Synthesis of cellulosic and nano-cellulosic aerogel from lignocellulosic materials for diverse sustainable applications: a review.

Cellulosic aerogels are sustainable, biodegradable, and ultra-light porous materials with three-dimensional networks having high specific surface area. Depending on the source of precursor materials, they are categorized into plant-based aerogel, bacterial cellulosic aerogel. Different types of aerogels are also produced from microcrystalline cellulose (MCC), nanocrystalline cellulose (NCC), cellulose microfibril (CMF) and cellulose nanofibril (CNF). Furthermore, inorganic and organic substances are embedded to produce hybrid aerogel or composite aerogel for the enhancement of its performance in various fields. Mixing, gelation, solvent exchange, and drying (e.g., super critical carbon dioxide or freeze drying) are the basic steps involved in cellulosic aerogel synthesis. Based on the composition of precursors during aerogel synthesis, cellulosic aerogels have broad applications in various fields such as adsorbents, electrodes, sensors, captive deionization materials, catalysts, drug delivery, thermal and sound insulating materials. This review provided consolidated information on: (i) classification of cellulosic aerogels based on the sources of raw materials, (ii) processes involved to produce the cellulosic aerogel, (iii) cellulosic aerogel synthesized from MCC, NCC, CMF and CNF, (iv) nano particle doped cellulosic aerogel, and (v) its application in various field with future perspectives.

Enhancement for the synthesis of bio-energy molecules (carbohydrates and lipids) in Desmodesmus subspicatus: experiments and optimization techniques.

Microalgae are regarded as renewable resources of energy, foods and high-valued compounds using a biorefinery approach. In the present study, we explored isolated microalgae (Desmodesmus subspicatus) for the production of bio-energy molecules (carbohydrate and lipid). Optimizations of media (BG-11) components have been made using the Taguchi orthogonal array (TOA) technique to maximize biomass, carbohydrate and lipid production. Optimized results showed that biomass, carbohydrates and lipid productivity increased by 1.3 times at optimal combinations of media components than standard BG-11 media. Further, the influence of various carbon and nitrogen sources as nutritional supplement with optimum media composition under different light intensities was investigated for productivity of carbohydrate and lipid. Results demonstrated that 1.5 times higher productivity of carbohydrate and lipids were achieved in the presence optimum BG-11 under a broad range of light intensities (84-504 µmol m-2 s-1). Among different nitrogen sources, glycine was found to give higher productivity (1.5 times) followed by urea. Use of the cellulose as a carbon source in the media significantly increases biomass (2.4 times), carbohydrates (2.3 times) and lipids (2.3 times) productivity. Investigations revealed that cultivating Desmodesmus subspicatus under optimum culture conditions has the potential for large-scale bio-ethanol and bio-diesel production.

Process intensification for the enhancement of growth and chlorophyll molecules of isolated Chlorella thermophila: A systematic experimental and optimization approach.

In our current work, we have optimized six physicochemical parameters (light intensity, light period, pH, inoculum size, culture period, and salt concentration) toward growth and chlorophyll synthesis using isolated fresh water microalgae Chlorella thermophila [contains ∼6% (w/w on dry biomass basis) chlorophyll]. Here, both experimental and computational [Taguchi orthogonal array (TOA), artificial neural network (ANN), and genetic algorithm (GA)] approaches were employed for the process intensification. Results revealed that the content of biomass and chlorophyll were enhanced by 118% and 95%, respectively, with productivity enhancement of 30% for biomass and 61% for chlorophyll from the optimization of physicochemical parameters. Further, optimum light intensity was found to be 128 µmol m-2 s-1 after conducting experiments in optimized chemical and physicochemical conditions, contributing to the enhancement of productivity of 46% for biomass and 106% for chlorophyll. Urea was found to be the most effective nitrogen source with an increase of 70% and 160% biomass and chlorophyll productivity, respectively. Moreover, sucrose as a carbon source contributed to an increase of 97% and 264% biomass and chlorophyll productivity.

Enhancement of growth and biomolecules (carbohydrates, proteins, and chlorophylls) of isolated Chlorella thermophila using optimization tools.

