Pretreatment of sweet sorghum bagasse for hydrogen production by Caldicellulosiruptor saccharolyticus

Pretreatment of sweet sorghum bagasse, an energy crop residue, with NaOH for the production of fermentable substrates, was investigated. Optimal conditions for the alkaline pretreatment of sweet sorghum bagasse were realized at 10% NaOH (w/w dry matter). A delignification of 46% was then observed, and improved significantly the efficiency of enzymatic hydrolysis. Under hydrolysis conditions without pH control, up to 50% and 41% of the cellulose and hemicellulose contained in NaOH-pretreated sweet sorghum bagasse were converted by 24 h enzymatic hydrolysis to soluble monomeric sugars. The extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus showed normal growth on hydrolysates of NaOH-pretreated biomass up to a sugar concentration of 20 g/L. Besides hydrogen, the main metabolic products detected in the fermentations were acetic and lactic acid. The maximal hydrogen yield observed in batch experiments under controlled conditions was 2.6 mol/mol C6 sugar. The maximal volumetric hydrogen production rate ranged from 10.2 to 10.6 mmol/(L h). At higher substrate concentrations the production of lactic acid increased at the expense of hydrogen production.     

The enhancement of hydrogen photoproduction in Chlorella protothecoides exposed to nitrogen limitation and sulfur deprivation

H₂ photoproduction, hydrogenase activities and PSII photochemical activities in Chlorella protothecoides under sulfur (S–) or nitrogen (N–) deprivation or simultaneous N-limitation and S-deprivation were studied. C. protothecoides pre-cultured in full nutrient TAP medium containing 7 mM NH₄Cl was found to produce a detectable but low level of H₂, once the cells were inoculated either in S-free or N-free medium. However, cells pre-grown in a low concentration of NH₄Cl (0.35 and 0.7 mM) generated a large amount of H₂ after transfer to N-limited and S-free medium. The maximal H₂ outputs of ∼233.7 and ∼129.1 ml/l were obtained within 100 h in the cultures exposed to S-deprived medium containing 0.35 mM and 0.7 mM NH₄Cl, with the average H₂ production rates being ∼2.19 and ∼1.37 ml/l/h, respectively. Our studies further indicated that N-limitation resulted in considerable starch accumulation, chlorophyll synthesis reduction, photosynthetic electron transfer block and oxygen evolving complex (OEC) injury, as well as attenuation in PSII oxygenic activity. Significant starch degradation was not observed during the H₂ photoevolution process. Attenuation of PSII O₂ evolution favored a rapid establishment of anaerobiosis for hydrogenase induction. Meanwhile, a constant high level of hydrogenase activities in C. protothecoides exposed to simultaneous N-limitation and S-deprivation were measured. Based on the above results, a possible mechanism of high H₂ photoproduction in C. protothecoides exposed to N-limitation and S-deprivation was discussed. Low net photosynthetic oxygenic rates, together with high hydrogenase activities were thought to contribute to the enhancement of H₂ photoproduction by C. protothecoides.     

The enhancement mechanism of hydrogen photoproduction in Chlorella protothecoides under nitrogen limitation and sulfur deprivation

The aim of this study was to understand the enhancement mechanism of H₂ photoproduction in Chlorella protothecoides under simultaneous nitrogen limitation and sulfur deprivation (LNS). Nitrogen limitation (LN) rather than sulfur deprivation significantly inhibited relative variable fluorescence at K-step (W_K) and J-step (V_J), photochemical efficiency of PSII (photosystem II), F_v/F_m, during the process of incubation in the light. Under such conditions, photosynthetic O₂ evolution decreased and the anaerobiosis was established after 12 h of incubation. The algae generated large amounts of H₂ under nitrogen limitation but generated only trace amounts under sulfur deprivation. Obviously, nitrogen limitation rather than sulfur deprivation was the decisive factor that induced H₂ photoproduction in C. protothecoides under LNS. The LNS culture generated much more H₂ than the LN culture in the presence of DCMU during incubation, suggesting that a PSII-independent electron source contributed many more electrons for transfer to hydrogenase in the LNS culture. PSII electron transport includes linear electron flow (LEF) and cyclic electron flow (CEF) of PSII in C. protothecoides. In the PSII-dependent electron source for H₂ photoproduction, PSII supplies electrons to hydrogenase through the LEF. The LNS culture showed much higher LEF and lower CEF than the LN culture during the H₂ photoproduction phase, as indicated by the large lower quantum yield of PSII electron transport (Φ_PSII) in the LNS culture in the presence of DCMU. Therefore, compared with nitrogen limitation, simultaneous nitrogen limitation and sulfur deprivation enhanced H₂ photoproduction in C. protothecoides mainly due to enhanced PSII-dependent and -independent electron sources.     

