Hydrogen production by the newly isolated Clostridium beijerinckii RZF-1108

A fermentative hydrogen-producing strain, RZF-1108, was isolated from a biohydrogen reactor, and identified as Clostridium beijerinckii on the basis of the 16S rRNA gene analysis and physiobiochemical characteristics. The effects of culture conditions on hydrogen production by C. beijerinckii RZF-1108 were investigated in batch cultures. The hydrogen production and growth of strain RZF-1108 were highly dependent on temperature, initial pH and substrate concentration. Yeast extract was a favorable nitrogen source for hydrogen production and growth of RZF-1108. Hydrogen production corresponded to cell biomass yield in different culture conditions. The maximum hydrogen evolution, yield and production rate of 2209 ml H₂/l medium, 1.97 mol H₂/mol glucose and 104.20 ml H₂/g CDW h⁻¹ were obtained at 9 g/l of glucose, initial pH of 7.0, inoculum volume of 8% and temperature of 35 °C, respectively. These results demonstrate that C. beijerinckii can efficiently produce H₂, and is another model microorganism for biohydrogen investigations.     

Biological hydrogen production by the algal biomass Chlorella vulgaris MSU 01 strain isolated from pond sediment

Chlorella vulgaris MSU 01 strain isolated from the sediment of the pond is able to produce molecular hydrogen in a clean way. To relate the dynamic coupling between the cultural conditions and biological responses, an original lab scale set up has been developed for hydrogen production. Different sources like mannitol, glucose, alanine, citric acid, aspartic acid, l-alanine, l-cysteine, sodium succinate and sodium pyruvate were used for algal media optimization. Corn stalk, from 1 to 5 g/L was tested for the effective algal growth and hydrogen production. The cell concentration of 1.6-19 g/L dry cell weight (DCW) was found at the 10th day. The kinetic parameters involved in the hydrogen production at 4 g/L corn stalk using the algal inoculum (50 mL) in the bioreactor volume (500 mL) was found to be with the hydrogen production potential (P_s) of 7.784 mL and production yield of (P_r) 5.534 mL respectively. The growth profile of the algal biomass at the above mentioned condition expressed the logistic model with R² 0.9988. The final pH of the broth was increased from 7.0 to 8.5-8.7. The anaerobic fermentation by C. vulgaris MSU 01 strain involved in the conversion process of complex carbon source has increased the H₂ evolution rate and higher butyrate concentration in the fermentate.     

Characterization of cellulolytic enzymes and bioH2 production from anaerobic thermophilic Clostridium sp. TCW1

A thermophilic anaerobic bacterium Clostridium sp. TCW1 was isolated from dairy cow dung and was used to produce hydrogen from cellulosic feedstock. Extracellular cellulolytic enzymes produced from TCW1 strain were identified as endoglucanases (45, 53 and 70 kDa), exoglucanase (70 kDa), xylanases (53 and 60 kDa), and β-glucosidase (45 kDa). The endoglucanase and xylanase were more abundant. The optimal conditions for H₂ production and enzyme production of the TCW1 strain were the same (60 °C, initial pH 7, agitation rate of 200 rpm). Ten cellulosic feedstock, including pure or natural cellulosic materials, were used as feedstock for hydrogen production by Clostridium strain TCW1 under optimal culture conditions. Using filter paper at 5.0 g/L resulted in the most effective hydrogen production performance, achieving a H₂ production rate and yield of 57.7 ml/h/L and 2.03 mol H₂/mol hexose, respectively. Production of cellulolytic enzyme activities was positively correlated with the efficiency of dark-H₂ fermentation.     

