Agaricus blazei as a Substrate for the Production of β-1,3-Glucanase by Trichoderma harzianum Rifai

Extracellular β-1,3-glucanase was produced by Trichoderma harzianum Rifai cultivated in the Agaricus blazei (Agaricus brasiliensis) extract as a substrate in submerged fermentation. A 22-central composite factorial design was developed using the time of culture (x1/day) and Agaricus blazei extract concentration (x2/(g/L)) as variables, and the results were analyzed using response surface methodology (RSM). The results showed that the Agaricus blazei extract concentration was the most important variable in the production of β-1,3-glucanase, and the maximum β-1,3-glucanase activity (0.77 U/mL) was obtained in one day of cultivation. The β-glucan present in the cell wall of Agaricus blazei mushroom proved to be a good substrate for inducing the production of specific β-1,3-glucanase by Trichoderma harzianum Rifai.

Production and Characterization of a Milk-clotting Protease Produced in Submerged Fermentation by the Thermophilic Fungus Thermomucor indicae-seudaticae N31

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Biochemical characterisation and application of lipases produced by Aspergillus sp on solid-state fermentation using three substrates

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bioconversion of Cellulose to Hydrogen by dark fermentation

Hydrogen is known as a clean energy resource. The biological production of hydrogen has been attracting attention as an environmentally friendly processs that does not consume fossil fuels. Cellulosic plant and waste materials are potential resources for fermentative hydrogen production. Cellulose is a linear biopolymer of glucose molecules, connected by β-1,4-glycosidic bonds. Enzymatic hydrolysis of cellulose requires the presence of cellulase. The present study aimed to investigate the efficiency of acid pretreatment on ruminal fluid in order to enrich H2 producing bacteria consortia to enhance biohydrogen rate and substrate removal efficiency. In this study, fermentative hydrogen producers were enriched on cellulose (2g/L) in a modificated Del Nery medium (DNM) at 37ºC and initial pH 7.0 using rumen fluid (10% v/v) as inoculum. To increase the hydrogen production it was added cellulose (10mL) to the medium. The gas products (mainly H2 and CO2) was analyzed by gas chromatography (Shimadzu GC 2010) using a thermal conductivity detector. The volatile fatty acids and ethanol were also detected by GC using a flame ionization detector. Cellulose degradation was quantified by using the phenolsulfuric acid method. Analysis showed that the biogas produced from the anaerobic fermentation contained only hydrogen and carbon dioxide, without detectable methane after acid pretreatment test. On DNM the hydrogen production started with 4 h (5,3 x 105 mmol H2/L) of incubation, and the maximum H2 concentration was observed with 34 h (7,1 x 106 mmol H2/L) of incubation. During the process, it was observed a predominance of acetic acid and butyric acid as well as a low production of acetone, ethanol and nbutanol in all experimental phases. Butyrate accounted for more than 77% of total. As a result of the accumulation of volatile fatty acids (VFAs), the pH value in anaerobic digestion system was reduced to 4,0. On microscopy analyses there were observed rods with endospores. The batch anaerobic fermentation assays performed on anaerobic mixed inoculum from rumen fluid demonstrated the feasibility of H2 generation utilizing cellulose as substrate. Based on the results, it can be concluded that the acid treatment was efficient to inhibit the methanogenic archaea cells present in rumen fluid. The rumen fluid cells present a potential route in converting renewable biomass such as cellulose into hydrogen energy.

Hygroscopic properties of solid agro-industrial by-products used in solid-state fermentation

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Mathematical modeling of a continuous alcoholic fermentation process in a two-stage tower reactor cascade with flocculating yeast recycle

Experiments of continuous alcoholic fermentation of sugarcane juice with flocculating yeast recycle were conducted in a system of two 0.22-L tower bioreactors in series, operated at a range of dilution rates (D (1) = D (2) = 0.27-0.95 h(-1)), constant recycle ratio (alpha = F (R) /F = 4.0) and a sugar concentration in the feed stream (S (0)) around 150 g/L. The data obtained in these experimental conditions were used to adjust the parameters of a mathematical model previously developed for the single-stage process. This model considers each of the tower bioreactors as a perfectly mixed continuous reactor and the kinetics of cell growth and product formation takes into account the limitation by substrate and the inhibition by ethanol and biomass, as well as the substrate consumption for cellular maintenance. The model predictions agreed satisfactorily with the measurements taken in both stages of the cascade. The major differences with respect to the kinetic parameters previously estimated for a single-stage system were observed for the maximum specific growth rate, for the inhibition constants of cell growth and for the specific rate of substrate consumption for cell maintenance. Mathematical models were validated and used to simulate alternative operating conditions as well as to analyze the performance of the two-stage process against that of the single-stage process.