Lipid production from sweet sorghum bagasse through yeast fermentation

Cryptococcus curvatus has great potential in fermenting unconditioned hydrolysates of sweet sorghum bagasse. With hydrolysates obtained by enzymatic hydrolysis of the solid pretreated by microwave with lime, the maximal yeast cell dry weight and lipid content were 10.83 g/l and 73.26%, respectively. For hydrolysates obtained in the same way but without lime, these two parameters were 15.50 g/l and 63.98%, respectively. During yeast fermentation, glucose and xylose were consumed simultaneously while cellobiose was released from the residual bagasse. The presence of lime, on one hand, made cellulose more accessible to enzymes as evidenced by higher total reducing sugar release compared to that without during enzymatic hydrolysis step; on the other hand, it caused the degradation of sugars to non-sugar chemicals during pretreatment step. As a result, higher lipid yield of 0.11 g/g bagasse or 0.65 ton/hectare of land was achieved from the pathway of microwave pretreatment and enzymatic hydrolysis while 0.09 g/g bagasse or 0.51 ton/hectare of land was attained from the process of lime-assisted microwave pretreatment followed by the same enzymatic saccharification.     

Application of a modified Anaerobic Digestion Model 1 version for fermentative hydrogen production from sweet sorghum extract by Ruminococcus albus

The aim of the present study was to evaluate the effectiveness of a developed, ADM1-based kinetic model for the hydrogen production process in batch and continuous cultures of the bacterium Ruminococcus albus grown on sweet sorghum extract as the sole carbon source. Although sorghum extract is known to contain at least two different sugars, i.e. sucrose and glucose, no biphasic growth was observed in batch cultures as such growth is reported to occur in cultures of R. albus with mixed substrates. Thus, taking into account that the main sugar of sweet sorghum extract is sucrose, batch experiments with different initial concentrations of sucrose were performed in order to estimate the growth kinetics of the bacterium on this substrate. The kinetic parameters used, concerning the endogenous metabolism of the bacterium as well as those concerning the effect of pH and hydrogen partial pressure (P_H2), were the same as those estimated in a previous study with glucose as carbon source. Subsequently, the experimental data of batch and continuous experiments with sweet sorghum extract were simulated based on the already developed, modified ADM1 model accounting for the use of sugar-based substrate. It was shown that the model which was developed on synthetic substrates was successful in adequately describing the behavior of the microorganism on a real substrate such as sweet sorghum extract and predicting the experimental results quite well with a deviation of the model predictions from the experimental results being between 5-18% for the hydrogen yield.     

Net energy analysis of the production of biodiesel and biogas from the microalgae: Haematococcus pluvialis and Nannochloropsis

Microalgae have been proposed as possible alternative feedstocks for the production of biodiesel because of their high photosynthetic efficiency. The high energy input required for microalgal culture and oil extraction may negate this advantage, however. There is a need to determine whether microalgal biodiesel can deliver more energy than is required to produce it. In this work, net energy analysis was done on systems to produce biodiesel and biogas from two microalgae: Haematococcus pluvialis and Nannochloropsis. Even with very optimistic assumptions regarding the performance of processing units, the results show a large energy deficit for both systems, due mainly to the energy required to culture and dry the microalgae or to disrupt the cell. Some energy savings may be realized from eliminating the fertilizer by the use of wastewater or, in the case of H. pluvialis, recycling some of the algal biomass to eliminate the need for a photobioreactor, but these are insufficient to completely eliminate the deficit. Recommendations are made to develop wet extraction and transesterification technology to make microalgal biodiesel systems viable from an energy standpoint.     