The production of multiple products from microalgae is essential for economic sustainability and the knowledge of optimum cultivation conditions for high growth and biomolecule synthesis of a microalgal strain is the prerequisite for its commercial production. In this work, optimization of nutrient concentrations for the cultivation of isolated Chlorella thermophila was performed by manipulating nine nutrients with the objectives of maximization of growth, carbohydrate, protein, and chlorophyll contents. Experiments were designed and effects of the parameters were studied using Taguchi orthogonal array (TOA). Experimental results of TOA were used for modeling artificial neural networks (ANN) followed by the optimization using genetic algorithm (GA) to find global optimal solutions. Results showed an increase of 36, 88, 36, and 88% for growth, carbohydrates, proteins, and chlorophylls, respectively, at optimal combinations of parameters given by TOA. Results obtained through the ANN-GA optimization were 9, 10, and 3% more compared to the TOA for biomass, carbohydrates, and chlorophylls, respectively with experimental verification. Nitrates and bicarbonate were found to play the most pivotal role in biomass and biomolecule synthesis of the isolated microalgal strain. Results of the current investigation can be used in the industrial scale-up for the production of multiple products using the biorefinery approach.

Priority-based multiple products from microalgae: review on techniques and strategies.

Microalgal biomass is composed of different valuable metabolites that can satisfy the requirements of renewable biofuels, alternative proteins, carbohydrates, and food grade natural colorants. Production of a specific product from microalgae has been proved to be economically infeasible on the commercial scale except for the production of high-value products (e.g. carotenoids and phycobiliproteins). Therefore, the simultaneous extraction of multiple products is essential to bring pragmatism for the production of biofuels, proteins, and carbohydrate derived products from microalgal biomass. In order to obtain multiple products, various strategies have been implemented using potential techniques of cell disruption and biomass fractionation based on the priorities of products. Conventional approaches of downstream processing have often proved to be inefficient in the case of integrated fractionation systems. This is attributable to the divergent nature of the intracellular metabolites of microalgae and their vulnerability toward the different chemicals and conditions of those downstream processes. However, three phase partitioning (TPP), aqueous two-phase separation, membrane separation, supercritical fluid extraction (SFE), and pressurized liquid extraction (PLE) are some of the advanced techniques which have been proved to be useful in this regard. Choice of cell disruption mechanisms is critical for several purposes, such as the selective release of metabolites into a suitable solvent, preservation of bioactivity of molecules and cost-savings. Unfortunately, consolidated report for the fractionation of priority-based products from microalgal biomass using these techniques is lacking. Therefore, in this review, we have critically discussed the different strategies for the priority-based multiple products by implementation of the advanced techniques.

Phycoremediation of As(III) and Cr(VI) by Desmodesmus subspicatus: Impact on growth and biomolecules (carbohydrate, protein, chlorophyll and lipid) - A dual mode investigation.

Microalgae are under research focus for the simultaneous production of biomolecules (e.g., carbohydrates, proteins, pigments and lipids) and bioremediation of toxic substances from wastewater. The current study explores the capability of indigenously isolated microalgae (Desmodesmus subspicatus) for the phycoremediation of As(III) and Cr(VI). Variation of biomolecules (carbohydrate, protein, lipid and chlorophyll) was investigated during phycoremediation. D. subspicatus survived up to the toxicity level of 10 mg/L for As(III) and 0.8 mg/L for Cr(VI). A 70% decline in carbohydrate accumulation was observed at 10 mg/L of As(III). An increased content of proteins (+ 28%) and lipids (+ 32%) within the cells was observed while growing in 0.5 and 0.2 mg/L of As(III) and Cr(VI) respectively. A decrease in carbohydrate accumulation was noted with increasing Cr(VI) concentration, and the lowest (- 44%) was recorded at 0.8 mg/L Cr(VI). D. subspicatus showed an excellent maximum removal efficiency for Cr(VI) and As(III) as 77% and 90% respectively.

Metabolic engineering for enhanced hydrogen production: a review.

