Valorizing Red Seaweed Spent Biomass into Reducing Sugars for β-Carotene Production by Rhodotorula paludigena

The present study evaluated first the characterization of Teff straw and then Box–Behnken design (BBD), and response surface methodology was adopted to optimize the parameters (hydrolysis temperature, dilute sulfuric acid concentration, solid to liquid ratio, and hydrolysis time) of dilute sulfuric acid hydrolysis of Teff straw in order to get a maximum yield of total reducing sugar (TRS). The chemical analysis of Teff straw revealed high amounts of cellulose (41.8 wt%), hemicellulose (38 wt%), and lignin (17 wt%). The morphological analysis using SEM showed that hydrolyzed Teff straw with dilute sulfuric acid has more pores and distorted bundles than those of raw Teff straw. XRD analysis also indicated that hydrolyzed Teff straw has higher crystallinity index and smaller crystallite size than raw Teff straw, which might be due to removal of hemicellulose, amorphous cellulose, and lignin components. Under the optimized conditions for dilute sulfuric acid hydrolysis of Teff straw (120°C, 4% v/v H2SO4 concentration, 1 : 20 solid to liquid ratio, and 55 min hydrolysis time), we have found a total reducing sugar yield of 26.65 mg/g. The results of validation experiment under the optimum conditions agreed well with model predictions.

Process to determine concentration of reducing sugar from oil palm fronds and rice straw has been developed. Reducing sugar of oil palm fronds and rice straw were determined to compare the hydrolysis conditions on the different temperature, acid concentrations and time. In this study, oil palm fronds and rice straw was pre-treated with 2M NaOH. Alkali treated material was then hydrolyzed with 1.0, 5.0 and 10.0 % (v/v) sulfuric acid at 111° C and 121° C for 15 minutes and 30 minutes. The results showed that the maximum reducing sugar concentration of oil palm fronds (81.11 ug/ml) was obtained at 121 ° C for 30 min with sulfuric acid concentration of 10.0 % (v/v), whereas the maximum reducing sugar of rice straw (78.80 ug/ml) was obtained at 111 ° C for 30 min with sulfuric acid concentration of 1.0 % (v/v). In comparison, rice straw showed high reducing sugar concentration at low concentration of sulfuric acid but at high temperature. In contrast, oil palm fronds showed high reducing sugar concentration at high concentration of sulfuric acid but at low temperature.

Alkaline pretreatment is used as a substrate prior to subsequent biological processes for palm oil mill effluent (POME) treatment. In the present study, Sodium hydroxide (NaOH) was used to recover reducing sugar from POME. Important process parameters, such as reaction time (min), reaction temperature (°C), and concentration (%) (w/v), were optimized using Box-Behnken Design in Response Surface Methodology (RSM). POME treated with NaOH yielded the reducing sugar of up to 3640.84 mg/L under the optimum conditions of 58.78 min, 77.06°C, and 2.58% (w/v) for reaction time, reaction temperature, and concentration of NaOH, respectively. The analysis of variance (ANOVA) indicated that the quadratic model for reducing sugar production had an R2 coefficient of 0.979. Conformity testing for optimum conditions proved the validity of the model, yielding reducing sugar at a 9.35% increase, relative to untreated POME. This study verified the importance of statistical tools such as RSM for enhanced reducing sugar production from the industrial waste stream (POME) and its usefulness and efficiency in energy conversion.

