Fungal delignification of lignocellulosic biomass improves the saccharification of cellulosics

The biological delignification of lignocellulosic feedstocks, Prosopis juliflora and Lantana camara was carried out with Pycnoporus cinnabarinus, a white rot fungus, at different scales under solid-state fermentation (SSF) and the fungal treated substrates were evaluated for their acid and enzymatic saccharification. The fungal fermentation at 10.0 g substrate level optimally delignified the P. juliflora by 11.89% and L. camara by 8.36%, and enriched their holocellulose content by 3.32 and 4.87%, respectively, after 15 days. The fungal delignification when scaled up from 10.0 g to 75.0, 200.0 and 500.0 g substrate level, the fungus degraded about 7.69–10.08% lignin in P. juliflora and 6.89–7.31% in L. camara, and eventually enhanced the holocellulose content by 2.90–3.97 and 4.25–4.61%, respectively. Furthermore, when the fungal fermented L. camara and P. juliflora was hydrolysed with dilute sulphuric acid, the sugar release was increased by 21.4-42.4% and the phenolics content in hydrolysate was decreased by 18.46 and 19.88%, as compared to the unfermented substrate acid hydrolysis, respectively. The reduction of phenolics in acid hydrolysates of fungal treated substrates decreased the amount of detoxifying material (activated charcoal) by 25.0–33.0% as compared to the amount required to reduce almost the same level of phenolics from unfermented substrate hydrolysates. Moreover, an increment of 21.1–25.1% sugar release was obtained when fungal treated substrates were enzymatically hydrolysed as compared to the hydrolysis of unfermented substrates. This study clearly shows that fungal delignification holds potential in utilizing plant residues for the production of sugars and biofuels.     

Cellulase production by Aspergillus niger in biofilm, solid-state, and submerged fermentations

Cellulase production by Aspergillus niger was compared in three different culture systems: biofilm, solid-state, and submerged fermentation. Biofilm and solid-state fermentations were carried out on perlite as inert support, and lactose was used as a carbon source in the three culture systems. In cryo-scanning electron microscopy, biofilm and solid-state cultures gave similar morphological patterns and confirmed that both spore first attachment and hyphal adhered growth are helped by the production of an adhesive extracellular matrix. Biofilm cultures produced higher cellulase activities than those in submerged and solid-state cultures (1,768, 1,165, and 1,174 U l⁻¹, respectively). Although biofilm cultures grew less than the other cultures, they produced significantly higher cellulase yields (370, 212, and 217 U g⁻¹ lactose, respectively) and volumetric productivities (24, 16, and 16 U l⁻¹ h⁻¹, respectively). Likewise, endoglucanase and xylanase activities were higher in biofilm cultures. Under the conditions tested, it seems that fungal attached growth on perlite may favor better enzyme production. Biofilms are efficient systems for cellulase production and may replace solid-state fermentation. Biofilm fermentation holds promise for further optimization and development. The results of this work reveal that fungal biofilms may be used for the commercial production of cellulase employing the technology developed for submerged fermentation at high cell densities.     

Dependence of the hydrolytic exoenzyme production level on the way of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> culturing

An 867 mutant has been derived from a B. subtilis 1560 strain capable of biofilm formation during stationary culturing. The mutant was able to form more elastic and resistant to mechanical stress biofilms that could be used in fermentation more than once. Analysis of the hydrolytic activity of the 1560 and 867 strains film and submerged cultures showed that the biofilm culturing permitted to enhance the levels of amylase by 1.5–2.4 times and that of glucanase by three times.     

