• Second Generation Biofuels
    Technologies and Background


There are two major pathways by which second generation biofuels biorefineries operate: through hydrolysis processes that aim to liberate sugars from the lignocellulosic polysaccharides (i.e. cellulose and hemicellulose), and through thermochemical processes that degrade more extensively the components of both polysaccharides and lignin. The various technologies available for the hydrolytic or thermochemical processing of biomass, along with their products and possible pre-treatment steps, are illustrated in the accompanying diagram:

Hydrolysis Biorefining Technologies

In the hydrolysis process cellulose and hemicellulose are hydrolysed (broken apart) in pure water through attack by the hydrogen atoms of the water molecule on these polysaccharides. This is a very slow reaction, particularly for cellulose due to its recalcitrance to hydrolysis, but it can be sped up using elevated temperatures and pressures, and catalysed by acids (concentrated or dilute) and highly selective enzymes such as cellulases. Most hydrolysis technologies also involve a pre-treatment step prior to the hydrolysis of the cellulose. There are a number of options for utilising the sugars liberated in hydrolysis, however most current activity is on the fermentation of these to products such as ethanol.

Acid Hydrolysis

The acid hydrolysis of lignocellulosic materials can take place using either concentrated or dilute acids. It was commercialised in the late 19th century and several dilute-acid facilities existed in the USA, Germany, Japan, and Russia by World War 1 while concentrated acid hydrolysis facilities were being built between 1937 and the late 1960's. However, these became uneconomic where fossil fuels were available. It is hoped that modern technologies will reinvigorate the industry.

Enzymatic Hydrolysis

Many current enzymatic hydrolysis technologies first employ a pre-treatment method for the hydrolysis of hemicellulose and to make a more digestible cellulose. Cellulase enzymes are then used to selectively hydrolyse the cellulose to glucose. This occurs under much milder process conditions than with dilute acid hydrolysis; hence the potential for sugar degradation is significantly decreased.

A cellulase is actually a mixture of many different enzymes each of which has a specific role in the hydrolysis of cellulose. For instance, endo-1,4-b-D-glucanases act internally within a cellulose chain at amorphous cellulose regions to cleave glyocisidc bonds, reducing the degree of polymerisation, while cellobiohydrolases degrade the cellulose chain from either the reducing or non-reducing ends and can cleave glycosidic bonds within crystalline cellulose regions, releasing cellobiose. Cellulases can be obtained from a variety of micro-organisms, including bacteria and fungi. However, aerobic fungi typically give higher growth rates and are the focus of most research.

Most current technologies produce enzymes in a separate tank to that for hydrolysis. Glucose is a poor carbon source for enzyme productivity since it favours the production of cellular mass. Inducers of cellulase include cellobiose, lactose, and sophorose. A cellulase production run lasts approximately one week after which the spent cell mass is disposed of.

There are numerous technologies proposed for the enzymatic hydrolysis of biomass. The most basic of these, Separate Hydrolysis and Fermentation (SHF), involves the enzymatic hydrolysis of cellulose followed by fermentation of the liberated hexoses. Pentose fermentation can occur after the distillation of the resulting beer, or with a separate stream taken from the hydrolysate of the pre-treatment process.

The more advanced enzymatic hydrolysis technologies combine the processes of hydrolysis and fermentation. For example, Simultaneous Saccharification and Fermentation (SSF) sees the hydrolysis of cellulose and the fermentation of the liberated sugars occur in the same reactor. A variant on SSF is Simulatneous Saccharification and Co-Fermentation (SSCF), where the glucose from cellulose and the pentoses from hemicellulose are fermented together (in the same reactor that is used for cellulose hydrolysis).

SSF and SSCF still require the yeast to be produced in a separate reactor, however Consolidated BioProcessing (CBP) involves all activities taking place in the same reactor, ideally with one micro-organism. CBP is considered to have the potential to offer the lowest costs for biofuels and chemicals via the enzymatic pathway.

Click here to read more about enzymatic hydrolysis, the related tests and analyses that we can undertake, and the custom processes that we can develop and optimise using enzymes.

Relevant Celignis Analysis Packages

Thermochemical Biorefining Technologies

Historically, the main means of gaining value from low value lignocellulosic materials has been to combust these for heat and/or electricity generation. This process typically operates at low efficiencies with biomass feedstocks and cannot compete economically with power production from fossil fuels or efficient renewables such as wind. Modern technologies, however, exist to produce more valuable gas, liquid and solid products from the thermal treatment of biomass. Two of the main technologies for this, pyrolysis and gasification, will be discussed below.

Pyrolysis of Biomass

Pyrolysis can be considered to be the thermal treatment of a material in the absence of oxygen that can also result in the production of three main products:

(1) a bio-oil that is produced from the condensable volatile components.
(2) non-condensable gases and;
(3) biochar, a residual solid component that does not decompose under the conditions employed.

Over time, refinements have allowed process conditions and the feedstock to be manipulated in order to achieve high yields of bio-oil, gases, or biochar, depending on the particular end product desired.


Bio-oil is a complex mixture of water, carboxylic acids and lignin- and carbohydrate-derived products that can deteriorate rapidly over time, with polymerisation reactions forming additional water and causing phase separations.

Due to the presence of water and abundance of oxygen, the heating value of the bio-oil is low compared to fossil oils and is only suitable as a heating fuel. It can, however, be used as a starting point towards a transport fuel if catalytic upgrading mechanisms are employed. Alternatively, there may be valuable chemicals that can be extracted from the bio-oil.