Fermentative hydrogen production from Laminaria japonica and optimization of thermal pretreatment conditions

As a sustainable biofuel feedstock, marine algae have superior aspects to terrestrial biomass such as less energy and water requirement for cultivation, higher CO₂ capture capacity, and negligible lignin content. In this study, various marine algae were tested for fermentative hydrogen production (FHP). Among them, Laminaria japonica exhibited the best performance, showing the highest H₂ yield of 69.1 mL H₂/g COD_added. It was attributed to its high carbohydrate content and main constituents of polysaccharides, laminarin and alginate, which were found to posses higher H₂ production potential than agar and carrageenan. To enhance the H₂ production from L. japonica, thermal pretreatment was applied at various conditions. At 170 °C and 20 min, H₂ yield was maximized to 109.6 mL H₂/g COD_added. The experimental results suggested that marine algae, especially L. japonica, could be used for FHP, and future works would be focused on gaining more energy from the H₂ fermentation effluent.     

Using a starch-rich mutant of Arabidopsis thaliana as feedstock for fermentative hydrogen production

A mutant plant (Arabidopsis thaliana), sex1-1 (starch excess 1-1), accumulating high starch content in leaves was created to serve as better biomass feedstock for a H₂-producing strain Clostridium butyricum CGS2, which efficiently utilizes starch for H₂ production but cannot assimilate cellulosic materials. The starch content of the mutant plant increased to 10.67 mg/fresh weight, which is four times higher than that of wild type plant. Using sex1-1 mutant plant as feedstock, C. butyricum CGS2 could produce 490.4 ml/l of H₂ with a H₂ production rate of 32.9 ml/h/l. The H₂ production performance appeared to increase with the increase in the concentration of mutant plant from 2.5 to 10 g/l. The highest H₂ to plant biomass yield was nearly 49 ml/g for the mutant plant. This study successfully demonstrated the feasibility of using a starch-rich mutant plant for more effective bioH₂ production with C. butyricum CGS2.     

Biohydrogen production by immobilized Chlorella sp. using cycles of oxygenic photosynthesis and anaerobiosis

Hydrogen production was studied using immobilized green alga Chlorella sp. through a two-stage cyclic process where immobilized cells were first incubated in oxygenic photosynthesis followed by anaerobic incubation for H₂ production in the absence of sulfur. Chlorella sp. used in this study was capable of generating H₂ under immobilized state in agar. The externally added glucose enhanced H₂ production rates and total produced volume while shortened the lag time required for cell adaptation prior to H₂ evolution. The rate of hydrogen evolution was increased as temperature increased, and the maximum evolution rate under 30 mM glucose was 183 mL/h/L and 238 mL/h/L at 37 °C and 40 °C, respectively. In order to continue repeated cycles of H₂ production, at least two days of photosynthesis stage should be allowed for cells to recover H₂ production potential and cell viability before returning to H₂ production stage again.     

Optimization of Spongiochloris sp. biomass production in the abattoir digestate

In the present study, the abattoir digestate was used as a culture medium for Spongiochloris sp. growth with added mineral components under optimized conditions in batch culture. Firstly, an Hadamard matrix was used to investigate the impact of certain influencing factors on the Spongiochloris sp. growth. Then, a fractional factorial design 2^7-4 was successfully employed to optimize the concentration of different added components to abattoir digestate for increased Spongiochloris sp. biomass production. The major influencing factors were NaHCO₃ and FeSO₄ at a level of 2000 mg/L and 5 mg/L, respectively. A high biomass production of 5.29 × 10⁶ cell/mL and an important content of chlorophyll a of 65.32 mg/L were obtained after 42 days of culture of Spongiochloris sp. on the defined abattoir medium at static conditions.     