Bioethanol production from tension and opposite wood of <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> using organosolv pretreatment and simultaneous saccharification and fermentation

During tree growth, hardwoods can initiate the formation of tension wood, which is a strongly stressed wood on the upper side of the stem and branches. In Eucalyptus globulus, tension wood presents wider and thicker cell walls with low lignin, similar glucan and high xylan content, as compared to opposite wood. In this work, tension and opposite wood of E. globulus trees were separated and evaluated for the production of bioethanol using ethanol/water delignification as pretreatment followed by simultaneous saccharification and fermentation (SSF). Low residual lignin and high glucan retention was obtained in organosolv pulps of tension wood as compared to pulps from opposite wood at the same H-factor of reaction. The faster delignification was associated with the low lignin content in tension wood, which was 15% lower than in opposite wood. Organosolv pulps obtained at low and high H-factor (3,900 and 12,500, respectively) were saccharified by cellulases resulting in glucan-to-glucose yields up to 69 and 77%, respectively. SSF of the pulps resulted in bioethanol yields up to 35 g/l that corresponded to 85–95% of the maximum theoretical yield on wood basis, considering 51% the yield of glucose to ethanol conversion in fermentation, which could be considered a very satisfactory result compared to previous studies on the conversion of organosolv pulps from hardwoods to bioethanol. Both tension and opposite wood of E. globulus were suitable raw materials for organosolv pretreatment and bioethanol production with high conversion yields.     

Biodelignification of lignocellulose substrates: An intrinsic and sustainable pretreatment strategy for clean energy production

Lignocellulosic biomass (LB) is a promising sugar feedstock for biofuels and other high-value chemical commodities. The recalcitrance of LB, however, impedes carbohydrate accessibility and its conversion into commercially significant products. Two important factors for the overall economization of biofuel production is LB pretreatment to liberate fermentable sugars followed by conversion into ethanol. Sustainable biofuel production must overcome issues such as minimizing water and energy usage, reducing chemical usage and process intensification. Amongst available pretreatment methods, microorganism-mediated pretreatments are the safest, green, and sustainable. Native biodelignifying agents such as Phanerochaete chrysosporium, Pycnoporous cinnabarinus, Ceriporiopsis subvermispora and Cyathus stercoreus can remove lignin, making the remaining substrates amenable for saccharification. The development of a robust, integrated bioprocessing (IBP) approach for economic ethanol production would incorporate all essential steps including pretreatment, cellulase production, enzyme hydrolysis and fermentation of the released sugars into ethanol. IBP represents an inexpensive, environmentally friendly, low energy and low capital approach for second-generation ethanol production. This paper reviews the advancements in microbial-assisted pretreatment for the delignification of lignocellulosic substrates, system metabolic engineering for biorefineries and highlights the possibilities of process integration for sustainable and economic ethanol production.     

Fungal-bacterial biofilms: their development for novel biotechnological applications

The attachment of microbes on biotic or abiotic surfaces to form biofilm structures has a great impact on biodegradation and biosynthesis in nature. Various interactions in such biofilms and their extracellular polymeric substances (EPS) layer make them considerably different in physiology and action, compared to that of their individual microbes in planktonic (free swimming) mode of growth. Expression of new genes is up-regulated in the biofilm cells, due in part to the cellular interactions, compared with the planktonic cells. Formation of fungal-bacterial biofilms (FBB) by bacterial colonization on biotic fungal surface gives the biofilm enhanced metabolic activities compared to monocultures, and perhaps multi-species bacterial or fungal biofilms on abiotic surfaces. Incorporation of a N₂-fixing rhizobial strain to the FBB to form fungal-rhizobial biofilms (FRB) has been shown to improve potential biofilm applications in N-deficient settings and in the production of biofilmed inocula for biofertilizers and biocontrol in plants. Their applications in agricultural and environmental settings, enzyme technology, drug discovery studies and energy research are being investigated. Thus, it has already been shown that the use of the FBB is a promising technology for many applications. This review deals with the different areas in which FBB/FRB have been seen to be applied with successful results as well as the numerous emerging avenues in which they show promising potential.     

Organic acid pretreatment of oil palm trunk: effect on enzymatic saccharification and ethanol production

Bioprocess and Biosystems Engineering - Effective lignocellulosic biomass saccharification is one of the crucial requirements of biofuel production via fermentation process. Organic acid...     