This study is the firts in Brazil to assess a simultaneous production of hemi(cellulolytic) enzymes and reducing sugars (RS) from Alstroemeria sp. stems and leaves by the fungus growth Pleurotus ostreatus PLO6 using solid state fermentation (SSF). The researching is pioneer to characterize brazilian flower waste and, from chemical view point, to taking advatange this biomass to obtain ART, regarding its subsequent conversion into second generation bioethanol. The chemical composition of this kind of waste was: moisture 10,1%, ash 7,5%, cellulose 36,1%, hemicelluloses 14,7% e lignin 15,2%. The elemental analysis was: carbon 42,8%, nitrogen 1,1%, hydrogen 5,4% and sulfur 1,3%. Besides, the metal concentrations were: cupper 20,0 mg/kg, iron 256,8 mg/kg, zinc 133,4 mg/kg, lead 0,0 mg/kg e manganese 105,22 mg/kg. The researching first stage (called preliminary) verified the simultaneous production of hemi(cellulolytic) enzimes and RS designing univariate experiments, where the response variables were (hemi)cellulolytic enzymatic activities and RS concentration, and the factor chosen was time between 0 and 14 days; performing quantification in duplicate each 24 hours. At this stage, the basidiomycete was able to produce endoglucanase, exoglucanase, -glucosidase, endoxylanase and -xylosidase enzymes, with maximum activities of 10,3 U/g (day 4), 74,5 U/g (day 5), 26,3 U/g (day 6), 20,5 U/g (day 9) and 94,9 U/g (day 11), respectively and the RS highest concetration got was 3,3 g/L at 12th day of fermentation. After to check the simultaneous production of enzymes and RS, a second stage was performed (called screening), which assesed effects of temperature (T, oC), substrate concentration (SC) and time (t, days) on enzimatic synthesis and RS. This phase, was developed with a statistical factorial design 23 in triplicate, where, the factor nivels were 24 and 32 oC, 20 and 30 % de SC, and 8 and 15 days, and the response variables were the same used at preliminary phase. In this part, with a significance level of 0,05, the effect more significant on enzymes (except to -xylosidase) and RS production was temperature; cellulolytic activities and RS were maximized during 24 oC, while hemicellulolytic enzymes were maximized at 32 oC. The last stage developed was RS optimization, it was performed based on central composite design (CCP), established acording to significance results obtained at screening phase. There, the highest production of RS predicted by the mathematical model was 4,35 g/L at 24,22 oC, 20,75% of SC and 8,94 days of fermentation; conditions very close to center point chosen (4,36 g/L at 24 oC, 20% of SC and 9 days of SSF) and the highest RS concentration achieved at screening stage (4,45 g/L with the same conditions of center point). The chemical composition of flower waste is inside the rank of cellulose, hemicelluloses and lignine, considered like alternative biomass to produce second generation bioethanol. According to content of carbon, nitrogen, cupper and manganese, Alstroemeria sp. waste were a suitable substrate that let good growth and developing of P. ostreatus PLO6 activities to synthesize enzymes that degrade lignocellulosic (LC) raw matrial. Also, results showed that hemicellulases synthesis was maximized at 32 oC, while cellulases and RS production was more favorable at 24 oC.

Microalgae biomass is a potential substrate for bioethanol production because (1) microalgae can exhibit a higher productivity than lignocellulosic crops; (2) cultivation of microalgae can avoid the competition between energy production and food supply when using energy crops; (3) microalgae, compared with terrestrial plants, have a low lignin content to facilitate the lignin removal. In order to generate bioethanol from microalgae biomass, cellulose in the microalgae biomass should be hydrolyzed by a variety of cellulases and the hydrolysates (reducing sugars) are subsequently fermented by yeast to produce ethanol. However, this traditional process, named separate hydrolysis and fermentation (SHF), revealed a great inhibition in the cellulase activity that is reduced by the accumulated reducing sugars. Nevertheless, it has been indicated that a simultaneous saccharification and fermentation (SSF) process can achieve a high ethanol yield than SHF process due to its maintenance of cellulase activity. Therefore, the aim of this study was to investigate the feasibility of bioethanol production from microalgae biomass by Cellulomonas sp. and Saccharomyces cerevisiae in a batch-mode SSF system. In this study, a commercial powder of Chlorella sp. biomass and a self-cultivated Graesiella sp. biomass, were used to estimate their bioethanol production. The effect of HCl or NaOH concentrations for hydrolysis pretreatment, composition of culture medium, microalgae biomass concentration, and the temperature for cellulose hydrolysis on the production of reducing sugars were all carried out, using the commercial powder of Chlorella sp. biomass as experimental substrate. On the other hand, the optimal conditions of temperature, pH value, and the initial amount of Saccharomyces cerevisiae inoculating biomass in the fermentation of glucose were examined. Considering the cellulase-producing bacterium, Cellulomonas sp. would consume the reducing sugars after the hydrolysis, the spent medium that contained the extracellular cellulase (crude cellulase) was used to understand the effect of above experimental parameters on the reducing sugar production and the inhibition of enzyme activity by hydrolysates. Finally, the efficiency of producing reducing sugar by the crude cellulase would be compared with that by a commercial cellulase. The results showed that the cellulose content of 55.18% in Graesiella sp. was observed in a 5-liter-photobioreactor cultivation, which can improve the cellulose content in 9.6 fold than a 200-milliliter-flask cultivation. For the hydrolysis of Chlorella sp. biomass by the crude cellulase, the maximum amount of reducing sugars was achieved in the Mandels Reese culture medium with 20 g/L inoculum at 30oC. With respect to the pretreatment procedure, treating the Chlorella sp. biomass with 2% NaOH and further mixing the treated biomass with neutralized supernatant from earlier base pretreatment can shorten the hydrolysis period and increase the reducing sugars production in 88%. In addition, the highest ethanol content of 43.0 g/L was obtained when the fermentation process was performed under 10 g/L of glucose with a inoculum of 0.07 g/L at 40oC and pH 6.5. Integrating those optimal parameters and utilizing the crude and commercial cellulase to hydrolyze the Graesiella sp. or Chlorella sp. biomass in both SHF and SSF processes, the results indicated that the commercial cellulase can yield reducing sugars of 0.6 g/L and ethanol of 4.09 g/L in the SHF process with unpretreated Chlorella sp. biomass. However, an obvious inhibition to endo-β-1, 4-gulcanase was observed and its enzyme activity decreased in 57% within this SHF experiment. On the contrary, using the crude cellulase in the SSF process showed a high ethanol production (6.59 g/L). Using the pretreated Chlorella sp. biomass as the substrate, the SSF process can improve the ethanol production in 3.03 fold than SHF process. Furthermore, hydrolysis of Graesiella sp. biomass produced more ethanol and exhibited a higher ethanol-producing rate than those of Chlorella sp. biomass no matter what kind of cellulase and process were performed.