Celignis offers a number of analysis packages for bio-oils, these are listed below:


Biochar has been used as a plant-growth promoter due to the porosity properties conferred by particular pore-sizes, and the ultrastructure that result from the pyrolysis process. Biochar also has use as a carbon sequestration tool since much of the char is recalcitrant to decomposition, hence a significant amount of the carbon added to soil is effectively locked away.

At Celignis we have an array of analytical techniques and equipment suitable for the characterisation of biochars. The data we provide will assist you in determining the value of your biochar and the best use for it. Feel free to contact us to learn more.

Gasification of Biomass

Biomass gasification differs from pyrolysis in that a limited amount of oxygen is allowed, either via the addition of air, oxygen, or steam. Gasification processes tend to provide a smaller range of products than pyrolysis processes and are geared more towards the production of combustible (e.g. hydrogen, carbon monoxide, methane, ethylene, etc) or reformable gases. Temperature, and the particular architecture of the gasifer employed, affect the distribution of these products. These gases can be used directly for energy or catalytically reformed into valuable chemicals for industry or transport.

Many potential products (such as Fischer-Tropsch diesels, alcohols, olefins, acetic acid etc.) can be synthesized from these gases with the synthesis depending on the ratio of carbon monoxide to hydrogen in the syngas. The amount of hydrogen (and hence carbon dioxide) produced can be increased via the water gas shift reaction which involves mixing steam with the syngas, since insufficient water vapour is usually released from the biomass.

Biocatalysis can also be used for the synthesis of chemicals, including ethanol, from syngas. Autotrophs use C1 compounds, such as CO, CO2 and methanol, for their carbon source and hydrogen as their energy source while unicarbonotrophs use C1 compounds alone for both these purposes. These microorganisms also utilise metals such as cobalt and nickel (which are contained in their enzymes) for the conversion of C1 compounds into value added products but they are less sensitive to many of the gas contaminants, such a sulphur, that poison metal-based catalysts. Also, the adjustment of the H2:CO ratio via the water gas shift reaction is not necessary where biological catalysts are employed and synthesis conditions can be much milder.

Representative Biorefining Technologies

Celignis has a number of formulae which are used to predict, based on sample compositional data, the potential product yields that could be obtained when processing biomass samples in a number of different biorefining technologies. These yields are presented online to customers, on the Celignis Database, in tabulated and graphical formats, providing that the appropriate analysis packages have been selected.

Samples for which data regarding the lignocellulosic sugar composition have been obtained (e.g Celignis Analysis Packages: P7 (Lignocellulosic Sugars), P9 (Lignocellulosic Constituents), P10 (Lignocellulosic Constituents and Extractives), and P11 (NIR Prediction Package) will have data for potential biofuel yields from 5 different representative hydrolysis technologies.

Samples for which data regarding the heating values have been obtained (e.g. Celignis Analysis Package P40 - Combustion Package) will have data for potential biofuel yields from 2 different representative gasification technologies.

Click here for more information on these representative technologies and for details on how the biofuel yields are calculated.

Publications on Advanced Biofuels By The Celignis Team

Haigh K.F, Petersen A.M, Gottumukkala, L, Mandegari M, Naleli, K, Gorgens J.F (2018) Simulation and comparison of processes for biobutanol production from lignocellulose via ABE fermentation, Biofuels, Bioproducts and Biorefining 12(6): 1023-1036

Six conceptual process scenarios for the production of biobutanol from lignocellulosic biomass through acetone?butanol?ethanol (ABE) fermentation, using reported data on process performances, were developed with ASPEN Plus® V8.2 software. The six scenarios covered three fermentation strategies, i.e. batch separate hydrolysis and fermentation (SHF), continuous SHF, and batch simultaneous saccharification and fermentation (SSF) integrated with gas stripping (GS). The two downstream processing options considered were double?effect distillation (DD) and liquid?liquid extraction and distillation (LLE&D). It was found that the SSF?GS/DD scenario was the most energy efficient with a liquid fuel efficiency of 24% and an overall efficiency of 31%. This was also the scenario with the best economic outcome, with an internal rate of return (IRR) of 15% and net present value (NPV) of US$387 million. The SSF?GS/DD scenario was compared to a similar molasses process, based on the product flow rates, and it was found that the molasses process was more energy efficient with a gross energy value (GEV) of 23?MJ?kg1 butanol compared to ?117?MJ?kg1 butanol for the lignocellulosic process. In addition, the molasses?based process was more profitable with an IRR of 36% compared to 21%. However, the energy requirements for the molasses process were supplied from fossil fuels, whereas for the lignocellulose processes a portion of the feedstock was diverted to provide process energy. Improved environmental performance is therefore associated with the lignocellulosic process.