Neochloris oleoabundans grown on anaerobically digested dairy manure for concomitant nutrient removal and biodiesel feedstock production

Microalgae have been investigated as a promising biodiesel feedstock; however, large-scale production is not currently cost-competitive with petroleum diesel, and its environmental impacts have received little attention. Using wastewater to supply nutrients for algal growth obviates synthetic fertilizer use, provides on-site nutrient removal, and reduces greenhouse gas emissions. In this work, anaerobically digested dairy manure was used to grow the oleaginous green alga Neochloris oleoabundans. In batch culture experiments with both synthetic media and anaerobic digester effluent, N. oleoabundans assimilated 90-95% of the initial nitrate and ammonium after 6 d and yielded 10-30% fatty acid methyl esters on a dry weight basis. Cellular lipid content and the N concentration in the growth media were inversely correlated. In addition, the proportion of polyunsaturated fatty acids (i.e. C16:3, C18:2, and C18:3) decreased with N concentration over time while the proportion of C18:1 fatty acid increased. Although N deficiency is likely the primary driver behind lipid accumulation, the influence of culture pH confounded results and requires further study. Other living microorganisms in the digester effluent were not observed to affect algal growth and lipid productivity, though the breakdown of organic nitrogen may have hindered lipid accumulation traditionally achieved through the manipulation of synthetic media. This work highlights the potential for waste-grown mono-algal cultures to produce high quality biodiesel while accomplishing simultaneous wastewater treatment.     

Bioethanol production from sweet sorghum: Evaluation of post-harvest treatments on sugar extraction and fermentation

Three experimental sweet sorghum varieties (M81, Topper and Theis) and three post-harvest conditions were evaluated for ethanol production: juices extracted by milling were obtained from the whole plant, plant without panicle, and stalk (plant without panicle and leaves), respectively. A linear relationship was found between the total fermentable sugar concentrations and Brix degrees of the juices, which can predict the potential ethanol yield by field analytical tests. The juice extractability presented different behavior among the sweet sorghum varieties with respect to the treatments studied. However such treatments did not affect the level of sugar concentration of the juices obtained and the fermentation efficiency. Topper and Theis showed the best performance in terms of ethanol concentration, fermentation efficiency and ethanol yield. The variety used and its post-harvest treatment should be appropriately selected in order to improve the ethanol production from sweet sorghum.     

Thermophilic biohydrogen production from the enzymatic hydrolysate of cellulose fraction of sweet sorghum bagasse by Thermoanaerobacterium thermosaccharolyticum KKU19: Optimization of media composition

The composition of media for thermophilic biohydrogen production from the enzymatic hydrolysate of cellulose fraction of sweet sorghum bagasse by Thermoanaerobacterium thermosaccharolyticum KKU19 were optimized in order to maximize the hydrogen production potential (P_s). Results from Plackett-Burman design indicated that FeSO₄, CaCl₂, NaHCO₃, and MgCl₂ had a significantly effect (P ≤ 0.05) on P_s. The optimum media composition obtained from the response surface methodology (RSM) with central composite design (CCD), using the hydrolysate at a total sugar concentration of 8.98 g/L, were (all in mg/L): FeSO₄, 1454.65; MgCl_2, 511.36; CaCl_2, 278.62; and NaHCO_3, 2186.41 in which the P_s of 2397 mL H₂/L were obtained. Verification experiment using the optimum media composition in a continuous stirred tank reactor indicated a highly reproducible result in which the P_s of 2608 mL H₂/L was achieved at a hydraulic retention time of 32 h. The results demonstrated that the media composition obtained from the batch experiment using RSM with CCD can be practically applied to continuously produce hydrogen from the hydrolysate with the least error.     