Hydrogen production by immobilized R. faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria E. harbinense B49

In order to increase the hydrogen yield from glucose, hydrogen production by immobilized Rhodopseudomonas faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria Ethanoligenens harbinense B49 was investigated. The soluble metabolites from dark-fermentation mainly were ethanol and acetate, which could be further utilized for photo-hydrogen production. Hydrogen production by B49 was noticeably affected by the glucose and phosphate buffer concentration. The maximum hydrogen yield (1.83 mol H₂/mol glucose) was obtained at 9 g/l glucose. In addition, we found that the ratio of acetate/ethanol (A/E) increased with increasing phosphate buffer concentration, which is favorable to further photo-hydrogen production. The total hydrogen yield during dark- and photo-fermentation reached its maximum value (6.32 mol H₂/mol glucose) using 9 g/l glucose, 30 mmol/l phosphate buffers and immobilized R. faecalis RLD-53. Results demonstrated that the combination of dark- and photo- fermentation was an effective and efficient process to improve hydrogen yield from a single substrate.     

Biohydrogen production in alkalithermophilic conditions: Thermobrachium celere as a case study

In the present work the hydrogenesis in the anaerobic alkalithermophilic bacterium Thermobrachium celere was studied. The impact of several factors on hydrogen production during glucose fermentation was investigated in batch conditions. The optimal hydrogen production occurred at pH_67 °C 8.2 with phosphate buffer concentration of 50 mM. Hydrogen yield reached the highest value of 3.36 mol H₂/mol glucose when the partial pressure in the gas headspace was reduced. Supplementation of nitrogen sources and iron affected hydrogen production. Under optimized conditions, the maximum H₂ accumulation and H₂ production rate were estimated to be respectively 124.3 mmol H₂/l culture and 20.7 mmol H₂/l/h. Considering the efficient and rapid hydrogen evolution, and the ability to grow in extreme environments, T. celere might be a good candidate for biohydrogen production in open (non-sterile) bioprocess system.     

Evaporative water loss and energy metabolic in two small mammals,voles (Eothenomys miletus) and mice (Apodemus chevrieri), in Hengduan mountains region

Evaporative water loss (EWL) and energy metabolism were measured at different temperatures in Eothenomys miletus and Apodemus chevrieri in dry air. The thermal neutral zone (TNZ) of E. miletus was 22.5–30 °C and that of A. chevrieri was 20–27.5 °C. Mean body temperatures of the two species were 35.75±0.5 and 36.54±0.61 °C. Basal metabolic rates (BMR) were 1.92±0.17 and 2.7±0.5 ml O₂/g h, respectively. Average minimum thermal conductance (C_m) were 0.23±0.08 and 0.25±0.06 ml O₂/g h °C. EWL in E. miletus and A. chevrieri increased with the increase in temperature; the maximal EWL at 35 °C was 4.78±0.6 mg H₂O/g h in E. miletus, and 5.92±0.43 mg H₂O/g h in A. chevrieri. Percentage of evaporative heat loss to total heat production (EHL/HP) increased with the increase in temperature; the maximal EHL/HP was 22.45% at 30 °C in E. miletus, and in A. chevrieri it was 19.96% at 27.5 °C. The results may reflect features of small rodents in the Hengduan mountains region: both E. miletus and A. chevrieri have high levels of BMR and high levels of total thermal conductance, compared with the predicted values based on their body masses, while their body temperatures are relatively low. EWL plays an important role in temperature regulation.     

Effects of light/dark cycle, mixing pattern and partial pressure of H2 on biohydrogen production by Rhodobacter sphaeroides ZX-5

The effects of light/dark cycle, mixing pattern and partial pressure of H₂ on the growth and hydrogen production of Rhodobacter sphaeroides ZX-5 were investigated. The results from light/dark cycle culture showed that little or no hydrogen production was observed during the dark periods, and the hydrogen production immediately recovered once illumination was resumed. Also, it was found that the optimum condition of shaking velocity was 120 rpm for hydrogen photo-fermentation. Meanwhile, shaking during H₂ production phase (i.e., cell growth stationary phase) of photo-fermentation played a crucial role on effectively enhancing the phototrophic hydrogen production, rather than that during cell exponential growth phase. The other factor evaluated was hydrogen partial pressure in the culture system. The substrate conversion efficiency increased from 86.07% to 95.56% along with the decrease of the total pressure in the photobioreactor from 1.082 × 10⁵ to 0.944 × 10⁵ Pa, which indicated that reduction of H₂ partial pressure by lowering the operating pressure substantially improved H₂ production in an anaerobic, photo-fermentation process.     