Bioethanol production from ammonia percolated wheat straw

This study examined the effectiveness of ammonia percolation pretreatment of wheat straw for ethanol production. Ground wheat straw at a 10% (w/v) loading was pretreated with a 15% (v/v) ammonia solution. The experiments were performed at treatment temperature of 50∼170°C and residence time of 10∼150 min. The solids treated with the ammonia solution showed high lignin degradation and sugar availability. The pretreated wheat straw was hydrolyzed by a cellulase complex (NS50013) and β-glucosidase (NS50010) at 45°C. After saccharification, Saccharomyces cerevisiae was added for fermentation. The incubator was rotated at 120 rpm at 35°C. As a result of the pretreatment, the delignification efficiency was > 70% (170°C, 30 min) and temperature was found to be a significant factor in the removal of lignin than the reaction time. In addition, the saccharification results showed an enzymatic digestibility of > 90% when 40 FPU/g cellulose was used. The ethanol concentration reached 24.15 g/L in 24 h. This paper reports a total process for bioethanol production from agricultural biomass and an efficient pretreatment of lignocellulosic material.     

Effects of Carbon Source, Carbon Concentration, and Chlorination on Growth Related Parameters of Heterotrophic Biofilm Bacteria

Abstract To investigate growth of heterotrophic biofilm bacteria, a model biofilm reactor was developed to simulate a drinking water distribution system. Controlled addition of three different carbon sources (amino acids, carbohydrates, and humics) at three different concentrations (500, 1,000, and 2,000 ppb carbon) in the presence and absence of chlorine were used in separate experiments. An additional experiment was run with a 1:1:2 mixture of the above carbon sources. Biofilm and effluent total and culturable cells in addition to total and dissolved organic carbon were measured in order to estimate specific growth rates (SGRs), observed yields, population densities, and bacterial carbon production rates. Bacterial carbon production rates (μg C/L day) were extremely high in the control biofilm communities (range = 295–1,738). Both growth rate and yield decreased with increasing carbon concentrations. Therefore, biofilm growth rates were zero-order with respect to the carbon concentrations used in these experiments. There was no correlation between growth rate and carbon concentration, but there was a significant negative correlation between growth rate and biofilm cell density (r=−0.637, p= 0.001 control and r=−0.57, p= 0.021 chlorinated biofilms). Growth efficiency was highest at the lowest carbon concentration (range = 12–4.5%, amino acids and humics respectively). Doubling times ranged from 2.3–15.4 days in the control biofilms and 1–12.3 days in the chlorinated biofilms. Growth rates were significantly higher in the presence of chlorine for the carbohydrates, humics, and mixed carbon sources (p= 0.004, < 0.0005, 0.013, respectively). The concept of r/K selection theory was used to explain the results with respect to specific growth rates and yields. Humic removal by the biofilm bacteria (78% and 56% for the control and chlorinated biofilms, respectively) was higher than previously reported literature values for planktonic bacteria. A number of control experiments indicated that filtration of drinking water was as effective as chlorination in controlling bacterial biofilm growth. Received: 26 March 1999; Accepted: 3 August 1999; Online Publication: 15 February 2000     

Lignocellulose conversion of ensiled Caragana korshinskii Kom. facilitated by Pediococcus acidilactici and cellulases

To explore the biofuel production potential of Caragana korshinskii Kom., Pediococcus acidilactici and an exogenous fibrolytic enzyme were employed to investigate the fermentation profile, structural carbohydrates degradation, enzymatic saccharification and the dynamics of bacterial community of C. korshinskii silage. After 60 d of ensiling, all additives increased the fermentation quality. The highest lactic and acetic acids and lowest non-protein nitrogen (NPN) and ammonia nitrogen (NH₃-N) were observed in P. acidilactici and Acremonium cellulase (PA + AC) treated silage. Additionally, all additives significantly increased the ferulic acid content and fibre degradability with the highest values obtained from PA + AC silage. The bacterial community in all silages was dominated by P. acidilactici throughout the entire fermentation process. The bacterial community was also modified by the silage additives exhibiting a relatively simple network of bacterial interaction characterized by a lower bacterial diversity in P. acidilactici (PA) treated silage. The highest 6-phospho-beta-glucosidase abundance was observed in PA-treated silage at the mid-later stage of ensiling. PA treatment exhibited lower structural carbohydrates degradation but performed better in lignocellulose conversion during enzymatic saccharification. These results indicated that pretreating C. korshinskii improved its silage quality and potential use as a lignocellulosic feedstock for the production of bio-product and biofuel.     