Bioethanol production from sweet potato by co-immobilization of saccharolytic molds and Saccharomyces cerevisiae

To investigate the bioethanol production from sweet potato, the saccharification and fermentation conditions of co-immobilization of saccharolytic molds (Aspergillus oryzae and Monascus purpureus) with Saccharomyces cerevisiae were analyzed. The immobilized yeast cells showed that at 10% glucose YPD (yeast extract peptone dextrose) the maximum fermentation rate was 80.23%. Viability of yeasts cells were 95.70% at a final ethanol concentration of 6%. Immobilization enhanced the ethanol tolerance of yeast cells. In co-immobilization of S. cerevisiae with A. oryzae or M. purpureus, the optimal hardening time of gel beads was between 15 and 60 min. Bioethanol production was 3.05-3.17% (v v⁻¹) and the Y_E/s (yield of ethanol production/starch consumption) was 0.31-0.37 at pH 4, 30 °C and 150 rpm during 13 days fermentation period. Co-immobilization of S. cerevisiae with a mixed cultures of A. oryzae and M. purpureus at a ratio of 2:1, the bioethanol production was 3.84% (v v⁻¹), and the Y_E/s was 0.39 for a 11 days incubation. However a ratio of A. oryzae and M. purpureus at 1:2 resulted a bioethanol production rate of 4.08% (v v⁻¹), and a Y_E/s of 0.41 after 9 days of fermentation.     

Step-change flow rate liquid hot water pretreatment of sweet sorghum bagasse for enhancement of total sugars recovery

A step-change flow rate liquid hot water (SCFLHW) process was developed with the objective of improving the total sugars recovery including xylose and glucose from sweet sorghum bagasse (SSB). Total xylose yield increased from 60% for batch system (4.25% w/v, 18 min, 184 °C) to about 80% at an initial flow rate of 20 ml/min for 8 min, followed by a rate of 10 ml/min for 10 min. It was hypothesized that the flowing water could enhance the mass transfer and improves the sugars recovery. Although little lignin was removed, the 72 h enzymatic digestibility of treated sample was enhanced by the increase of flow rate. The lignin droplet redeposited on the surface of residual solids might play a crucial role in determining the enzymatic digestibility. The total recovery of sugars from SSB, after the pretreatment (first with the flow rate of 20 ml/min for 8 min, then 10 ml/min for 10 min) and 72 h enzymatic digestion, reached 83.7%, which is superior to the recovery using the SO₂-steam pretreatment or ammonia fiber expansion.     

A potent lipid producing isolate of Epicoccum purpurascens AUMC5615 and its promising use for biodiesel production

Oils of oleaginous microorganisms are a powerful alternative to vegetable oils for biodiesel production. In this study, the fungus Epicoccum purpurascens AUMC5615 isolated from Egypt showed a potent high lipid content (80% lipid) when grown on 4% sucrose in submerged culture under continuous illumination. Under dark submerged conditions the lipid content has drastically decreased to 12%. In light static conditions, the lipid content was 70%; however, the net lipid yield was significantly lower than that of light submerged cultures because of the decrease in growth under light static conditions in comparison to light submerged cultures. Under dark static conditions the lipid content of the fungus has declined to 30%. These results indicate that light plays a crucial role in the lipid accumulation whereas submersion enhances the growth of the fungus. Concomitantly, the highest yield of carotenoids was obtained under light submerged conditions followed respectively by light static, dark submerged and dark static. This synchronized increase in carotenoids content might be implicated in protecting the high lipid pools in the fungus from peroxidation. Growing the fungus on 4% of crude molasses resulted in a net lipid production of 26.8 g per liter under light submerged conditions. The determination of fatty acids by GC/MS revealed that the major constituents are four saturated fatty acids, hexadecanoic, n-decanoic, dodecanoic and octadecanoic acids. These saturated fatty acids would give valuable stability properties of such fungal biodiesel. The current investigation opens the scope for the possible use of this promising fungal isolate in biodiesel production.     