Gottumukkala L.D, Haigh K, Gorgens J (2017) Trends and advances in conversion of lignocellulosic biomass to biobutanol: microbes, bioprocesses and industrial viability, Renewable and Sustainable Energy Reviews 76: 963-973

Biobutanol has gained attention as an alternative renewable transportation fuel for its superior fuel properties and widespread applications in chemical industry, primarily as a solvent. Conventional butanol fermentation has drawbacks that include strain degeneration, end-product toxicity, by-product formation, low butanol concentrations and high substrate cost. The complexity of Clostridium physiology and close control between sporulation phase and ABE fermentation has made it demanding to develop industrially potent strains. In addition to the isolation and engineering of superior butanol producing bacteria, the development of advanced cost-effective technologies for butanol production from feedstock like lignocellulosic biomass has become the primary research focus. High process costs associated with complex feedstocks, product toxicity and low product concentrations are few of the several bioprocess challenges involved in biobutanol production. The article aims to assess the challenges in lignocellulosic biomass to biobutanol conversion and identify key process improvements that can make biobutanol commercially viable.

Gottumukka L.D, Haigh K, Collard F.X, Van Rensburg E, Gorgens J (2016) Opportunities and prospects of biorefinery-based valorisation of pulp and paper sludge, Bioresource technology 215: 37-49

The paper and pulp industry is one of the major industries that generate large amount of solid waste with high moisture content. Numerous opportunities exist for valorisation of waste paper sludge, although this review focuses on primary sludge with high cellulose content. The most mature options for paper sludge valorisation are fermentation, anaerobic digestion and pyrolysis. In this review, biochemical and thermal processes are considered individually and also as integrated biorefinery. The objective of integrated biorefinery is to reduce or avoid paper sludge disposal by landfilling, water reclamation and value addition. Assessment of selected processes for biorefinery varies from a detailed analysis of a single process to high level optimisation and integration of the processes, which allow the initial assessment and comparison of technologies. This data can be used to provide key stakeholders with a roadmap of technologies that can generate economic benefits, and reduce carbon wastage and pollution load.

Boshoff A, Gottumukka L.D, Van Rensburg E, Gorgens J (2016) Paper sludge (PS) to bioethanol: Evaluation of virgin and recycle mill sludge for low enzyme, high-solids fermentationl, Bioresource technology 23: 103-111

Paper sludge (PS) from the paper and pulp industry consists primarily of cellulose and ash and has significant potential for ethanol production. Thirty-seven PS samples from 11 South African paper and pulp mills exhibited large variation in chemical composition and resulting ethanol production. Simultaneous saccharification and fermentation (SSF) of PS in fed-batch culture was investigated at high solid loadings and low enzyme dosages. Water holding capacity and viscosity of the PS influenced ethanol production at elevated solid loadings of PS. High viscosity of PS from virgin pulp mills restricted the solid loading to 18% (w/w) at an enzyme dosage of 20 FPU/gram dry PS (gdPS), whereas an optimal solid loading of 27% (w/w) was achieved with corrugated recycle mill PS at 11 FPU/gdPS. Ethanol concentration and yield of virgin pulp and corrugated recycle PS were 34.2 g/L at 66.9% and 45.5 g/L at 78.2%, respectively.

Robus C.L.L, Gottumukkala, L.D, Van Rensburg E, Gorgens J.F. (2016) Feasible process development and techno-economic evaluation of paper sludge to bioethanol conversion: South African paper mills scenario, Renewable energy 92: 333-345

Paper sludge samples collected from recycling mills exhibited high ash content in the range of 54.59%–65.50% and glucose concentrations between 21.97% and 31.11%. Washing the sludge reduced the total ash content to between 10.7% and 19.31% and increased the concentration of glucose, xylose and lignin. Samples were screened for ethanol production and fed-batch simultaneous saccharification and fermentation (SSF) was optimised for the washed samples that resulted in highest and lowest ethanol concentrations. Maximum ethanol concentrations of 57.31 g/L and 47.72 g/L (94.07% and 85.34% of the maximum theoretical yield, respectively) was predicted for high and low fermentative potential samples, respectively, and was experimentally achieved with 1% deviation. A generic set of process conditions were established for the conversion of high ash-containing paper sludge to ethanol. Techno-economic analysis based on three different revenue scenarios, together with Monte Carlo analysis revealed 95% probability of achieving IRR values in excess of 25% at a paper sludge feed rate of 15 t/d. Feed rates of 30 t/d and 50 t/d exhibited a cumulative probability of 100%. This study presents the technical feasibility and economic viability of paper mills expansion towards bioethanol production from paper sludge.

Gottumukkala L.D. Gorgens J.F (2016) Biobutanol production from lignocellulosics, Biofuels Production and future perspectives, Taylor & Francis group

Next-generation biofuels from renewable sources have gained interest among research investigators, industrialists, and governments due to major concerns on the volatility of oil prices, climate change, and depletion of oil reserves. Biobutanol has drawn signicant attention as an alternative transportation fuel due to its superior fuel properties over ethanol. e advantages of butanol are its high energy content, better blending with gasoline, less hydroscopic nature, lower volatility, direct use in convention engines, low corrosiveness, etc. Butanol production through (acetone, butanol, and ethanol) ABE fermentation is a well-established process, but it has several drawbacks like feedstock cost, strain degeneration, product toxicity, and low product concentrations. Lignocellulosic biomass is considered as the most abundant, renewable, low-cost feedstock for biofuels. Production of butanol from lignocellulosic biomass is more complicated due to the recalcitrance of feedstock and inhibitors generated during the pretreatment and hydrolysis process. Advanced fermentation and product recovery techniques are being researched to make biobutanol industrially viable.