The recalcitrance of lignocellulosic feedstock needs to be altered for to produce fuels and chemicals. Pretreatment is used to enhance the reactivity of cellulose and the digestibility of biomass, resulting in the effective generation of fermentable sugars. Potassium hydroxide (KOH) is particularly effective at selectively removing lignin from biomass without excessively degrading cellulose and hemicellulose. Moreover, KOH is generally less corrosive than sodium hydroxide (NaOH), leading to lower maintenance costs for pretreatment equipment. In present study, Napier grass was utilized as the substrate for reducing sugar production. Proximate analysis indicated that Napier grass contains approximately 28.50±0.12% hemicellulose, 34.15±0.08% cellulose and 26.41±0.04% lignin. With the use of the Box-Behnken Design (BBD) method, pretreatment conditions were improved. The ideal conditions for KOH pretreatment of Napier grass were determined to be 6% KOH, 180 °C temperature, and a pretreatment time of 120 min. Higher yields of reducing sugars (43.29 g/L) were achieved by this optimized condition. By analyzing the experimental data, ANOVA helps in developing a reliable model that predicts the ideal conditions for increasing reducing sugar yield. Desirability functional studies were employed in optimization to identify ideal conditions that satisfy multiple criteria simultaneously, and the design was validated by trial experiments to ensure accuracy. Desirability studies confirmed that the optimal yield of reducing sugars, approximately 43.72 g/L, was achieved with 6.79% KOH concentration, at a temperature of 178.4°C, and a pretreatment duration of 119.6 min. The results are closely resembles the experimental values predicted by the response surface model. Substrates pretreated with higher KOH concentrations yielded more ethanol (72 g/L) from Saccharomyces cerevisiae in SSF tests compared to those pretreated with lower KOH concentrations.

Background Cellulase is the most employed industrial enzyme in biological conversion of many cellulosic wastes. In this work, economic cellulase production by fungi in solid-state fermentation (SSF) by using solid wastes of medicinal plants was studied. Optimization of growth conditions for production of cellulase was the main target of this study. Objective The current study aimed to isolate and screen fungal isolates that have the ability to produce enzymes to degrade solid wastes of medicinal plant process and optimization of growth factors that affect cellulase production. Materials and methods Thirty-five fungal isolates were isolated from different sources by plating and screened for their cellulase activities using Czapek–Dox broth medium amended with 1% cellulose. Cellulase production by tested fungal isolates was carried out through utilization of olive (Olea europaea), black seeds (Nigella sativa), and castor bean (Ricinus communis) cakes in SSF. Optimization of the cellulase productivity was performed by Plackett–Burman design (PBD) and Box–Behnken design. Results and conclusion Out of the isolated 35 fungi, only 12 (34%) produced cellulase in SSF using olive, black seeds (Nigella), and castor bean cakes. Out of these fungal isolates, only 4, that is, no. 1, 7, 10, and 17 were superior in reducing sugar production from olive cakes (13.04, 15.61, 17.03, and 12.85 mg/ml), respectively. While four fungal isolates no. (1, 7, 7, and 10) were active producers of reducing sugars from black seeds (15.45, 18.96, 20, and 18.08 mg/ml), respectively. Only a fungal isolate no. 7 gave high reducing sugars (15.34 mg/ml) in castor cake SSF. The most potent fungal isolate (no. 10) produced 20 mg/ml of reducing sugars using black seed cakes as substrate for SSF. The potential fungal isolate was identified as Aspergillus terreus (OQ085169) based on the extracted fungal DNA that was amplified by PCR using specific internal-transcribed spacer primer (ITS1/ITS4). The PCR products were sequenced and compared with the other related sequences in GenBank (NCBI). The screening of seven factors using PBD showed that only three variables: pH, incubation time, and aeration rate (rpm) affected significantly cellulase production. Box–Behnken design was used to estimate the optimal level of the selected variables based on the results of the PBD. All variables increased significantly cellulase using A. terreus (OQ085169). The P value was very low (0.0207) that indicated the significant, high correlation between the predicted and actual values (R 2=0.98), this indicating 98% of the variation in the cellulase activity was owing to the selected independent variables.