Potential of microalgae oil from Dunaliella tertiolecta as a feedstock for biodiesel

Alternative, non-food based biomass fuel feedstock development is vital for our national security, economy and the environment. Microalgae are among the most promising of these alternatives. Microalgal cell growth rates and metabolic products are affected by a combination of environmental parameters. In this work, the influences of light source, light intensity, CO₂ concentration, and photoperiod on the growth of Dunaliella tertiolecta (D. tertiolecta) were studied. The effects of these environmental parameters on the lipid content and fatty acid composition of D. tertiolecta were also investigated. Red light-emitting diodes (LEDs), white LEDs, and fluorescent lights were all found to be effective for algal growth. Increasing light intensity resulted in significantly more rapid algal growth, and increasing the period of light also significantly increased biomass productivity. Similar growth rates were observed for 2%, 4%, and 6% CO₂-concentrations. The different light sources and intensities were found to have no significant effect on FAME composition of D. tertiolecta. Methyl linolenate and methyl palmitate were found to be the major components of FAME produced from D. tertiolecta oil. D. tertiolecta and its derived oils should be a suitable feedstock for biofuel production.     

Technoeconomic analysis of an integrated microalgae photobioreactor, biodiesel and biogas production facility

As fossil fuel prices increase and environmental concerns gain prominence, the development of alternative fuels from biomass has become more important. Biodiesel produced from microalgae is becoming an attractive alternative to share the role of petroleum. Currently it appears that the production of microalgal biodiesel is not economically viable in current environment because it costs more than conventional fuels. Therefore, a new concept is introduced in this article as an option to reduce the total production cost of microalgal biodiesel. The integration of biodiesel production system with methane production via anaerobic digestion is proved in improving the economics and sustainability of overall biodiesel stages. Anaerobic digestion of microalgae produces methane and further be converted to generate electricity. The generated electricity can surrogate the consumption of energy that require in microalgal cultivation, dewatering, extraction and transesterification process. From theoretical calculations, the electricity generated from methane is able to power all of the biodiesel production stages and will substantially reduce the cost of biodiesel production (33% reduction). The carbon emissions of biodiesel production systems are also reduced by approximately 75% when utilizing biogas electricity compared to when the electricity is otherwise purchased from the Victorian grid. The overall findings from this study indicate that the approach of digesting microalgal waste to produce biogas will make the production of biodiesel from algae more viable by reducing the overall cost of production per unit of biodiesel and hence enable biodiesel to be more competitive with existing fuels.     

High-yield biohydrogen production from biodiesel manufacturing waste by Thermotoga neapolitana

Efficient conversion of glycerol waste from biodiesel manufacturing processes into biohydrogen by the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 was investigated. Biohydrogen production by T. neapolitana was examined using the batch cultivation mode in culture medium containing pure glycerol or glycerol waste as the sole substrate. Pre-treated glycerol waste showed higher hydrogen (H₂) production than untreated waste. Nitrogen (N₂) sparging and pH control were successfully implemented to maintain the culture pH and to reduce H₂ partial pressure in the headspace for optimal growth rate and to enhance hydrogen production from the glycerol waste. It was found that hydrogen production increased from 1.24 ± 0.06 to 1.98 ± 0.1 mol-H₂ mol⁻¹ glycerol_consumed by optimising N₂ sparging and pH control. We observed that in medium containing 0.05 M HEPES, with three cycles of N₂ sparging, the H₂ yield increased to 2.73 ± 0.14 mol-H₂ mol⁻¹ glycerol_consumed, which was 2.22-fold higher than the non-N₂ sparged H₂ yield (1.23 ± 0.06 mol-H₂ mol⁻¹ glycerol_consumed).     

Land use and second-generation biofuel feedstocks: The unconsidered impacts of Jatropha biodiesel in Rajasthan,India

Governments around the world see biofuels as a common solution to the multiple policy challenges posed by energy insecurity, climate change and falling farmer incomes. The Indian government has enthusiastically adopted a second-generation feedstock – the oilseed-bearing shrub, Jatropha curcas – for an ambitious national biodiesel program. Studies estimating the production capacity and potential land use implications of this program have typically assumed that the ‘waste land’ slated for Jatropha production has no economic value and that no activities of note will be displaced by plantation development. Here we examine the specific local impacts of rapid Jatropha plantation development on rural livelihoods and land use in Rajasthan, India. We find that in Jhadol Tehsil, Jatropha is planted on both government and private land, and has typically displaced grazing and forage collection. For those at the socioeconomic margins, these unconsidered impacts counteract the very benefits that the biofuel programs aim to create. The Rajasthan case demonstrates that local land-use impacts need to be integrated into decision-making for national targets and global biofuel promotion efforts.     