Potential application of Ganoderma lucidum in solid state fermentation of primary sludge and wheat straw

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Production of α-amilase by solid state fermentation by Rhizopus oryzae

A thermotolerant strain of Rhizopus oryzae was grown in three agro-industrial by-products: brewers’ rice, corn grits and wheat bran. Different substrates, cultivation time, moisture content, additional nitrogen sources, pH and temperature of incubation were evaluated aiming to optimize growing conditions. The highest enzymatic activity was observed after 24 h of cultivation using wheat bran as substrate with the following salt solutions: NH4NO3, MgSO4.7H2O and (NH4)2SO4 0.1% at temperature of 35°C. It was observed that changes in the pH range 4.0-6.0 did not significantly affect α-amylase activity. The optimum operation conditions were 75°C and pH 4.5. The enzymes remained stable at 75°C in the absence of substrate for 25 min.

Preliminary Characterization of Novel Extra-cellular Lipase from Penicillium crustosum Under Solid-State Fermentation and its Potential Application for Triglycerides Hydrolysis

Current studies about lipase production involve the use of agro-industrial residues and newly isolated microorganisms aimed at increasing economic attractiveness of the process. Based on these aspects, the main objective of this work is to perform the partial characterization of enzymatic extracts produced by a newly isolated Penicillium crustosum in solid-state fermentation. Lipase extract presented optimal temperature and pH of 37 A degrees C and 9-10, respectively. The concentrated enzymatic extract showed more stability at 25 A degrees C and pH 7. The enzymes kept 100% of their enzymatic activity until 60 days of storage at 4 and -10 A degrees C. The stability under calcium salts indicated that the hydrolytic activity presented decay with the increase of calcium concentration. The specificity under several substrates indicated good enzyme activities in triglycerides from C4 to C18.

Fermentation medium and oxygen transfer conditions that maximize the xylose conversion to ethanol by Pichia stipitis

The xylose conversion to ethanol by Pichia stipitis was studied. In a first step, the necessity of supplementing the fermentation medium with urea. MgSO(4) x 7H(2)O, and/or yeast extract was evaluated through a 2(3) full factorial design. The simultaneous addition of these three nutritional sources to the fermentation medium, in concentrations of 2.3, 1.0, and 3.0 g/l, respectively, showed to be important to improve the ethanol production in detriment of the substrate conversion to cell. In a second stage, fermentation assays performed in a bioreactor under different K(L)a (volumetric oxygen transfer coefficient) conditions made possible understanding the influence of the oxygen transfer on yeast performance, as well as to define the most suitable range of values for an efficient ethanol production. The most promising region to perform this bioconversion process was found to be between 2.3 and 4.9 h(-1), since it promoted the highest ethanol production results with practically exhaustion of the xylose from the medium. These findings contribute for the development of an economical and efficient technology for large scale production of second generation ethanol. (C) 2011 Elsevier Ltd. All rights reserved.

Produzione di bioidrogeno in dark fermentation da scarti dell'industria agroalimentale mediante l'impiego di batteri ipertermofili