Hydrogen gas exhibits potential as a sustainable fuel for the future. Therefore, many attempts have been made with the aim of producing high yields of hydrogen gas through renewable biological routes. Engineering of strains to enhance the production of hydrogen gas has been an active area of research for the past 2 decades. This includes overexpression of hydrogen-producing genes (native and heterologous), knockout of competitive pathways, creation of a new productive pathway, and creation of dual systems. Interestingly, genetic mutations in 2 different strains of the same species may not yield similar results. Similarly, 2 different studies on hydrogen productivities may differ largely for the same mutation and on the same species. Consequently, here we analyzed the effect of various genetic modifications on several species, considering a wide range of published data on hydrogen biosynthesis. This article includes a comprehensive metabolic engineering analysis of hydrogen-producing organisms, namely Escherichia coli, Clostridium, and Enterobacter species, and in addition, a short discussion on thermophilic and halophilic organisms. Also, apart from single-culture utilization, dual systems of various organisms and associated developments have been discussed, which are considered potential future targets for economical hydrogen production. Additionally, an indirect contribution towards hydrogen production has been reviewed for associated species.

Exploring the Influence of pH on the Dynamics of Acetone-Butanol-Ethanol Fermentation.

Clostridium acetobutylicum is an anaerobic bacterium that is extensively studied for its ability to produce butanol. Over the past two decades, various genetic and metabolic engineering approaches have been used to investigate the physiology and regulation system of the biphasic metabolic pathway in this organism. However, there has been a relatively limited amount of research focused on the fermentation dynamics of C. acetobutylicum. In this study, we developed a pH-based phenomenological model to predict the fermentative production of butanol from glucose using C. acetobutylicum in a batch system. The model describes the relationship between the dynamics of growth and the production of desired metabolites and the extracellular pH of the media. Our model was found to be successful in predicting the fermentation dynamics of C. acetobutylicum, and the simulations were validated using experimental fermentation data. Furthermore, the proposed model has the potential to be extended to represent the dynamics of butanol production in other fermentation systems, such as fed-batch or continuous fermentation using single and multi-sugars.

Transition from synthetic to alternative media for microalgae cultivation: A critical review.

In recent decades, microalgae have drawn attention as a most feasible alternative and sustainable feedstock for biofuel production. However, laboratory-scale and pilot-scale studies revealed that producing only biofuels through the microalgal route is economically unfeasible. The high cost of synthetic media is one concern, and low-cost alternative cultivation media would replace synthetic media to culture microalgae for economic benefit. This paper critically consolidated the advantages of alternative media over synthetic media for microalgae cultivation. A comparative analysis of the compositions of synthetic and alternative media was made to evaluate the potential use of alternative media in microalgae cultivation. Investigations on microalgae cultivation using alternative media derived from different waste materials, such as domestic, farm, agricultural, industrial, etc., are highlighted. Vermiwash is another alternative media that contains essential micro and macronutrients required for the cultivation of microalgae. Two prime techniques, such as mix-waste culture media and recycling culture media, may provide more economic benefit for the large-scale production of microalgae.

Valorization of waste biomass for synthesis of carboxy-methyl-cellulose as a sustainable edible coating on fruits: A review.

The coating on fruits and vegetables increases the shelf-life by providing protection against their spoilage. The existing petroleum-based coating materials have considerable health threats. Edible coating materials prepared with the cellulose derivative extracted from the waste biomass could be a sustainable alternative and environment friendly process to increase the shelf-life periods of the post-harvest crops. Selection of suitable waste biomass and extraction of cellulose are the critical steps for the synthesis of cellulose-based edible film. Conversion of extracted cellulose into cellulosic macromolecular derivatives such as carboxy-methyl-cellulose (CMC) is vital for synthesizing edible coating formulation. Applications of sophisticated tools and methods for the characterization of the coated fruits would be helpful to determine the efficiency of the coating material. In this review, we focused on: i) criteria for the selection of suitable waste biomass for extraction of cellulose, ii) pretreatment and extraction process of cellulose from the different waste biomasses, iii) synthesis processes of CMC by using extracted cellulose, iv) characterizations of CMC as food coating materials, v) various formulation techniques for the synthesis of the CMC based food coating materials and vi) the parameters which are used to evaluate the shelf-life performance of different coated fruits.

Chemical hazard prediction and hypothesis testing using quantitative adverse outcome pathways.