LP Christopher; VP Zambare, AV Zambare, H. Kumar, L Malek (2015) A thermo-alkaline lipase from a new extremophile Geobacillus thermodenitrificans AV5 with potential application in biodiesel production, Journal of Chemical Technology and Biotechnology 90(11): 2007-2016


BACKGROUND A thermophilic lipase?producing Geobacillus thermodenitrificans strain AV?5 was isolated from the Mushroom Spring of Yellowstone National Park in WY, USA and studied as a source of lipase for transesterification of vegetable oils to biodiesel. RESULTS A maximum activity of 330 U mL?1 was produced on 2% (v/v) waste cooking oil at 50 °C, pH 8, aeration rate of 1 vvm and agitation speed of 400 rpm. However, the higher lipase productivity (14.04 U mL?1 h?1) was found at a volumetric oxygen transfer coefficient (kLa) value of 18.48 h?1. The partially purified lipase had a molecular weight, temperature and pH optimum of 50 kDa, 65 °C and pH 9, respectively, and was thermo?alkali stable: at 70 °C, it retained 81% activity and 45% stability; at pH 10 it lost only 15% and 2.6% of its maximum activity and stability, respectively. Enzyme kinetic studies with p?nitrophenyl laurate as substrate revealed high substrate specificity (km of 0.440 mmol L?1) and kinetic activity (vmax of 556 nmol mL min?1) of lipase. CONCLUSIONS The kLa was found to be highly dependent on aeration and agitation rates. Following optimization of fermentation medium and parameters, a 7.5?fold increase in lipase production by G. thermodenitrificans was attained. The lipase activity and substrate specificity (as km) are among the highest reported in the literature for bacterial lipases. It was demonstrated that the enzyme can produce biodiesel from waste cooking oil with a conversion yields of 76%. © 2015 Society of Chemical Industry

Gottumukkala L.D, Sukumaran R.K. Mohan S.V. Valappil S.K. Sarkar O, Pandey A (2015) Rice straw hydrolysate to fuel and volatile fatty acid conversion by Clostridium sporogenes BE01: bio-electrochemical analysis of the electron transport mediators involved, Green chemistry 17(5): 3047-3058

Clostridium sporogenes BE01, a non-acetone forming butanol producer, can produce hydrogen and volatile fatty acids (VFAs) during butanol fermentation from rice straw hydrolysate. Bio-electrochemical analysis revealed the changes that occurred in the redox microenvironment and electron transport mediators during fermentation at different pH and CaCO3 concentrations. CaCO3 played a very important role in enhancing the production of hydrogen, volatile fatty acids and solvents by stimulating the changes in the electron transport system. The electron transport system mediated by NAD/NADH, flavins, Fe–S clusters, protein bound FAD, and cytochrome complex in C. sporogenes BE01 was analysed by cyclic voltammetry (CV). Electrokinetic analysis revealed that the favorability for redox reactions increased with an increase in pH, and the polarization resistance reduced significantly with CaCO3 supplementation.

Lew P. Christopher, Hemanathan Kumar, Vasudeo P. Zambare (2014) Enzymatic biodiesel: Challenges and opportunities, Applied Energy 119: 497-520


The chemical-catalyzed transesterification of vegetable oils to biodiesel has been industrially adopted due to its high conversion rates and low production time. However, this process suffers from several inherent drawbacks related to energy-intensive and environmentally unfriendly processing steps such as catalyst and product recovery, and waste water treatment. This has led to the development of the immobilized enzyme catalyzed process for biodiesel production which is characterized by certain environmental and economical advantages over the conventional chemical method. These include room-temperature reaction conditions, elimination of treatment costs associated with recovery of chemical catalysts, enzyme re-use, high substrate specificity, the ability to convert both free fatty acids and triglycerides to biodiesel in one step, lower alcohol to oil ratio, avoidance of side reactions and minimized impurities, easier product separation and recovery; biodegradability and environmental acceptance. This paper provides a comprehensive review of the current state of advancements in the enzymatic transesterification of oils. A thorough analysis of recent biotechnological progress is presented in the context of present technological challenges and future developmental opportunities aimed at bringing the enzyme costs down and improving the overall process economics towards large scale production of enzymatic biodiesel. As the major obstacles that impede industrial production of enzymatic biodiesel is the enzyme cost and conversion efficiency, this topic is addressed in greater detail in the review. A better understanding and control of the underpinning mechanisms of the enzymatic biodiesel process would lead to improved process efficiency and economics. The yield and conversion efficiency of enzymatic catalysis is influenced by a number of factors such as the nature and properties of the enzyme catalyst, enzyme and whole cell immobilization techniques, enzyme pretreatment, biodiesel substrates, acyl acceptors and their step-wise addition, use of solvents, operating conditions of enzymatic catalysis, bioreactor design. The ability of lipase to catalyze the synthesis of alkyl esters from low-cost feedstock with high free fatty acid content such as waste cooking oil, grease and tallow would lower the cost of enzymatic biodiesel. Discovery and engineering of new and robust lipases with high activity, thermostability and resistance to inhibition are needed for the establishment of a cost-effective enzymatic process. Opportunities to create a sustainable and eco-friendly pathway for production of enzymatic biodiesel from renewable resources are discussed.

