Cellulolytic Enzymes Production via Solid-State Fermentation: Effect of Pretreatment Methods on Physicochemical Characteristics of Substrate

We investigated the effect of pretreatment on the physicochemical characteristics—crystallinity, bed porosity, and volumetric specific surface of soybean hulls and production of cellulolytic enzymes in solid-state fermentation of Trichoderma reesei and Aspergillus oryzae cultures. Mild acid and alkali and steam pretreatments significantly increased crystallinity and bed porosity without significant change inholocellulosic composition of substrate. Crystalline and porous steam-pretreated soybean hulls inoculated with T. reesei culture had 4 filter paper units (FPU)/g-ds, 0.6 IU/g-ds β-glucosidase, and 45 IU/g-ds endocellulase, whereas untreated hulls had 0.75 FPU/g-ds, 0.06 IU/g-ds β-glucosidase, and 7.29 IU/g-ds endocellulase enzyme activities. In A. oryzae steam-pretreated soybean hulls had 47.10 IU/g-ds endocellulase compared to 30.82 IU/g-ds in untreated soybean hulls. Generalized linear statistical model fitted to enzyme activity data showed that effects of physicochemical characteristics on enzymes production were both culture and enzyme specific. The paper shows a correlation between substrate physicochemical properties and enzyme production

Structural heat treatments against Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae): effect of flour depth, life stage and floor.

The effect of high temperatures (50-60°C) and two levels of sanitation (~0.5 and 43 g of flour), on mortality of eggs, young larvae, old larvae, pupae, and adults of the red flour beetle, Tribolium castaneum, were evaluated during heat treatment of a pilot flour mill at Kansas State University. The mill was heated once during 13-14 May 2009 and once during 25-26 August 2009. Each of the heat treatments lasted 24 h. Bioassay boxes, with life stages of T. castaneum and temperature sensors confined in small compartments, were placed in 25 locations across all five mill floors. Temperature data showed that the mean time to 50°C based on the two treatments ranged from 10.39 to 17.18 h, and the mean time above 50°C ranged from 6.01 to 13.63 h. The mean maximum temperatures attained ranged from 50.7 to 61.4°C. In general, temperatures were lower in compartments with 43 g of flour when compared with compartments with 0.5 g of flour. Temperatures were also lower on the first floor than on the remaining floors. In box bioassays, essentially none of the life stages survived the 24 h heat treatment (99-100% mortality), except on the first floor. The survival of insects, especially on the first floor, is related to how quickly temperatures reached 50°C and how long temperatures were held between 50 and 60°C, and the maximum temperatures attained at a given location. There were only small differences in mortality between the two levels of sanitation. These results show that heat treatment of flour mills can control all life stages of T. castaneum in 24 h. Keywords: Tribolium castaneum, Heat treatment, Sanitation, Life stages, Methyl bromide alternative

The Use of Co-Culturing in Solid Substrate Cultivation and Possible Solutions to Scientific Challenges

This perspective systematically summarizes the use of solid substrate co‐cultures in agriculture, food, plant, and industrial biotechnology applications. The summarization is organized by organism, i.e. fungus, bacteria, yeast and then co‐cultivation of either two or three organisms. Generally, in solid substrate co‐culture, the organisms synergistically penetrate and degrade the solid substrate, thereby increasing product yield and productivity over a monoculture. Efforts to increase co‐culture performance include optimizing process parameters (pH, temperature, moisture, and oxygen demand) and defining the acceptable types of substrate. Scientific challenges exist in understanding the interactions between microbial stains, such as viability, suite of products, and bio‐transformations. The perspective details possible solutions to these challenges and highlights future research directions for co‐cultures using either solid or liquid fermentation

