekstraksi berpengaruh secara kuadratik terhadap respon kadar vanillin dan yield vanillin. Hasil solusi optimal diperoleh pada perlakuan dengan volume pelarut etil asetat sebesar 101,1 ml dan lama ekstraksi selama 123,5 menit. Hasil prediksi solusi optimal diperoleh kadar vanillin 0,014% dan yield vanillin 7,302 μg/g dengan ketepatan 92,8%. Validasi yang dilakukan berdasarkan pada prediksi solusi optimal diperoleh respon kadar vanillin 0,013% dan yield vanillin 6,950 μg/g. Hasil validasi kadar dan yield vanillin tersebut memiliki perbedaan masing-masing 4,081% dan 4,826% lebih rendah jika dibandingkan dengan prediksi pada solusi optimal.Kata kunci: etil asetat, kadar vanillin, yield vanillin

Penentuan Isolat Bakteri Asidogenik yang Mampu Menghasilkan Total Asam Tertinggi dari Limbah Cair Tahu

Abstrak Penelitian ini bertujuan untuk mendapatkan bakteri asidogenik yang mampu memproduksi total asam tertinggi dari limbah cair tahu. Uji kemampuan asidogenik meggunakan Rancangan Acak Kelompok pola faktorial dengan menggunakan dua perlakuan yaitu persentase inokulum (5%, 10%, dan 15%) dan lama waktu inkubasi (24 jam, 48 jam, dan 72 jam). Parameter yang diukur meliputi kadar total asam tertitrasi, pH, kadar gula reduksi, dan total bakteri asidogenik. Hasil penelitian menunjukkan karakterisasi morfologi koloni isolat terpilih (isolat 1031) berwarna putih tulang, berbentuk bulat, permukaan timbul, dan tepiannnya rata dan karakterisasi morfologi sel isolat 1031 adalah Gram negatif dan berbentuk batang. Berdasarkan hasil karakterisasi biokimia, isolat 1031 memiliki probabilitas kedekatan dengan Hafnia alvei biogp 1 sebesar 94,53%. Persentase inokulum dan lama waktu inkubasi berpengaruh terhadap produksi total asam tertitrasi. Perlakuan terbaik adalah kombinasi perlakuan A3B1 (persentase inokulum 15% dan waktu inkubasi 24 jam), dengan nilai total asam tertitrasi 2,70%, nilai pH 6,33, kadar gula reduksi 0,07%, dan jumlah populasi bakteri asidogenik 1,1 x 108 CFU/ml. Kata kunci: asidogenesis, bakteri asidogenik, limbah cair tahu, dan total asam teritrasi Abstract This research had an objective to find an acidogenic bacteria which was able to produce the highest organic acid from tofu wastewater. Test of acidogenic capabilty used Randomized Block Design arranged in factorial by using two treatments, inoculum percentage (5%, 10%, and 15%) and incubation time (24 hours, 48 hours, and 72 hours). Parameters measured were production of titration acidity total, pH, degree of sugar reduction, and acidogenic bacteria total. Result showed identification of colony morphology of selected isolate (isolat 1031) were bone-white, round shape, raised surface (elevation), flat edge and cell morphology were Gram-negative bacillusshaped. Based on the biochemical identification showed isolate 1031 was Hafnia alvei biogp 1 with a probability value of 94.53%. Inoculum percentage and incubation time had significant effect in production of titration acidity total. The best treatment combination was A3B1 (inoculum percentage of 15% and incubation time in 24 hours). A3B1 treatment produced titration acidity total 2,70%, pH 6,33, degree of sugar reduction 0,07%, and acidogenic bacteria total 1,1x108 CFU/ml. Keywords: acidogenesis, acidogenic bacteria, titration acidity total, and tofu wastewate

OPTIMASI KONDISI EKTRAKSI VANILLIN HASIL DEGRADASI LIGNOSELULOSA BAGAS TEBU MENGGUNAKAN RESPONSE SURFACE METHOD (RSM)