Thomas L, Joseph A, Gottumukkala L.D. (2014) Xylanase and cellulase systems of Clostridium sp.: an insight on molecular approaches for strain improvement, Bioresource technology 158: 343-350

Bioethanol and biobutanol hold great promise as alternative biofuels, especially for transport sector, because they can be produced from lignocellulosic agro-industrial residues. From techno-economic point of view, the bioprocess for biofuels production should involve minimal processing steps. Consolidated bioprocessing (CBP), which combines various processing steps such as pretreatment, hydrolysis and fermentation in a single bioreactor, could be of great relevance for the production of bioethanol and biobutanol or solvents (acetone, butanol, ethanol), employing clostridia. For CBP, Clostridium holds best promise because it possesses multi-enzyme system involving cellulosome and xylanosome, which comprise several enzymes such as cellulases and xylanases. The aim of this article was to review the recent developments on enzyme systems of clostridia, especially xylanase and cellulase with an effort to analyse the information available on molecular approaches for the improvement of strains with ultimate aim to improve the efficiencies of hydrolysis and fermentation.

Gottumukkala L.D, Parameswaran B, Valappil S.K, Pandey A (2014) Growth and butanol production by Clostridium sporogenes BE01 in rice straw hydrolysate: kinetics of inhibition by organic acids and the strategies for their removal, Biomass Conversion and Biorefinery 4(3): 277-283

Growth inhibition kinetics of a novel non-acetone forming butanol producer, Clostridium sporogenes BE01, was studied under varying concentrations of acetic and formic acids in rice straw hydrolysate medium. Both the organic acids were considered as inhibitors as they could inhibit the growth of the bacterium, and the inhibition constants were determined to be 1.6 and 0.76 g/L, respectively, for acetic acid and formic acid. Amberlite resins—XAD 4, XAD 7, XAD 16, and an anion exchange resin—Seralite 400 were tested for the efficient removal of these acidic inhibitors along with minimal adsorption of sugars and essential minerals present in the hydrolysate. Seralite 400 was an efficient adsorbent of acids, with minimal affinity towards minerals and sugars. Butanol production was evaluated to emphasize the effect of minerals loss and acids removal by the resins during detoxification.

Hayes, D. J. M. (2013) Report on Optimal Use of DIBANET Feedstocks and Technologies, DIBANET WP5 Report84 pages


The DIBANET process chain, as a result of its patented pre-treatment stage, has significantly increased the yields of levulinic acid, formic acid, and furfural beyond what was considered to be the state of the art. By fractionating lignocellulosic biomass into its three main polymers (cellulose, hemicellulose, lignin) it has also allowed for lignin to be recovered and sold as a higher-value product. These developments have meant that the amount of acid hydrolysis residues (AHRs) that have been produced are significantly (up to 88%) less than in the Biofine process. These AHRs are required to provide process heat for DIBANET. Direct combustion is the most efficient means for doing this. If such combustion does not occur and the AHRs are instead used in other processes, e.g. pyrolysis and gasification, then more biomass will need to be purchased to fuel the core DIBANET process. The AHRs have not been proven to be superior to virgin biomass when put through these thermochemical processes. Indeed, many of the results from DIBANET Work Package 4 indicate the opposite. Hence, given that DIBANET, and the modelling of its optimal configuration, is designed on the basis of an integrated process, centred on the core element of the acid hydrolysis of biomass, then combustion is the only viable end use for the AHRs. Given that realisation, the focus of this modelling Deliverable is on what the optimal configuration of the process chain would be regarding the three core stages (pretreatment, hydrolysis, and the esterification of levulinic acid with ethanol). It has been demonstrated that a scenario incorporating only the first stage can be profitable in its own right and allow for commercial development at much lower capital costs. In this instance bagasse is a much more attractive feedstock, compared with Miscanthus, due to its higher pentose content.

Integrating the second stage increases capital costs but improves the net present value. The esterification step is somewhat capital intensive but an integrated DIBANET biorefinery that incorporates all three stages can still be highly profitable providing the furfural is sold at its current market price and the lignin is sold rather than used as a fuel for process needs. Indeed, the DIBANET process should not be considered only in the context of biofuels but as a true biorefinery that produces lower value fuels (e.g. ethyl-levulinate) in addition to high value chemicals and bio-products (e.g. furfural and lignin).

The energy and carbon balances of the various DIBANET scenarios have been investigated and are highly positive with values significantly superior to those for the energy-intensive Biofine process. A socioeconomic survey has also been carried out and has shown that there can be a positive effect on employment, both direct and indirect, particularly when Miscanthus is used as the feedstock. The DIBANET integrated process also holds up well when its environmental and social performances are ranked for a range of important parameters.

The development of the core DIBANET IP towards commercial deployment appears to be warranted, based on data provided from the models developed. Indeed, these models present possible scenarios whereby even demonstration-scale DIBANET facilities could operate at significant profits and provide healthy returns on the capital invested.

Gottumukkala, L. D, Valappi, S. K. (2013) Biobutanol production from rice straw by a non acetone producing Clostridium sporogenes BE01, Bioresource Technology 145: 182-187

Biobutanol from lignocellulosic biomass has gained much attention due to several advantages over bioethanol. Though microbial production of butanol through ABE fermentation is an established technology, the use of lignocellulosic biomass as feedstock presents several challenges. In the present study, biobutanol production from enzymatic hydrolysate of acid pretreated rice straw was evaluated using Clostridium sporogenes BE01. This strain gave a butanol yield of 3.43 g/l and a total solvent yield of 5.32 g/l in rice straw hydrolysate supplemented with calcium carbonate and yeast extract. Hydrolysate was analyzed for the level of inhibitors such as acetic acid, formic acid and furfurals which affect the growth of the organism and in turn ABE fermentation. Methods for preconditioning the hydrolysate to remove toxic end products were done so as to improve the fermentation efficiency. Conditions of ABE fermentation were fine tuned resulting in an enhanced biobutanol reaching 5.52 g/l.