Hydrolysis of lignocellulosic feedstock by novel cellulases originating from Pseudomonas sp. CL3 for fermentative hydrogen production

A newly isolated indigenous bacterium Pseudomonas sp. CL3 was able to produce novel cellulases consisting of endo-β-1,4-d-glucanase (80 and 100 kDa), exo-β-1,4-d-glucanase (55 kDa) and β-1,4-d-glucosidase (65 kDa) characterized by enzyme assay and zymography analysis. In addition, the CL3 strain also produced xylanase with a molecular weight of 20 kDa. The optimal temperature for enzyme activity was 50, 45, 45 and 55 °C for endo-β-1,4-d-glucanase, exo-β-1,4-d-glucanase, β-1,4-d-glucosidase and xylanase, respectively. All the enzymes displayed optimal activity at pH 6.0. The cellulases/xylanase could hydrolyze cellulosic materials very effectively and were thus used to hydrolyze natural agricultural waste (i.e., bagasse) for clean energy (H₂) production by Clostridiumpasteurianum CH4 using separate hydrolysis and fermentation process. The maximum hydrogen production rate and cumulative hydrogen production were 35 ml/L/h and 1420 ml/L, respectively, with a hydrogen yield of around 0.96 mol H₂/mol glucose.     

Effects of NH4–N concentrations and gradient redox level on growth and allied biochemical parameters of Elodea nuttallii (Planch.)

Aquatic plants frequently encounter multiple stresses under natural conditions. Nuttall's water weed, Elodea nuttallii (Planch.) is a submerged aquatic macrophyte which has flexible ability to use different nutrient sources from various environments. However, recently the growth of E. nuttallii has been declining in waters of Japan and in the Chesapeake Bay, a large estuary in the United States. In the present experiment, we studied growth and survival capabilities of the plant under a gradient of redox conditions; from highly oxic (+400 to +440 mV) to extremely reduced (−180 to −120 mV) conditions. Reduced environment was prepared by adding glucose to growth medium and nitrogen gas bubbling, while the oxic environment was brought about by atmospheric air bubbling. In comparison to the oxic environment, growth rate and carbon–nitrogen content of the plants were significantly affected negatively at hypoxic and anoxic conditions. In hypoxic and anoxic environments, indole acetic acid (IAA), tissue nitrogen and chlorophyll levels were down-regulated, whereas hydrogen peroxide (H₂O₂), indole acetic acid oxidase (IAAO) and peroxidase (POD) levels were up-regulated. It was also found that high NH₄–N concentrations (10–40 ppm) affect the growth rate and biochemical parameters of the plant; however, in hypoxic and anoxic treatments the effects were more severe. We conclude that E. nuttallii is poorly tolerant to hypoxia/anoxia. Moreover, oxygen stress combined with high ammonium concentration act as important factors influencing distribution and abundance of this species.     

Metabolism,thermoregulation and evaporative water loss in the Chaotung Vole (Eothenomys olitor) in Yunnan-Kweichow Plateau in summer

Proper adjustment of thermoregulatory mechanisms ensures the survival of mammals when they are subjected to seasonal changes in their natural environment. To understand the physiological and ecological adaptations of Eothenomys olitor, we measured their metabolic rate, thermal conductance, body temperature (T_b) and evaporative water loss at a temperature range of 5–30 °C in summer. The thermal neutral zone (TNZ) of E. olitor was 20–27.5 °C, and the mean body temperature was 35.81±0.15 °C. Basal metabolic rate (BMR) was 2.81±0.11 ml O₂/g h and mean minimum thermal conductance (C_m) was 0.18±0.01 ml O₂/g h °C. Evaporative water loss (EWL) in E. olitor increased when the ambient temperature increased. The maximal evaporative water loss was 6.74±0.19 mg H₂O/g h at 30 °C. These results indicated that E. olitor have relatively high BMR, low body temperature, low lower critical temperature, and normal thermal conductance. EWL plays an inportant role in temperature regulation. These characteristics are closely related to the living habitat of the species, and represent its adaptive strategy to the climate of the Yunnan-Kweichow Plateau, a low-latitude, high-altitude region where annual temperature fluctuations are small, but daily temperature fluctuations are greater.     