La presente tesi di dottorato ha come argomento la produzione d’idrogeno per via fermentativa sfruttando il metabolismo anaerobico di particolari batteri estremofili del genere Thermotoga. In questo lavoro, svolto in seno al progetto Bio-Hydro, sfruttando reattori batch da 116 mL, è stato selezionato il ceppo migliore di Thermotoga fra i quatto ceppi testati: T. neapolitana. Una volta individuato il candidato batterico migliore è stato individuato il valore ottimale di pH (8.5 a t.amb) per la produzione d’idrogeno. Un intenso lavoro è stato svolto sul medium di coltura permettendone la minimizzazione e rendendolo così economicamente sostenibile per il suo utilizzo nel reattore da 19L; in questo caso il glucosio è stato completamente sostituito con due sottoprodotti agroindustriali individuati in precedenza, il melasso di barbabietola e il siero di latte. Sono stati poi eliminati i gravosi micronutrienti e le vitamine. È stata sfruttata la capacità di T. neapolitana di produrre biofilm e sono stati testati 4 diversi supporti in vetro sinterizzato e ceramici, tali test hanno permesso di individuare Biomax come supporto migliore. Sono stati svolti studi sul metabolismo di T. neapolitana volti ad individuare le concentrazioni inibenti di ogni substrato testato, l’inibizione da prodotto (idrogeno) e l’inibizione da ossigeno. Tutte queste prove hanno dato le conoscenze di base per la conduzione di esperienze su reattore da 19L. L’innovativo reattore di tipo SPCSTR è stato interamente studiato, progettato e costruito presso il DICMA. dell’Università di Bologna. La conduzione di esperienze batch su SPCSTR ha dato la possibilità di verificare il funzionamento del nuovo tipo d’impianto. Presso il Wageningen UR (NL), è stata svolta la selezione del miglior ceppo di Caldicellulosisruptor fra 3 testati e del miglior supporto per la produzione d’idrogeno; è stato poi costruito testato e condotto in continuo l’innovativo reattore CMTB.

Mikrobiologie der Fermentation in NawaRo-Biogasanlagen unter besonderer Berücksichtigung des Abbaus von Propionsäure

In NawaRo-Biogasanlagen (BGA) kann es durch das Angebot an leicht fermentierbaren Kohlenstoff¬quel¬len zu einer bakteriell bedingten Übersäuerung durch unerwünschte kurzkettige Fettsäuren kommen. Häufiger kommt es zur Akkumulation von Propionsäure. Methanogene Archaea können bei niedrigen pH-Werten nicht mehr wachsen. Somit kann der gesamte Prozess der mikrobiellen Bildung von Biogas zum Erliegen kom¬men, was für die Biogasbetreiber zu erheblichen finanziellen Verlusten führt. Das Ziel dieser Disserta¬tion war die Aufklärung der anaeroben bakteriellen Population, die in Biogasanlagen Propionsäure ab¬bauen kann. Aus Propionat entsteht dabei Acetat und Wasserstoff. Da dieser anaerobe Prozess endergon verläuft, kann Propionsäure anaerob nur abgebaut werden, wenn der Wasserstoffpartialdruck niedrig ge¬halten wird. Diese Aufgabe erfüllen in Biogasanalgen methanogene Archaea. Die sog. sekundären Gärer leben somit in synthropher Kultur mit methanogenen Archaea.rnIn dieser Arbeit wurden die Mikroorganismen von Propionsäure-abbauenden Anreicherungskulturen aus vier NawaRo-BGA‘s identifiziert und ihr Substrat- und Produktspektrum analysiert. Die Anreicherungskul¬turen wurden vom Prüf- und Forschungsinstitut e. V. in Pirmasens zur Verfügung gestellt. Durch Analyse der bakteriellen 16S rDNA-Sequenzen der erhaltenen stabilen Propionsäure-abbauenden Mischkulturen wurde gezeigt, dass sich unter den Bakterien hauptsächlich Verwandte von den Clostridiales, aber auch Bacteroides sp., δ-, ε- so¬wie γ-Proteobakterien, Spirochäten, Synergistales und ungewöhnlicher Weise auch Thermotogales befanden. Aus Propionsäure-abbauenden Mischkulturen und aus Fermentern mesophiler NawaRo-Biogasanlagen wurden anaerobe Bakterien und methanogene Archaea angereichert und isoliert. Es wurden aus den Propionsäure-abbauenden Mischkulturen Stämme in Reinkultur erhalten, die entsprechend der 16S rDNA-Analyse als Clostridium sartagoforme Stamm Ap1a520 und Proteiniphilum acetatigenes Stamm Fp1a520 identifiziert wurden. Sowohl aus Fermentern und Nachgärern von drei NawaRo-BGA‘s als auch aus zwei Laborfermentern des Leibniz-Instituts für Agrartechnik in Potsdam-Bornim e.V. (ATB) wurden Reinkulturen von methanogenen Archaea erhalten. Diese konnten den Species Methanobacterium formicicum, Metha¬noculleus bourgensis, Methanosarcina barkeri, Methanosarcina mazei, Methanosarcina sp., Methanosaeta concilii und Methanomethylovorans sp. zugeordnet werden. Damit wurden in dieser Arbeit unter anderem die typischen bisher nur durch molekularbiologische Methoden identifizierten Species methanogener Ar¬chaea aus unterschiedlichen Fermentern in Reinkultur erhalten. Dabei wurde gezeigt, dass die specifically amplified polymorphic DNA-PCR (SAPD-PCR) eine geeignete Methode darstellt, Stämme der gleichen Art methanogener Archaea voneinander zu unterscheiden. Die Methanproduktion der kultivierten methanoge¬nen Archaea wurde gaschromatographisch analysiert. Es zeigte sich, dass die hydrogenotrophe Metha¬nogenese der effizientere und ergiebigere Weg zur Bildung von Methan ist. Mit der Bestimmung der Zellzahl des Isolates Methanoculleus bourgensis Stamm TAF1.1 bei gleichzeitiger Messung der Methanbildung wurde gezeigt, dass die Methanbildung nicht zwangsläufig mit dem Wachstum korreliert. Ne-ben Pflanzenfasern beinhalteten das hergestellte Reaktorfiltrat in den Kultivierungsansätzen Acetat, die essentielle Aminosäure Valin und den Zuckeralkohol Glycerol. Gezielte Misch¬kul¬turen von sekundären Gärern mit methanogenen Isolaten ergaben einen fördernden Einfluss auf diese Bak¬terien durch hydrogenotrophe Archaea. Diese Bakterien bauten Substrate ab oder bildeten Produkte, die sie unter den gegebenen Bedingungen ohne hydrogenotrophe Archaea nicht umsetzen konnten.