Gottumukkala L.D, Parameswaran B, Valappil S.K, Mathiyazhakan, K (2013) Biobutanol production from rice straw by a non acetone producing Clostridium sporogenes BE01, Bioresource technology 145: 182-187

Biobutanol from lignocellulosic biomass has gained much attention due to several advantages over bioethanol. Though microbial production of butanol through ABE fermentation is an established technology, the use of lignocellulosic biomass as feedstock presents several challenges. In the present study, biobutanol production from enzymatic hydrolysate of acid pretreated rice straw was evaluated using Clostridium sporogenes BE01. This strain gave a butanol yield of 3.43 g/l and a total solvent yield of 5.32 g/l in rice straw hydrolysate supplemented with calcium carbonate and yeast extract. Hydrolysate was analyzed for the level of inhibitors such as acetic acid, formic acid and furfurals which affect the growth of the organism and in turn ABE fermentation. Methods for preconditioning the hydrolysate to remove toxic end products were done so as to improve the fermentation efficiency. Conditions of ABE fermentation were fine tuned resulting in an enhanced biobutanol reaching 5.52 g/l.

V.P. Zambare, Lew P. Christopher (2012) Optimization of enzymatic hydrolysis of corn stover for improved ethanol production, Energy Exploration & Exploitation 30(2): 193-205


Response surface methodology (RSM) was used to optimize the enzymatic hydrolysis of corn stover (CS), an abundant agricultural residue in the USA. A five-level, three-variable central composite design (CCD) was employed in a total of 20 experiments to model and evaluate the impact of pH (4.1–6.0), solids loadings (6.6–23.4%), and enzyme loadings (6.6?23.4 FPU g?1 DM) on glucose yield from thermo-mechanically extruded CS. The extruded CS was first hydrolyzed with the crude cellulase of Penicillium pinophilum ATCC 200401 and then fermented to ethanol with Saccharomyces cerevisiae ATCC 24860. Although all three variables had a significant impact, the enzyme loadings proved the most significant parameter for maximizing the glucose yield. A partial cubic equation could accurately model the response surface of enzymatic hydrolysis as the analysis of variance (ANOVA) showed a coefficient of determination (R2 ) of 0.82. At the optimal conditions of pH of 4.5, solids loadings of 10% and enzyme loadings of 20 FPU g?1 DM, the enzymatic hydrolysis of pretreated CS produced a glucose yield of 57.6% of the glucose maximum yield which was an increase of 10.4% over the non-optimized controls at zero-level central points. The predicted results based on the RSM regression model were in good agreement with the actual experimental values. The model can present a rapid means for estimating lignocellulose conversion yields within the selected ranges.

Vasudeo Zambare, Archana Zambare, Debmalya Barh, Lew Christopher (2012) Optimization of enzymatic hydrolysis of prairie cordgrass for improved ethanol production, Journal of Renewable and Sustainable Energy 4(3): 1-8


Prairie cordgrass (PCG), Spartina pectinata, is considered an energy crop with potential for bioethanol production in North America. The focus of this study was to optimize enzymatic hydrolysis of PCG at higher solids loadings using a thermostable cellulase of a mutant Penicillium pinophilum ATCC 200401. A three variable, five-level central composite design of response surface methodology (RSM) was employed in a total of 20 experiments to model and evaluate the impact of pH (4.1–6.0), solids loadings (6.6%–23.4%), and enzyme loadings (6.6–23.4 FPU/g dry matter, DM) on glucose yield from a thermo-mechanically extruded PCG. The extruded PCG was first hydrolyzed with the crude P. pinophilum cellulase and then fermented to ethanol with Saccharomyces cerevisiae ATCC 24860. Although all three variables had a significant impact, the enzyme loadings proved the most significant parameter for maximizing the glucose yield. A partial cubic equation could accurately model the response surface of enzymatic hydrolysis as the analysis of variance showed a coefficient of determination (R2) of 0.89. At the optimal conditions of pH of 4.5, solids loadings of 10% and enzyme loadings of 20 FPU/g DM, the enzymatic hydrolysis of pretreated PCG produced a glucose yield of 76.1% from the maximum yield which represents an increase of 15% over the non-optimized controls at the zero-level central points. The predicted results based on the RSM regression model were in good agreement with the actual experimental values. The model can present a rapid means for estimating lignocellulose conversion yields within the selected ranges. Furthermore, statistical optimization of solids and enzyme loadings of enzymatic hydrolysis of biomass may have important implications for reduced capital and operating costs of ethanol production.