Optimization of Vanillin Extraction Conditions from Lignocellulose Degradation of Sugarcane Bagasse using the Response Surface Method (RSM) Sugarcane bagasse is an agricultural waste containing lignocellulose and has the potential to be processed into high-value chemicals such as vanillin. The degradation of sugarcane bagasse lignocellulose can be carried out biologically by the white rot fungus Phanerochaete chrysosporium. This study aims to obtain optimal extraction conditions in the form of ethyl acetate solvent volume and extraction time, using the response surface method (RSM). Two optimized factors were the volume of ethyl acetate (71.72; 80; 100; 120; and 128.28 mL) and the extraction time (35.16; 60; 120; 180; 204.84 minutes). The response variables were the concentration (%) and yield of vanillin (µg g–1). The research on the optimization of the response of vanillin levels and vanillin yield was carried out at 14 days incubation with the highest average total soluble phenol (TSP) value of 0.101 mg g–1. The optimal condition of ethyl acetate volume of 109.730 mL with an extraction time of 137.302 minutes was predicted to produce vanillin levels and yields of 0.0078% and 8.9089 g g–1, respectively, with an accuracy value of 93.4%. Based on the verification results, the optimal vanillin concentration and yield were 0.0077% and 8.9805 g g–1, respectively. Bagas tebu merupakan limbah pertanian yang mengandung lignoselulosa dan berpotensi diolah menjadi bahan kimia bernilai tinggi seperti vanillin. Degradasi lignoselulosa bagas tebu dapat dilakukan secara biologis oleh jamur pelapuk putih Phanerochaete chrysosporium. Penelitian ini bertujuan mendapatkan kondisi ekstraksi optimal berupa volume pelarut etil asetat dan lama waktu ekstraksi, menggunakan response surface method (RSM). Dua faktor yang dioptimasi adalah volume etil asetat (71,72; 80; 100; 120; dan 128,28 mL) dan lama waktu ekstraksi (35,16; 60; 120; 180; 204,84 menit). Variabel respons adalah kadar (%) dan yield vanillin (µg g–1). Penelitian optimasi respons kadar vanillin dan yield vanillin dilakukan pada inkubasi 14 hari dengan nilai total soluble phenol (TSP) rata-rata tertinggi 0,101 mg g–1. Kondisi optimal volume etil asetat 109,730 mL dengan waktu ekstraksi 137,302 menit diprediksi menghasilkan kadar dan yield vanillin sebesar 0,0078% dan 8,9089 µg g–1 dengan nilai ketepatan 93,4%. Berdasar hasil verifikasi, konsentrasi dan yield vanillin yang optimal masing-masing adalah 0,0077% dan 8,9805 µg g–1

PENGARUH PENAMBAHAN CUSO4 TERHADAP AKTIVITAS ENZIM SCHYZOPHYLLUM COMMUNE PADA DEGRADASI LIGNIN LIMBAH KULIT KAKAO

Kulit coklat merupakan sumber lignoselulosa dari limbah pertanian dengan kandungan lignin mencapai 14.7 ± 0.35% (w/w). Lignin merupakan senyawa aromatik kompleks heteropolymer yang tidak mudah dipecah secara hidrolitik, sehingga jamur pelapuk seringkali digunakan untuk mempermudah proses degradasi lignin. Pada penelitian ini digunakan jamur pelapuk putih (S. commune). Selama proses pemecahan lignin, S. commune memanfaatkan enzim peroksidase dan lakase. Akan tetapi, produksi enzim lignolitik pada jamur pelapuk putih dalam jumlah kecil, sehingga diperlukan penambahan inducer untuk meningkatkan aktivitas enzim lignolitik. Tujuan penelitian ini adalah untuk mengetahui pengaruh penambahan CuSO4 terhadap aktivitas enzim MnP dan Lakase pada S. commune yang berperan dalam proses pemecahan lignin sehingga dapat dihasilkan berbagai macam senyawa kimia bernilai tinggi. Tembaga dengan konsentrasi 0 mM; 0.5 mM; 1.5 mM; dan 2.5 mM ditambahkan pada S. commune dan di inkubasi selama 5 minggu. Hasil kemudian diekstrak dan diuji aktivitas enzim MnP dan Lakase menggunakan spektofotometri. Hasil menunjukkan aktivitas enzim lignolitik S. commune terbesar yaitu 0.48 IU.L-1 untuk enzim MnP pada minggu ke-4 dengan penambahan konsentrasi CuSO4 1.5 mM dan 0.18 IU.L-1 untuk enzime lakase pada minggu ke-3 dengan penambahan konsentrasi CuSO4 0.5 mM dan 1.5 m

The white-rot fungus, Phanerochaete chrysosporium, under combinatorial stress produces variable oil profiles following analysis of secondary metabolites

Aims: The effects of combinatorial stress on lipid production in Phanerochaete chrysosporium remains understudied. This species of white‐rot fungi was cultivated on solid‐state media whilst under variable levels of known abiotic and biotic stressors to establish the effect upon fungal oil profiles. Methods and Results: Environmental stressors induced upon the fungus included: temperature; nutrient limitation; and interspecies competition to assess impact upon oil profiles. Fatty acid type and concentration was determined using analytical methods of Gas Chromatography and Mass Spectrometry. Growth rate under stress was established using High Performance Liquid Chromatography with ergosterol as the biomarker. Fungi grown on solid‐state agar were able to simultaneously produce short and long‐chain fatty acids which appeared to be influenced by nutritional composition as well as temperature. Addition of nitrogen supplements increased the growth rate, but lipid dynamics remained unchanged. Introducing competition‐induced stress had significantly altered the production of certain fatty acids beyond that of the monoculture whilst under nutrient‐limiting conditions. Linoleic acid concentrations, for example, increased from an average of 885 ng/μl at monoculture towards 13820 ng/μl at co‐culture, following 7 days of incubation. Conclusions: Interspecies competition produced the most notable impact on lipid production for solid‐state media cultivated fungi whilst the addition of nitrogen supplementation presented growth and lipid accumulation to be uncorrelated. Combinatorial stress therefore influences the yield of overall lipid production as well as the number of intermediate fatty acids produced, deriving similar oil profiles to the composition of vegetable and fish oils. Significance and Impact of Study: Fungal secondary metabolism remains highly sensitive following combinatorial stress. The outcome impacts the research towards optimising fungal oil profiles for biomass and nutrition. Future investigations on fungal stress tolerance mechanisms need to address these environmental factors throughout the experimental design