Current efforts in chemical safety are focused on utilizing human in vitro or alternative animal data in biological pathway context. However, it remains unclear how biological pathways, and toxicology data developed in that context, can be used to quantitatively facilitate decision-making.  The objective of this work is to determine if hypothesis testing using Adverse Outcome Pathways (AOPs) can provide quantitative chemical hazard predictions.  Current methods for predicting hazards of chemicals in a biological pathway context were extensively reviewed, specific case studies examined and computational modeling used to demonstrate quantitative hazard prediction based on an AOP. Since AOPs are chemically agnostic, we propose that AOPs function as hypotheses for how specific chemicals may cause adverse effects via specific pathways. Three broad approaches were identified for testing the hypothesis with AOPs, semi-quantitative weight of evidence, probabilistic, and mechanistic modeling. We then demonstrate how these approaches could be used to test hypotheses using high throughput in vitro data and alternative animal data. Finally, we discuss standards in development and documentation that would facilitate use in a regulatory context. We conclude that quantitative AOPs provide a flexible hypothesis framework for predicting chemical hazards. It accommodates a wide range of approaches that are useful at many stages and build upon one another to become increasingly quantitative.

Quantification of cell size distribution as applied to the growth of Corynebacterium glutamicum.

It is known that the cell size is related to the physiological state of a cell. Therefore, cell size distribution directly reflects the average physiological properties of the cell culture. Cell size distribution can be enumerated by image analysis, flow cytometry and coulter counter. In this study, image analysis was used to characterize the cell size distribution during the growth of Corynebacterium glutamicum and was further analyzed by a distribution function. The parameters of the distribution function indicate the mean value and spread of the distribution. Analysis demonstrated that the maximum specific growth rate was higher (0.67h(-1)) for the growth obtained through serial dilution of seed as compared to growth from a normal seed culture (0.53h(-1)). This was due to a greater percentage of the cell population being in the state of division for the growth through serial dilution in the mid-log phase. The measurement of the cell size distribution demonstrated that the average cell size decreased during the course of growth. The distribution function was also used to enumerate the average specific growth rate of both the conditions of the culture. The demonstrated methodology can be used to predict an average growth property of a cell culture.

Development of Spectrophotometric Method for the Analysis of Multi-component Carbohydrate Mixture of Different Moieties.

Present study is a critical analysis and subsequent development of an analytical tool to measure the total sugar concentration in a carbohydrate mixture comprising both hexose and pentose. For this purpose, individual sugars were measured and standardized with anthrone reagent prepared in an ice-cold 98 % sulphuric acid followed by 3 min of boiling. Furthermore, regression analysis was performed after mathematical manipulation with the individual standards to formulate a linear relation between the absorbance of the mixture and its concentration, which satisfies Beer's law. It was found that the correlation coefficient for the equation is 0.973, when confidence interval was set at 0.95. The validation was done with a synthetic mixture of concentrations at 0.17 and 0.22 g/L (as range was ensured between 0.1 and 0.3 g/L) and also with the carbohydrate mixture as the prehydrolyzate obtained after the pretreatment of banana stem, which showed around 94.1 % accuracy and higher sensitivity with the cellulose present in the mixture. Thus, the method is evident to quantify the total sugars accurately obtained from hydrolyzed lignocellulosic biomass.

Analysis of optimal phenotypic space using elementary modes as applied to Corynebacterium glutamicum.