Hemaiswarya S., Raja R., Carvalho Isabel S., Ravikumar R., Vasudeo Zambare and Debmalya Barh (2012) An Indian scenario on renewable and sustainable energy sources with emphasis on algae, Applied Microbiology and Biotechnology 96: 1125-1135


India is the fifth largest primary energy consumer and fourth largest petroleum consumer after USA, China, and Japan. Despite the global economic crisis, India’s economy is expected to grow at 6 to 8 %/year. There is an extreme dependence on petroleum products with considerable risks and environmental issues. Petroleum-derived transport fuels are of limited availability and contribute to global warming, making renewable biofuel as the best alternative. The focus on biogas and biomass-based energy, such as bioethanol and biohydrogen, will enhance cost-effectiveness and provide an opportunity for the rural community. Among all energy sources, microalgae have received, so far, more attention due to their facile adaptability to grow in the photobioreactors or open ponds, high yields, and multiple applications. Microalgae can produce a substantial amount of triacylglycerols as a storage lipid under photooxidative stress or other adverse environmental conditions. In addition to renewable biofuels, they can provide different types of high-value bioproducts added to their advantages, such as higher photosynthetic efficiency, higher biomass production, and faster growth compared to any other energy crops. The viability of first-generation biofuels production is, however, questionable because of the conflict with food supply. In the future, biofuels should ideally create the environmental, economic, and social benefits to the communities and reflect energy efficiency so as to plan a road map for the industry to produce third-generation biofuels.

V.P. Zambare, A. Bhalla, K. Muthukumarappan, R. Sani, L. Christopher (2011) Bioprocessing of agricultural residues to ethanol utilizing a cellulolytic extremophile, Extremophiles 15: 611-618


A recently discovered thermophilic isolate, Geobacillus sp. R7, was shown to produce a thermostable cellulase with a high hydrolytic potential when grown on extrusion-pretreated agricultural residues such corn stover and prairie cord grass. At 70°C and 15–20% solids, the thermostable cellulase was able to partially liquefy solid biomass only after 36 h of hydrolysis time. The hydrolytic capabilities of Geobacillus sp. R7 cellulase were comparable to those of a commercial cellulase. Fermentation of the enzymatic hydrolyzates with Saccharomyces cerevisiae ATCC 24860 produced ethanol yields of 0.45–0.50 g ethanol/g glucose with more than 99% glucose utilization. It was further demonstrated that Geobacillus sp. R7 can ferment the lignocellulosic substrates to ethanol in a single step that could facilitate the development of a consolidated bioprocessing as an alternative approach for bioethanol production with outstanding potential for cost reductions.

Zambare, V. P., Zambare, A. V., Muthukumarappan, K., Christopher, L. P. (2011) Potential of thermostable cellulases in the bioprocessing of switchgrass to ethanol, BioResources 6(2): 2004-2020


Switchgrass (Panicum virgatum), a perennial grass native to North America, is a promising energy crop for bioethanol production. The aim of this study was to optimize the enzymatic saccharification of thermo-mechanically pretreated switchgrass using a thermostable cellulase from Geobacillus sp. in a three-level, four-variable central composite design of response surface methodology. Different combinations of solids loadings (5 to 20%), enzyme loadings (5 to 20 FPU g-1 DM), temperature (50 to 70 oC), and time (36 to 96 h) were investigated in a total of 30 experiments to model glucose release from switchgrass. All four factors had a significant impact on the cellulose conversion yields with a high coefficient of determination of 0.96. The use of higher solids loadings (20%) and temperatures (70 oC) during enzymatic hydrolysis proved beneficial for the significant reduction of hydrolysis times (2.67-times) and enzyme loadings (4-times), with important implications for reduced capital and operating costs of ethanol production. At 20% solids, the increase of temperature of enzymatic hydrolysis from 50 oC to 70 oC increased glucose concentrations by 34%. The attained maximum glucose concentration of 23.52 g L-1 translates into a glucose recovery efficiency of 46% from the theoretical yield. Following red yeast fermentation, a maximum ethanol concentration of 11 g L-1 was obtained, accounting for a high glucose to ethanol fermentation efficiency of 92%. The overall conversion efficiency of switchgrass to ethanol was 42%.

Vasudeo Zambare, Archana Zambare, Kasiviswanathan Muthukumarappan, Lew Christopher (2011) Biochemical characterization of thermophilic lignocellulose degrading enzymes and their potential for biomass bioprocessing, International Journal of Energy and Environment 2(1): 99-112


A thermophilic microbial consortium (TMC) producing hydrolytic (cellulolytic and xylanolytic) enzymes was isolated from yard waste compost following enrichment with carboxymethyl cellulose and birchwood xylan. When grown on 5% lignocellulosic substrates (corn stover and prairie cord grass) at 600 C, the thermophilic consortium produced more xylanase (up to 489 U/l on corn stover) than cellulase activity (up to 367 U/l on prairie cord grass). Except for the carboxymethyl cellulose-enriched consortium, thermo-mechanical extrusion pretreatment of these substrates had a positive effect on both activities with up to 13% and 21% increase in the xylanase and cellulase production, respectively. The optimum temperatures of the crude cellulase and xylanase were 600 C and 700 C with half-lives of 15 h and 18 h, respectively, suggesting higher thermostability for the TMC xylanase. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis of the crude enzyme exhibited protein bands of 25-77 kDa with multiple enzyme activities containing 3 cellulases and 3 xylanases. The substrate specificity declined in the following descending order: avicel>birchwood xylan>microcrystalline cellulose>filter paper>pine wood saw dust>carboxymethyl cellulose. The crude enzyme was 77% more active on insoluble than soluble cellulose. The Km and Vmax values were 36.49 mg/ml and 2.98 U/mg protein on avicel (cellulase), and 22.25 mg/ml and 2.09 U/mg protein, on birchwood xylan (xylanase). A total of 50 TMC isolates were screened for cellulase and xylanase secretion on agar plates. All single isolates showed significantly lower enzyme activities when compared to the thermophilic consortia. This is indicative of the strong synergistic interactions that exist within the thermophilic microbial consortium and enhance its hydrolytic capabilities. It was further demonstrated that the thermostable enzyme-generated lignocellulosic hydrolyzates can be fermented to bioethanol by a recombinant strain of Escherichia coli. This could have important implications in the enzymatic breakdown of lignocellulosic biomass for the establishment of a robust and cost-efficient process for production of cellulosic ethanol. To the best of our knowledge, this work represents the first report in literature on biochemical characterization of lignocellulose-degrading enzymes from a thermophilic microbial consortium.