Ex vivo modelling of drug efficacy in a rare metastatic urachal carcinoma

Background Ex vivo drug screening refers to the out-of-body assessment of drug efficacy in patient derived vital tumor cells. The purpose of these methods is to enable functional testing of patient specific efficacy of anti-cancer therapeutics and personalized treatment strategies. Such approaches could prove powerful especially in context of rare cancers for which demonstration of novel therapies is difficult due to the low numbers of patients. Here, we report comparison of different ex vivo drug screening methods in a metastatic urachal adenocarcinoma, a rare and aggressive non-urothelial bladder malignancy that arises from the remnant embryologic urachus in adults. Methods To compare the feasibility and results obtained with alternative ex vivo drug screening techniques, we used three different approaches; enzymatic cell viability assay of 2D cell cultures and image-based cytometry of 2D and 3D cell cultures in parallel. Vital tumor cells isolated from a biopsy obtained in context of a surgical debulking procedure were used for screening of 1160 drugs with the aim to evaluate patterns of efficacy in the urachal cancer cells. Results Dose response data from the enzymatic cell viability assay and the image-based assay of 2D cell cultures showed the best consistency. With 3D cell culture conditions, the proliferation rate of the tumor cells was slower and potency of several drugs was reduced even following growth rate normalization of the responses. MEK, mTOR, and MET inhibitors were identified as the most cytotoxic targeted drugs. Secondary validation analyses confirmed the efficacy of these drugs also with the new human urachal adenocarcinoma cell line (MISB18) established from the patient’s tumor. Conclusions All the tested ex vivo drug screening methods captured the patient’s tumor cells’ sensitivity to drugs that could be associated with the oncogenic KRASG12V mutation found in the patient’s tumor cells. Specific drug classes however resulted in differential dose response profiles dependent on the used cell culture method indicating that the choice of assay could bias results from ex vivo drug screening assays for selected drug classes

Screening of winery and olive mill wastes for lignocellulolytic enzyme production from Aspergillus species by solid-state fermentation

Wastes from olive oil and wine industries (as exhausted grape marc, vineshoot trimmings, two-phase olive mill waste, vinasses, and olive mill wastewater) were evaluated for lignocellulolytic enzyme production (as endocellulases, endoxylanases, and feruloyl esterases) by solid-state fermentation (SSF) with Aspergillus niger, Aspergillus ibericus, and Aspergillus uvarum. To study the effect of different solid medium composition and time in enzyme production, a PlackettBurman experimental design was used. Variables that had a higher positive effect in lignocellulolytic enzyme production were urea, time, and exhausted grape marc. The maximum values of enzymatic activity per unit of substrate dry mass were found with A. niger for feruloyl esterase. Enzymatic extracts from SSF with A. niger achieved maximum feruloyl esterase activity (89.53 U/g) and endoxylanase activity (3.06 U/g) and with A. uvarum for endocellulase activity (6.77 U/g). The enzyme cocktails obtained in the SSF extracts may have applications in biorefinery industries.Jose Manuel Salgado is grateful for the postdoctoral fellowship (EX-2010-0402) of the Education Ministry of Spanish Government. Luis Abrunhosa was supported by the grant SFRH/BPD/43922/2008 from Fundacao para a Ciencia e Tecnologia-FCT, Portugal

Growth kinetics and modelling of S. cerevisiae (NCYC 431) during de-lignified waste banana fermentation and chemical characterization

S. cerevisiae (NCYC 431) growth kinetics was studied using mathematical models in order to ascertain the optimum operational parameters for banana waste fermentation. Chapman-Richards model was used to describe yeast growth kinetics under varying pH and temperatures and the results were compared to Bergter and Andrew models. Alkaline-delignification of the wastes was done to solubilize lignin prior fermentation. This is because lignin is a complex organic plant compound that has been reported not to be degraded easily by many microorganisms. From the results temperatures 22–28 °C and pH 4.5–5.6 were noted as optimum for yeast growth on delignified waste bananas (DWB). Chapman model results were close to Bergter and Andrew models with very low RMSE. Delignification was noted to aid yeast growth with higher microbial populations (log10 cfu/g) registered with DWB samples as compared to non-delignified waste bananas (NDWB). Also, chemical characterization of the DWB and NDWB indicated higher proteins and lipids in the former than the latter by 3 and 4% respectively. This suggested the possible use of the upgraded wastes as chicken feed supplements. Higher minerals in DWB of 8.6% also suggested the possible use of the waste as a nutrient-rich fertilizer

Direct use of spent mushroom substrate from Pleurotus pulmonarius as a readily delignified feedstock for cellulase production

The feasibility of spent mushroom substrate (SMS) as an alternative fermentation feedstock for cellulase production has been demonstrated in this work. Utilization of SMS as a substrate has been attempted widely due to its high cellulose content and readily available in smaller particle size. On top of that, the availability of delignified SMS by the action of Pleurotus pulmonarius during mushroom cultivation offers another benefit to its use whereby no chemical pretreatment would be required prior to fermentation. The recovery of crude laccase and manganese peroxidase from delignified SMS were found to be 3 and 1.4 U/g, respectively. Further to this, the cellulase production from SMS by Trichoderma asperellum UPM 1 under solid state fermentation was optimized by applying central composite design, resulted in increment of 1.4-fold in CMCase (171.21 U/g) and 1.5-fold in β-glucosidase (6.83 U/g), with the optimum temperature of 27.5 °C, initial moisture content 81% and initial pH of fermentation 4.5. Therefore, this study showed that the direct utilization of SMS is feasible for promising cellulase production by T. asperellum UPM 1