Fruit peel waste (FPW) is a renewable and cheaper source of fermentable sugars for biofuel production. In this research, three types of FPW which are banana peel, orange peel, and pineapple peel were investigated for reducing sugar generation using microwave pretreatment. Pineapple peel waste was found to produce the highest yield of total reducing sugar compared to banana peel and orange peel, with the value of 10.58 g/L. The optimization condition for microwave pretreatment was studied by using the Central Composite Design under Response Surface Methodology. Microwave power (180 – 450 watt), time (3 – 7 minutes) and biomass loading (5 -15 % w/v) were the parameters used to optimize the reducing sugar yield from pineapple peel. The optimum condition for microwave pretreatment was found at biomass loading of pineapple peel of 15% (w/v), power of 450 watt with 5 minutes of treatment time with the concentration of 12.52 g/L. It was apparent that the structure of hemicellulose, cellulose, and lignin of the pineapple peel was affected by the microwave pretreatment condition based on Fourier Transform Infrared spectroscopy (FTIR) and Scanning Electron Microscope (SEM) analysis. FTIR showed alteration in a functional group while the SEM micrographs showed a marked change on the pretreated sample when compared with the untreated sample. In conclusion, microwave pretreatment is a promising approach for reducing sugar production from the agricultural residue such as FPW.

Modelling of the high pressure–temperature effects on naringin hydrolysis based on response surface methodology

Polyhydroxybutyrate (PHB) is a suitable biodegradable alternative to non-renewable petroleum-based plastics. Readily available agricultural lignocellulosic residues such as banana pseudostem can be utilized as a substrate to reduce the production cost of PHB and improve its feasibility for commercialization. Accordingly, pretreatment of banana pseudostem is needed to efficiently convert the substrate to PHB. In this study, optimization of two pretreatment methods for banana pseudostem – namely, steam explosion and sequential steam explosion – dilute acid pretreatment – were optimized to improve the digestibility of the biomass and consequently increase the production of reducing sugars in the hydrolysate during enzymatic saccharification. Response surface methodology (RSM)-designed experiments showed that among all factors investigated, for both pretreatment methods, the steam explosion temperature had the strongest positive impact on reducing sugar production. Optimum conditions of steam explosion pretreatment were 219.31 °C steam explosion temperature and 10 min of pretreatment time, producing 7.33 g/L (48.87% yield) of reducing sugars in the enzymatic hydrolysate. For sequential pretreatment, optimum conditions were 220 °C steam explosion temperature, 135 °C dilute acid temperature, 44 min of dilute acid reaction time, and 1.57% w/v H2SO4, with a corresponding reducing sugar concentration of 13.02 g/L (86.79% yield). A 90% increase in reducing sugar yield was observed after dilute acid pretreatment of steam-exploded banana pseudostem. Using the hydrolysate from sequentially-pretreated banana pseudostem, PHB (2.64 g/L) was successfully synthesized after 12 h of bacterial fermentation. Hence, sequential pretreatment was proven effective in producing enzymatic hydrolysates from banana pseudostem for PHB production.

Robusta coffee ( Coffee robusta L. ) pulp waste has been known for its high cellulose and hemicellulose content which potentially could be utilized as a source of a reducing sugar feedstock. Unfortunately, it contains inhibitors such as lignin, tannin, caffeine, and total polyphenols that can inhibit the enzymatic hydrolysis process. Therefore, coffee pulp waste needs pre-treatment prior to its utilization in reducing sugar production. To optimize the pre-treatment condition, the two-stage of pre-treatment process was carried out using 0.2 M sulfuric acid and then organosolv using ethanol. Subsequently, the optimization was done using Response Surface Methodology (RSM), 2 3 full factorial design, with the following input variables: ethanol concentration, temperature, and duration of pre-treatment. This study was subjected to determine the optimum conditions for organosolv pre-treatment which resulted in inhibitor removal and high concentration of reducing sugar. The hydrolysis process was carried out for 60 hours using a mixture of enzymes with and without the addition of Tween 80 as a surfactant. The result indicated that the lignin removal percentage increased from 0.85% (w/w) to 16.905% (w/w) towards the remaining lignin grams, with the change of crystallinity index of cellulose from 17.23% into 16.43%. The concentration of obtained reducing sugar with the addition of Tween 80 was 2.402 mg/ml, 1.6 times higher than that obtained without the addition of Tween 80.