Vasudeo Zambare, Archana Zambare, Lew Christopher (2011) Enzymatic hydrolysis of hemicellulose from corn stover and prairie cordgrass, Advances in Biotech Research, Microbiologist Society and Education Book Publisher & Distributor
Singhania R.R, Sukumaran R.K, Rajasree K.P, Mathew A, Gottumukkala L.D, Pandey A (2011) Properties of a major ?-glucosidase-BGL1 from Aspergillus niger NII-08121 expressed differentially in response to carbon sources, Process Biochemistry 46(7): 1521-1524

Aspergillus niger NII-08121/MTCC 7956 exhibited differences in expression of ?-glucosidase (BGL) in response to carbon sources provided in the medium. Activity staining with methyl umbelliferyl ?-d-glucopyranoside (MUG) indicated that four different isoforms of BGL were expressed when A. niger was grown under submerged fermentation with either lactose or cellulose, whereas only two were expressed when wheat bran or rice straw was used as the carbon source. Among the four isoforms of BGL expressed during lactose supplementation, two were found to retain 92% and 82% activity respectively in presence of 250 mM glucose in the MUG assay. The major ?-glucosidase (BGL1) was purified to homogeneity by electro elution from a Native PAGE gel. The purified 120 kDa protein was active at 50 °C and was stable for 48 h without any loss of activity. The optimum pH and temperature were 4.8 and 70 °C respectively.

Sukumaran R.K, Gottumukkala L.D, Rajasree K.P, Alex D, Pandey A (2011) Butanol fuel from biomass: Revisiting ABE fermentation, Biofuels, Elsevier

ABE (Acetone-Butanol-Ethanol) fermentations were next only to ethanol fermentations and used to be a major industry until 1960s. Later, biological route for butanol production lost its importance owing to competition from petrochemical route, and today there is a renewed interest in ABE fermentation due to increased concerns over petroleum depletion and the increased pollution due to burning of petroleum fuels. Though the ABE fermentation process used to be operational decades back, the same technologies are not applicable today due to the lack of cost effectiveness and the nonavailability of conventional raw materials. The most feasible feedstock for butanol seems to be lignocellulose, but the problems plaguing bioethanol are also applicable for biobutanol. However, the future for biobutanol seemsbright since the Clostridia that produce ABE are capable of utilizing a range of carbon sources for growth and solvent production and also are not inhibited by the sugar degradation products generated during biomass pretreatment are being developed. Meanwhile, in the short term, advanced fermentation technologies are being developed by the expert groups which tackle problems such as low cell density, viability, and solvent sensitivity by modulations in the methods of carbon feeding, mode of culture, and in situ removal and recovery of solvents. These efforts may be developed into commercially viable technologies.

Parameswaran, B, Raveendran S, Singhania, R.R, Surender V, L Devi, Nagalakshmi S, Kurien N, Sukumaran R.K, Pandey A. (2010) Bioethanol production from rice straw: an overview, Bioresource technology 101(13): 4767-4774

Rice straw is an attractive lignocellulosic material for bioethanol production since it is one of the most abundant renewable resources. It has several characteristics, such as high cellulose and hemicelluloses content that can be readily hydrolyzed into fermentable sugars. But there occur several challenges and limitations in the process of converting rice straw to ethanol. The presence of high ash and silica content in rice straw makes it an inferior feedstock for ethanol production. One of the major challenges in developing technology for bioethanol production from rice straw is selection of an appropriate pretreatment technique. The choice of pretreatment methods plays an important role to increase the efficiency of enzymatic saccharification thereby making the whole process economically viable. The present review discusses the available technologies for bioethanol production using rice straw.

Aswathy U.S, Sukumaran R.K, Devi G.L, Rajasree K.P, Singhania R.R. (2010) Bio-ethanol from water hyacinth biomass: an evaluation of enzymatic saccharification strategy, Bioresource technology 101(3): 925-930

Biomass feedstock having less competition with food crops are desirable for bio-ethanol production and such resources may not be localized geographically. A distributed production strategy is therefore more suitable for feedstock like water hyacinth with a decentralized availability. In this study, we have demonstrated the suitability of this feedstock for production of fermentable sugars using cellulases produced on site. Testing of acid and alkali pretreatment methods indicated that alkali pretreatment was more efficient in making the sample susceptible to enzyme hydrolysis. Cellulase and ?-glucosidase loading and the effect of surfactants were studied and optimized to improve saccharification. Redesigning of enzyme blends resulted in an improvement of saccharification from 57% to 71%. A crude trial on fermentation of the enzymatic hydrolysate using the common baker’s yeast Saccharomyces cerevisiae yielded an ethanol concentration of 4.4 g/L.