A comparison of ergosterol and PLFA methods for monitoring the growth of ligninolytic fungi during wheat straw solid state cultivation

Ergosterol, total phospholipid fatty acid (PLFA) and linoleic acid (18:2n-6) have all been used to determine fungal growth. This paper compares these methods to assess the growth of four different saprotrophic fungal species during solid state cultivation using a wheat straw substrate that have not been compared or measured previously. Ergosterol production appeared to track the mycelia growth well but its production differed considerably between fungi. This means that a specific conversion factor needs to be determined and applied for any given fungus. In comparison, measurements of total PLFA and linoleic acid only showed promise for determining the growth of Postia placenta due to the positive correlation with ergosterol measurements. In contrast, the other fungi tested (Phanerochaete chrysosporium, Serpula lacrymans and Schizophyllum commune) showed either no correlation or in some cases a negative correlation using this assay. The novel findings highlight the variation in fungal fatty acid between species, culture conditions and durations of incubation; suggesting that measurement of linoleic acid is only usable in specific cases. These findings provide important consideration for the study of fungi growing in solid substrates and suggest that the use of PLFA might bias diversity indices

Extraction of vanillin following bioconversion of rice straw and its optimization by response surface methodology

Value-added chemicals, including phenolic compounds, can be generated through lignocellulosic biomass conversion via either biological or chemical pretreatment. Currently vanillin is one of the most valuable of these products that has been shown to be extractable on an industrial scale. This study demonstrates the potential of using rice straw inoculated with Serpula lacrymans, which produced a mixture of high value bio-based compounds including vanillin. Key extraction conditions were identified to be the volume of solvent used and extraction time, which were optimized using response surface methodology (RSM). The vanillin compounds extracted from rice straw solid state fermentation (SSF) was confirmed through LC-ESI MS/MS in selective ion mode. The optimum concentration and yield differed depending on the solvent, which was predicted using 60 mL ethyl acetate for 160 min were 0.408% and 3.957 μg g−1 respectively. In comparison, when ethanol was used, the highest concentration and yields of vanillin were 0.165% and 2.596 μg g−1. These were achieved using 40 mL of solvent, and extraction time increased to 248 min. The results confirm that fungal conversion of rice straw to vanillin could consequently offer a cost-effect alternative to other modes of production

BNC production of siwalan neer and sugarcane molasses as novel media sources

Bacterial nanocellulose (BNC) is a unique biopolymer with remarkable purity and material properties that distinguish it from plant-derived cellulose by its absence of lignin and hemicellulose. Recent research has focused on using organic waste materials as alternative substrates to reduce the cost of BNC production. This study examines the efficacy of Siwalan neer and sugarcane molasses as novel media for cultivating Komagataeibacter xylinus by comparing their performance with that of Hestrin and Schramm (HS) medium. The BNC harvested from these alternative media was rigorously characterized using scanning electron microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), and Thermogravimetric Analysis (TGA) to evaluate its structural and thermal properties. Notably, the use of Siwalan neer and sugarcane molasses as growth substrates enabled K. xylinus to produce BNC at a yield of 3.83 g/L, significantly exceeding the yield from the HS medium, which was 1.46 g/L. These findings highlight the potential of leveraging alternative substrates to enhance BNC production yield, cost-effectiveness, and environmental sustainability

Integrated biorefinery approach : the generation of bioproducts vanillin and biomethane through a sequential bioconversion of lignocellulose from Oil Palm Empty Fruit Bunch (OPEFB)

The efficient utilization of all of the key lignocellulosic biomass residues is important for the development of an integrated biorefineries uassing this as its feedstock. In this study, a process involving a simultaneous pre-treatement process with anaerobic digestion (AD) were implemented for processing oil palm empty fruit bunches (OPEFB) in order to produced both vanillin and methane. Before and after treatment with the dry rot fungus (Serpula lacrymans) the biomass was analyzed for total reducing sugar (TRS), total soluble phenols (TSP), pH, and weighed. Change in the physical characteristic and morphology of untreated and OPEFB following fungal culture was observed using scanning electron microscopy (SEM). The biomethane potential (BMP) of pretreated OPEFB significantly increased (62.07%) compared to untreated sample and in addition. The amount of vanillin extracted reached 3.48ug mL−1. This study therefore confirms that multiple products can effectively be isolated using a fungal preatreatment of lignocellulosic OPEFB followed by anerobic fermentation of biomass residual thus maximizing the potential returns and reducing environmentally impacts compared to other pretreatments