Effect of heterogeneous structure in mechanically unstressed biofilms on overall growth

In contrast to their name, biofilms are not always flat and homogeneous but instead often exhibit complex structural heterogeneity. It has been suggested that nonhomogeneous geometry is selected in order to increase biofilm growth rate. A previous study (Dockery and Klapper (2002) SIAM J. Appl. Math., 62, 853–869) of a model biofilm system in a static bulk fluid demonstrated that under some circumstances a flat biofilm-bulk fluid interface is linearly unstable to perturbation due to growth induced forces. Computations indicated that subsequent nonlinear evolution results in fingers and mushrooms of biofilm similar to structures observed in actual biofilms. However, the important complementary issue of biological functionality was not considered. Here a weakly nonlinear analysis of the simple growing biofilm layer model in Dockery and Klapper (2002, SIAM J. Appl. Math., 62, 853–869) is presented. It is argued that, at least in the case of biofilms free of external mechanical stress, overall growth is in fact generally inhibited by the presence of growing perturbations in the linear stage. Hence a more complex explanation of function is necessary.     

Variation in Cell Wall Composition and Accessibility in Relation to Biofuel Potential of Short Rotation Coppice Willows

Short rotation coppice (SRC) willow is currently emerging as an important dedicated lignocellulosic energy crop in the UK. However, investigation into the variation between species and genotypes in their suitability for liquid transport biofuel processing has been limited. To address this, four traits relevant to biofuel processing (composition, enzymatic saccharification, response to pretreatment and projected ethanol yields) were studied in 35 genotypes of willow including Europe’s leading SRC willow cultivars. Large, genotype-specific variation was observed for all four traits. Significant positive correlations were identified between the accessibility of glucan to enzymatic saccharification before and after pretreatment as well as glucose release and xylose release via acid hydrolysis during pretreatment. Of particular interest is that the lignin content of the biomass did not correlate with accessibility of glucan to enzymatic saccharification. The genotype-specific variations identified have implications for SRC willow breeding and for potential reductions in both the net energy expenditure and environmental impact of the lignocellulosic biofuel process chain. The large range of projected ethanol yields demonstrate the importance of feedstock selection based on an ideotype encompassing the performance of both field biomass growth and ease of conversion.     

Implementation of fed-batch strategies for vitamin K (menaquinone-7) production by Bacillus subtilis natto in biofilm reactors

Recent studies show the essential health benefits associated with vitamin K, especially menaquinone-7 (MK-7). These benefits include reducing risks of cardiovascular diseases, osteoporosis, and even cancer. However, MK-7 production on an industrial level is only possible through bacterial fermentation and also current static fermentation strategies are not potent enough with difficulties to scale up. Biofilm reactors, however, may be a practical alternative. Biofilm reactors provide a controlled environment for the microorganisms to form mature and robust biofilms that enable them to produce value-added products with enhanced efficiencies. In this study, fed-batch addition of glucose and glycerol were investigated to the base media in biofilm reactors, as carbon source addition seemed crucial in batch fermentations. Results indicated that fed-batch strategies can be significantly effective in glucose-based medium, increasing the end-product concentrations to 28.7 ± 0.3 mg/L of MK-7 which was 2.3 fold higher than the level produced in suspended-cell bioreactors and renders the biofilm reactors as a potential replacement for static fermentation strategies. Moreover, morphological changes of B. subtilis were tracked during the 12-day long fermentation runs and finally, SEM investigations confirmed significant biofilm and extracellular matrices formed on the plastic composite support (PCS) in the biofilm reactors. In conclusion, biofilm reactors especially with fed-batch fermentation regimes seem to be an effective tool for MK-7 production at industrial scales.