BACKGROUND: Quantification of the metabolic network of an organism offers insights into possible ways of developing mutant strain for better productivity of an extracellular metabolite. The first step in this quantification is the enumeration of stoichiometries of all reactions occurring in a metabolic network. The structural details of the network in combination with experimentally observed accumulation rates of external metabolites can yield flux distribution at steady state. One such methodology for quantification is the use of elementary modes, which are minimal set of enzymes connecting external metabolites. Here, we have used a linear objective function subject to elementary modes as constraint to determine the fluxes in the metabolic network of Corynebacterium glutamicum. The feasible phenotypic space was evaluated at various combinations of oxygen and ammonia uptake rates. RESULTS: Quantification of the fluxes of the elementary modes in the metabolism of C. glutamicum was formulated as linear programming. The analysis demonstrated that the solution was dependent on the criteria of objective function when less than four accumulation rates of the external metabolites were considered. The analysis yielded feasible ranges of fluxes of elementary modes that satisfy the experimental accumulation rates. In C. glutamicum, the elementary modes relating to biomass synthesis through glycolysis and TCA cycle were predominantly operational in the initial growth phase. At a later time, the elementary modes contributing to lysine synthesis became active. The oxygen and ammonia uptake rates were shown to be bounded in the phenotypic space due to the stoichiometric constraint of the elementary modes. CONCLUSION: We have demonstrated the use of elementary modes and the linear programming to quantify a metabolic network. We have used the methodology to quantify the network of C. glutamicum, which evaluates the set of operational elementary modes at different phases of fermentation. The methodology was also used to determine the feasible solution space for a given set of substrate uptake rates under specific optimization criteria. Such an approach can be used to determine the optimality of the accumulation rates of any metabolite in a given network.

Elementary mode analysis reveals that Clostridium acetobutylicum modulates its metabolic strategy under external stress.

Clostridium acetobutylicum is a strict anaerobe which exhibits two distinct steps in its metabolic network. In the first step, sugars are oxidized to organic acids (acetic and butyric). This is accompanied with growth. The acids produced in the first phase are re-assimilated into solvents (acetone, butanol, and ethanol) in the second phase of metabolism. The two phases are hence called acidogenesis and solventogenesis, respectively. In this work, using Elementary Mode Analysis (EMA), we quantify fluxes through Elementary Modes under different physical and chemical conditions. Our analysis reveals that, in response to external stresses, the organism invokes Elementary Modes which couple acidogenesis and solventogenesis. This coupling leads to the organism exhibiting characteristics of both, acidogenesis and solventogenesis at the same time. Significantly, this coupling was not invoked during any "unstressed" conditions tested in this study. Overall, our work highlights the flexibility in Clostridium acetobutylicum to modulate its metabolism to enhance chances of survival under harsh conditions.

Role of extracellular cues to trigger the metabolic phase shifting from acidogenesis to solventogenesis in Clostridium acetobutylicum.

Clostridium acetobutylicum exhibits a two-step metabolic pathway where substrates are first converted to organic acids accompanied by a decrease in pH. The acids are then assimilated to organic solvents. The transition from the acid-producing (acidogenesis) to the solvent-producing phase (solventogenesis) is controlled by integration of a number of cellular and environmental cues, whose precise mode of action are not well understood. In this study, a series of batch experiments were performed to understand the impact of extracellular cues in regulating the dynamics of acidogenesis and solventogenesis. It is demonstrated that the two phases operate independently of each other and the growth phase of the cell, i.e. the cues controlling a phase are not linked to the status of the other phase or the growth phase of the cell. Kinetic experiments demonstrated that there exist two previously uncharacterized negative feedback loops controlling the amounts of acids produced in the acidogenesis phase.

Lysine overproducing Corynebacterium glutamicum is characterized by a robust linear combination of two optimal phenotypic states.

A homoserine auxotroph strain of Corynebacterium glutamicum accumulates storage compound trehalose with lysine when limited by growth. Industrially lysine is produced from C. glutamicum through aspartate biosynthetic pathway, where enzymatic activity of aspartate kinase is allosterically controlled by the concerted feedback inhibition of threonine plus lysine. Ample threonine in the medium supports growth and inhibits lysine production (phenotype-I) and its complete absence leads to inhibition of growth in addition to accumulating lysine and trehalose (phenotype-II). In this work, we demonstrate that as threonine concentration becomes limiting, metabolic state of the cell shifts from maximizing growth (phenotype-I) to maximizing trehalose phenotype (phenotype-II) in a highly sensitive manner (with a Hill coefficient of 4). Trehalose formation was linked to lysine production through stoichiometry of the network. The study demonstrated that the net flux of the population was a linear combination of the two optimal phenotypic states, requiring only two experimental measurements to evaluate the flux distribution. The property of linear combination of two extreme phenotypes was robust for various medium conditions including varying batch time, initial glucose concentrations and medium osmolality.

Fathead minnow steroidogenesis: in silico analyses reveals tradeoffs between nominal target efficacy and robustness to cross-talk.