ABSTRACTBioethanol is considered the cleanest liquid fuel used as a substitute for depleting fossil fuels. Various technologies have been introduced to form bioethanol from lignocellulosic biomass. Seed pods of Bombax ceiba, which are produced and wasted in large amount annually, were used as a source of cellulose. In this study, response surface methodology was used to explore the effects of KOH concentrations, substrate loading, and residence time on cellulose exposure and liberation of reducing sugars (RS), total sugars (TS), and total phenolic compounds from seed pods of B. ceiba. Box–Behnken design with three variables and three levels showed maximum release of total phenolic compounds (394.04 mg/ml) and RS (50.06 mg/ml) corresponding to 3% KOH concentration, 15% substrate level with residence time of 8 h at 121°C, and maximum cellulose exposure (64%), and TS (206.65 mg/ml) liberation was observed at 5% KOH concentration and 10% substrate level at same temperature for same soaking time. While at room temperature maximum cellulose exposed (46%), TS (146.1480 mg/ml), total phenol (300.3901 mg/ml), and RS (9.0075 mg/ml) were observed at 3% KOH, 15% substrate concentration, and 8-h residence time. These results suggested that thermochemical pretreatment is more effective than chemical pretreatment alone. The second-order polynomial equation using analysis of variance was employed for analyzing the results.

Saccharification of sugarcane bagasse by magnetic carbon-based solid acid pretreatment and enzymatic hydrolysis

The main aim of this study was to evaluate the optimum conditions for extracting the total reducing sugar content for bioethanol production using spirulina algae. The spirulina algae was pretreated using microwave-assisted acid hydrolysis, and the parameters were optimized using response surface methodology (RSM). The selected independent parameters were microwave power (250-350 W), sulfuric acid concentration (1-7%), and time duration (1-5min). The results showed that a maximum reducing sugar concentration of 3.8mg/mL was produced at optimum conditions. ANOVA and R-squared (R2) value (99.87%) show the model was significant (p value is < 0.0001). Additionally, a study on optimization and modeling was conducted utilizing response surface methodology (RSM) as well as artificial neural networks (ANN) to evaluate the impact of temperature (30-40°C), concentration of inoculum (1-5g/L), and fermentation duration (12-45h). This comparative assessment showed that the highest ethanol concentration of 1.824g/L was achieved under optimal conditions of 30°C, 5g/L inoculum concentration, and 28.5h duration, as determined by the high-performance liquid chromatography method. Finally, it is suggested that the RSM approach demonstrated superior performance with a higher R2 value (97.42%), p value is < 0.0001 (significant), and a lower mean square error (MSE) of 0.0065 compared to the ANN model.

This study was aimed to investigate the optimal condition of ethanol production that has 2 major stages: acid hydrolysis and fermentation processes. These processes came from low quality sweet potato (LQSP) which was destroyed by the sweet potato weevil. The main compositions of LQSP were starch and fiber which consist of 55.25 and 10.29 %, respectively. In this case, the starch can be hydrolyzed to reduce the sugar, followed by the fermentation of the reduced sugar to ethanol. For this experiment, the effecting factors on acid hydrolysis of LQSP and the ethanol fermentation condition were optimized by S. cerevisiae using Response Surface Methodology (RSM) with Box-Behnken design in order to maximize ethanol yield. It was found that the maximum reducing sugar concentration of 390.99 ± 5.35 g/L was obtained from the hydrolysis condition with 1 % (v/v) of sulfuric acid and 25 % (w/v) of LQSP. Accordingly, the effects of ammonium sulphate content (0.05 - 0.15 %), pH (4.5 - 5.5) and inoculum content (5 - 10 %) on ethanol production was determined by RSM using Box-Behnken experiment design with a total 17 sets of all trials. The results were found that the maximum experimental ethanol productivity of 5.98 g/L was obtained from the condition at 0.05 % of ammonium sulphate, pH 5.5 and 5.0 % of inoculum size to 90 mL LQSP based medium and incubated at 30 °C for 48 h. In addition, the scale-up of ethanol production was studied in 9 L fermenter which provided the maximum ethanol yield of 5.04 g/L. Therefore, it can be concluded that LQSP had a potential as a substrate for ethanol production.