     

Comparison of the efficiency of bacterial and fungal laccases in delignification and detoxification of steam-pretreated lignocellulosic biomass for bioethanol production

This study evaluates the potential of a bacterial laccase from Streptomyces ipomoeae (SilA) for delignification and detoxification of steam-exploded wheat straw, in comparison with a commercial fungal laccase from Trametes villosa. When alkali extraction followed by SilA laccase treatment was applied to the water insoluble solids fraction, a slight reduction in lignin content was detected, and after a saccharification step, an increase in both glucose and xylose production (16 and 6%, respectively) was observed. These effects were not produced with T. villosa laccase. Concerning to the fermentation process, the treatment of the steam-exploded whole slurry with both laccases produced a decrease in the phenol content by up to 35 and 71% with bacterial and fungal laccases, respectively. The phenols reduction resulted in an improved performance of Saccharomyces cerevisiae during a simultaneous saccharification and fermentation (SSF) process, improving ethanol production rate. This enhancement was more marked with a presaccharification step prior to the SSF process.     

Enhanced saccharification kinetics of sugarcane bagasse pretreated in 1-butyl-3-methylimidazolium chloride at high temperature and without complete dissolution

Dissolution of bagasse with 1-butyl-3-methylimidazolium chloride at high temperatures (110-160 °C) is investigated as a pretreatment process for saccharification and fermentation based biofuel production. Material balances are reported and used along with enzymatic saccharification data to identify optimum pretreatment conditions (150 °C for 90 min). At all pretreatment temperatures, dissolved and reprecipitated material is enriched in cellulose, has a low crystallinity and the cellulose component is easily and quantitatively hydrolysed (100%, 3h, 15 FPU). At pretreatment temperatures ≤ 150 °C, the undissolved material has only slightly lower crystallinity than the starting. At pretreatment temperatures ≥ 150 °C, the undissolved material has low crystallinity and when combined with the dissolved material has a saccharification rate and extent similar to completely dissolved material. Complete dissolution is not necessary to maximise saccharification efficiency at temperatures ≥ 150 °C.     

白腐真菌Pleurotus sajor-caju预处理对红麻秸秆发酵乙醇的影响

为研究微生物法预处理对红麻秸秆中木质素的降解及后续的红麻纤维素酶促糖化和发酵效率的影响,将白腐真菌Pleurotus sajor-caju接种在红麻秸秆培养基上固态培养,对红麻秸秆进行预处理。经P. sajor-caju培养25~35 d后,有效转化红麻秸秆中的木质素,转化率最高可达50.20％,并提高红麻纤维素的酶促水解效率,糖化率达69.33％~78.64％,与对照组相比提高了3.5~4.1倍。以微生物法预处理后的红麻秸秆样品为底物的同步糖化发酵实验表明,发酵72 h,发酵液中乙醇浓度达到18.35~     

Substrate-Related Factors Affecting Enzymatic Saccharification of Lignocelluloses: Our Recent Understanding

Enzymatic saccharification of cellulose is a key step in conversion of plant biomass to advanced biofuel and chemicals. Many substrate-related factors affect saccharification. Rather than examining the role of each individual factor on overall saccharification efficiency, this study examined how each factor affects the three basic processes of a heterogeneous biochemistry reaction: (1) substrate accessibility to cellulose—the roles of component removal and size reduction by pretreatments, (2) substrate and cellulase reactivity limited by component inhibition, and (3) reaction conditions—substrate-specific optimization. Our in-depth analysis of published literature work, especially those published in the last 5 years, explained and reconciled some of the conflicting results in literature, especially the relative importance of hemicellulose vs. lignin removal and substrate size reduction on enzymatic saccharification of lignocelluloses. We concluded that hemicellulose removal is more important than lignin removal for creating cellulase accessible pores. Lignin removal is important when alkaline-based pretreatment is used with limited hemicellulose removal. Partial delignification is needed to achieve satisfactory saccharification of lignocelluloses with high lignin content, such as softwood species. Rather than using passive approaches, such as washing and additives, controlling pretreatment or hydrolysis conditions, such as pH, to modify lignin surface properties can be more efficient for reducing or eliminating lignin inhibition to cellulase, leading to improved lignocellulose saccharification.     