BACKGROUND: Interpreting proteomic and genomic data is a major challenge in predictive ecotoxicology that can be addressed by a systems biology approach. Mathematical modeling provides an organizational platform to consolidate protein dynamics with possible genomic regulation. Here, a model of ovarian steroidogenesis in the fathead minnow, Pimephales promelas, (FHM) is developed to evaluate possible transcriptional regulation of steroid production observed in microarray studies. RESULTS: The model was developed from literature sources, integrating key signaling components (G-protein and PKA activation) with their ensuing effect on steroid production. The model properly predicted trajectory behavior of estradiol and testosterone when fish were exposed to fadrozole, a specific aromatase inhibitor, but failed to predict the steroid hormone behavior occurring one week post-exposure as well as the increase in steroid levels when the stressor was removed. In vivo microarray data implicated three modes of regulation which may account for over-production of steroids during a depuration phase (when the stressor is removed): P450 enzyme up-regulation, inhibin down-regulation, and luteinizing hormone receptor up-regulation. Simulation studies and sensitivity analysis were used to evaluate each case as possible source of compensation to endocrine stress. CONCLUSIONS: Simulation studies of the testosterone and estradiol response to regulation observed in microarray data supported the hypothesis that the FHM steroidogenesis network compensated for endocrine stress by modulating the sensitivity of the ovarian network to global cues coming from the hypothalamus and pituitary. Model predictions of luteinizing hormone receptor regulation were consistent with depuration and in vitro data. These results challenge the traditional approach to network elucidation in systems biology. Generally, the most sensitive interactions in a network are targeted for further elucidation but microarray evidence shows that homeostatic regulation of the steroidogenic network is likely maintained by a mildly sensitive interaction. We hypothesize that effective network elucidation must consider both the sensitivity of the target as well as the target's robustness to biological noise (in this case, to cross-talk) when identifying possible points of regulation.

A phenomenological model to represent the kinetics of growth by Corynebacterium glutamicum for lysine production.

Corynebacterium glutamicum is commonly used for lysine production. In the last decade, several metabolic engineering approaches have been successfully applied to C. glutamicum. However, only few studies have been focused on the kinetics of growth and lysine production. Here, we present a phenomenological model that captures the growth and lysine production during different phases of fermentation at various initial dextrose concentrations. The model invokes control coefficients to capture the dynamics of lysine and trehalose synthesis. The analysis indicated that maximum lysine productivity can be obtained using 72 g/L of initial dextrose concentration in the media, while growth was optimum at 27 g/L of dextrose concentration. The predictive capability was demonstrated through a two-stage fermentation strategy to enhance the productivity of lysine by 1.5 times of the maximum obtained in the batch fermentation. Two-stage fermentation indicated that the kinetic model could be further extended to predict the optimal feeding strategy for fed-batch fermentation.

Elementary mode analysis to study the preculturing effect on the metabolic state of Lactobacillus rhamnosus during growth on mixed substrates.

Quantification of metabolism through elementary modes offers insights into the working of a metabolic network. We have determined the fluxes of elementary modes through linear optimization using the stoichiometry of the elementary modes as a constraint. We apply this methodology to obtain insights into the effect of preculturing on growth of Lactobacillus rhamnosus on medium containing mixed substrates. L. rhamnosus, a microaerophilic organism, produces flavor compounds such as diacetyl and acetoin during growth on glucose and citrate. The uptake of citrate has been shown to be sensitive to preculturing states of the cells. Elementary modes demonstrated that citrate was utilized by the organism as a sole carbon source. Further, both glucose and citrate was catabolized by this organism through aerobic and anaerobic routes. The flux analysis indicated that only 21 elementary modes were operational during growth of L. rhamnosus on glucose and citrate. Glucose specifically accounted for 6 elementary modes, while the remaining 15 involved citrate as substrate. The modes associated with glucose were mainly operational when cells were precultured on glucose. It was observed that all the 21 modes contributed to the fluxes when the cells were precultured on citrate. The NADH recycling through lactate formation and oxygen uptake were dependent on the preculturing state. The analysis also demonstrated that preculturing on citrate yielded better productivity of diacetyl and acetoin.