Organosolv pretreatment of Liriodendron tulipifera and simultaneous saccharification and fermentation for bioethanol production

An acid-free organosolv process was proposed to overcome the problems caused by acid catalyst in organosolv process, thereby producing ethanol from Liriodendron tulipifera effectively. Although relative lignin contents were above 20%, enzymatic conversion increased significantly to 65% at all conditions, and thus correlation between lignin and enzymatic conversion could not be explained using relative lignin content. Enzymatic conversion increased significantly above 65% regardless of temperature, which suggests the organosolv pretreatment with sodium hydroxide can be performed at lower temperature. FE-SEM showed that the process made the structure loose and broke down biomass through lignin dissolution. Wrinkle formation by alkaline swelling was also observed and it might increase surface area. Although pore-volume increased slightly, it was not the sole key factor for the organosolv pretreatment with sodium hydroxide. Increase in surface area and enzyme adsorption enhanced the enzymatic hydrolysis. Ethanol of 96% could be produced theoretically and it suggested that the acid-free organosolv process was an effective pretreatment method for bioethanol production from L. tulipifera.     

High quality biodiesel from yellow oleander (Thevetia peruviana) seed oil

Yellow oleander (Thevetia peruviana Schum.) seed oil has been investigated to produce biodiesel. Transesterification of the oil to biodiesel was carried out in methanol by batch reaction using a heterogeneous catalyst derived from the trunk of Musa balbisiana Colla (one variety of banana plant). 96 wt.% of the oil is converted to biodiesel at 32 °C in 3 h. The wt.% composition of the biodiesel is methyl oleate 43.72, methyl palmitate 23.28, methyl linoleate 19.85, methyl stearate 10.71 and methyl arachidate 2.41. Fuel properties conform to standards set for ASTM D6751, EN 14214, BS II and BS III, and in certain aspects better. The biodiesel is free from sulfur and has exhibited a high cetane number of 61.5.     

Batch and continuous biohydrogen production from starch hydrolysate by Clostridium species

In this study, hydrogen gas was produced from starch feedstock via combination of enzymatic hydrolysis of starch and dark hydrogen fermentation. Starch hydrolysis was conducted using batch culture of Caldimonas taiwanensis On1 able to hydrolyze starch completely under the optimal condition of 55 °C and pH 7.5, giving a yield of 0.46–0.53 g reducing sugar/g starch. Five H₂-producing pure strains and a mixed culture were used for hydrogen production from raw and hydrolyzed starch. All the cultures could produce H₂ from hydrolyzed starch, whereas only two pure strains (i.e., Clostridium butyricum CGS2 and CGS5) and the mixed culture were able to ferment raw starch. Nevertheless, all the cultures displayed higher hydrogen production efficiencies while using the starch hydrolysate, leading to a maximum specific H₂ production rate of 116 and 118 ml/g VSS/h, for Cl. butyricumCGS2 and Cl. pasteurianum CH5, respectively. Meanwhile, the H₂ yield obtained from strain CGS2 and strain CH5 was 1.23 and 1.28 mol H₂/mol glucose, respectively. The best starch-fermenting strain Cl. butyricum CGS2 was further used for continuous H₂ production using hydrolyzed starch as the carbon source under different hydraulic retention time (HRT). When the HRT was gradually shortened from 12 to 2 h, the specific H₂ production rate increased from 250 to 534 ml/g  VSS/h, whereas the H₂ yield decreased from 2.03 to 1.50  mol H₂/mol glucose. While operating at 2 h HRT, the volumetric H₂ production rate reached a high level of 1.5 l/h/l.