Concentration profiles in batch fermentation of hydrolyzed lignocellulose

Experimental data was obtained for profiling changes in concentrations of two inhibiting compounds in batch fermentation of a synthesized liquor resembling hydrolyzed lignocellulose, a furan (furfural) and a phenolic compound (vanillin), along with standard fermentation data, i.e. substrate, biomass and ethanol concentrations. The initial inhibitor concentrations and fermentation temperatures in the 18 experiments were varied according to a two-level complete center composite experimental design. Based upon these observed variations in the fermentative behavior, the fermentation kinetics were modeled, as published in the corresponding article, including microbial conversion rates of the inhibitive compounds into their less toxic derivatives.

Treatment of tropical forages with exogenous fibrolytiic enzymes: effects on chemical composition and in vitro rumen fermentation

The effects of three treatments of fibrolytic enzymes (cellulase from Trichoderma longibrachiatum (CEL), xylanase from rumen micro-organisms (XYL) and a 1:1 mixture of CEL and XYL (MIX) on the in vitro fermentation of two samples of Pennisetum clandestinum (P1 and P2), two samples of Dichanthium aristatum (D1 and D2) and one sample of each Acacia decurrens and Acacia mangium (A1 and A2) were investigated. The first experiment compared the effects of two methods of applying the enzymes to forages, either at the time of incubation or 24 h before, on the in vitro gas production. In general, the 24 h pre-treatment resulted in higher values of gas production rate, and this application method was chosen for a second study investigating the effects of enzymes on chemical composition and in vitro fermentation of forages. The pre-treatment with CEL for 24 h reduced (p < 0.05) the content of neutral detergent fibre (NDF) of P1, P2, D1 and D2, and that of MIX reduced the NDF content of P1 and D1, but XYL had no effect on any forage. The CEL treatment increased (p < 0.05) total volatile fatty acid (VFA) production for all forages (ranging from 8.6% to 22.7%), but in general, no effects of MIX and XYL were observed. For both P. clandestinum samples, CEL treatment reduced (p < 0.05) the molar proportion of acetate and increased (p < 0.05) that of butyrate, but only subtle changes in VFA profile were observed for the rest of forages. Under the conditions of the present experiment, the treatment of tropical forages with CEL stimulated their in vitro ruminal fermentation, but XYL did not produce any positive effect. These results showed clearly that effectiveness of enzymes varied with the incubated forage and further study is warranted to investigate specific, optimal enzyme-substrate combinations.