Evaluation of productive biofilms for continuous lactic acid production

In white biotechnology research, the putative superiority of productive biofilms to conventional biotransformation processes based on planktonic cultures has been increasingly discussed in recent years. In the present study, we chose lactic acid production as a model application to evaluate biofilm potential. A pure culture of Lactobacillus bacteria was grown in a tubular biofilm reactor. The biofilm system was cultivated monoseptically in a continuous mode for more than 3 weeks. The higher cell densities that could be obtained in the continuous biofilm system compared with the planktonic culture led to a significantly increased space-time yield. The productivity reached 80% of the maximum value 10 days after start-up and no subsequent decline was observed, confirming the suitability of the system for long-term fermentation. The analysis of biofilm performance revealed that productivity increases with the flow velocity. This is explained by the reduced retention time of the liquid phase in the reactor, and, thus, a minor pH drop caused by the released lactic acid. At low flow velocities, the pH drops to a value where growth and production are significantly inhibited. The biofilm was visualized by magnetic resonance imaging to analyze biofilm thickness. To deepen the understanding of the biofilm system, we used a simple model for cell growth and lactic acid production.     

An industrial level system with nonisothermal simultaneous solid state saccharification,fermentation and separation for ethanol production

To alleviate the problems of low substrate loading, nonisothermal, end-product inhibition of ethanol during the simultaneous saccharification and fermentation, a nonisothermal simultaneous solid state saccharification, fermentation, and separation (NSSSFS) process was investigated; one novel pilot scale nonisothermal simultaneous solid state enzymatic saccharification and fermentation coupled with CO₂ gas stripping loop system was invented and tested. The optimal pretreatment condition of steam-explosion was 1.5 MPa for 5 min in industrial level. In the NSSSFS, enzymatic saccharification and fermentation proceeded at around 50 °C and 37 °C, respectively, and were coupled together by the hydrolyzate loop; glucose from enzymatic saccharification was timely consumed by yeast, and the formed ethanol was separated online by CO₂ gas stripping coupled with adsorption of activated carbon; the solids substrate loading reached 25%; ethanol yields from 18.96% to 30.29% were obtained in fermentation depending on the materials tested. Based on the pilot level of 300 L fermenter, a novel industrial-level of 110 m³ solid state enzymatic saccharification, fermentation and ethanol separation plant had been successfully established and operated. The NSSSFS was a novel and feasible engineering solution to the inherent problems of simultaneous saccharification and fermentation, which would be used in large scale and in industrial production of ethanol.     

Saccharification of foodwastes using cellulolytic and amylolytic enzymes fromTrichoderma harzianum FJ1 and its kinetics

The study was targeted to saccharify foodwastes with the cellulolytic and amylolytic enzymes obtained from culture supernatant ofTrichoderma harzianum FJ1 and analyze the kinetics of the saccharification in order to enlarge the utilization in industrial application.T. harzianum FJ1 highly produced various cellulolytic (filter paperase 0.9, carboxymethyl cellulase 22.0, β-glucosidase 1.2, Avicelase 0.4, xylanase 30.8, as U/mL-supernatant) and amylolytic (α-amylase 5.6, β-amylase 3.1, glucoamylase 2.6, as U/mL-supernatant) enzymes. The 23–98 g/L of reducing sugars were obtained under various experimental conditions by changing FPase to between 0.2–0.6 U/mL and foodwastes between 5–20% (w/v), with fixed conditions at 50°C, pH 5.0, and 100 rpm for 24 h. As the enzymatic hydrolysis of foodwastes were performed in a heterogeneous solid-liquid reaction system, it was significantly influenced by enzyme and substrate concentrations used, where the pH and temperature were fixed at their experimental optima of 5.0 and 50°C, respectively. An empirical model was employed to simplify the kinetics of the saccharification reaction. The reducing sugars concentration (X, g/L) in the saccharification reaction was expressed by a power curve (X=K·t ⁿ) for the reaction time (t), where the coefficient,K andn, were related to functions of the enzymes concentrations (E) and foodwastes concentrations (S), as follow:K=10.894 Ln(E·S ²)-56.768,n=0.0608·(E/S)^−0.2130. The kinetic developed to analyze the effective saccharification of foodwastes composed of complex organic compounds could adequately explain the cases under various saccharification conditions. The kinetics results would be available for reducing sugars production processes, with the reducing sugars obtained at a lower cost can be used as carbon and energy sources in various fermentation industries.