Multi-objective optimization of bioethanol production during cold enzyme starch hydrolysis in very high gravity cassava mash

Cold enzymatic hydrolysis conditions for bioethanol production were optimized using multi-objective optimization. Response surface methodology was used to optimize the effects of α-amylase, glucoamylase, liquefaction temperature and liquefaction time on S. cerevisiae biomass, ethanol concentration and starch utilization ratio. The optimum hydrolysis conditions were: 224 IU/g_starch α-amylase, 694 IU/g_starch glucoamylase, 77 °C and 104 min for biomass; 264 IU/g_starch α-amylase, 392 IU/g_starch glucoamylase, 60 °C and 85 min for ethanol concentration; 214 IU/g_starch α-amylase, 398 IU/g_starch glucoamylase, 79 °C and 117 min for starch utilization ratio. The hydrolysis conditions were subsequently evaluated by multi-objectives optimization utilizing the weighted coefficient methods. The Pareto solutions for biomass (3.655-4.380 × 10⁸ cells/ml), ethanol concentration (15.96-18.25 wt.%) and starch utilization ratio (92.50-94.64%) were obtained. The optimized conditions were shown to be feasible and reliable through verification tests. This kind of multi-objective optimization is of potential importance in industrial bioethanol production.     

Responses of Gmelina arborea, a tropical deciduous tree species, to elevated atmospheric CO2: Growth, biomass productivity and carbon sequestration efficacy

The photosynthetic response of trees to rising CO₂ concentrations largely depends on source-sink relations, in addition to differences in responsiveness by species, genotype, and functional group. Previous studies on elevated CO₂ responses in trees have either doubled the gas concentration (>700 μmol mol⁻¹) or used single large addition of CO₂ (500-600 μmol mol⁻¹). In this study, Gmelina arborea, a fast growing tropical deciduous tree species, was selected to determine the photosynthetic efficiency, growth response and overall source-sink relations under near elevated atmospheric CO₂ concentration (460 μmol mol⁻¹). Net photosynthetic rate of Gmelina was ∼30% higher in plants grown in elevated CO₂ compared with ambient CO₂-grown plants. The elevated CO₂ concentration also had significant effect on photochemical and biochemical capacities evidenced by changes in F_V/F_M, ABS/CSm, ET₀/CSm and RuBPcase activity. The study also revealed that elevated CO₂ conditions significantly increased absolute growth rate, above ground biomass and carbon sequestration potential in Gmelina which sequestered ∼2100 g tree⁻¹ carbon after 120 days of treatment when compared to ambient CO₂-grown plants. Our data indicate that young Gmelina could accumulate significant biomass and escape acclimatory down-regulation of photosynthesis due to high source-sink capacity even with an increase of 100 μmol mol⁻¹ CO₂.     

Burrow ventilation and associated porewater irrigation by the polychaete Marenzelleria viridis

Burrow ventilation of benthic infauna generates water currents that irrigate the interstices of the sediments surrounding the burrow walls. Such activities have associated effects on biogeochemical processes affecting ultimately important ecosystem processes. In this study, the ventilation and irrigation behavior of Marenzelleria viridis, an invasive polychaete species in Europe, was analyzed using different approaches. M. viridis showed to perform two types of ventilation: (1) muscular pumping of water out of the burrow and (2) cilia pumping of water into the burrow. Flowmeter measurements presented muscular pumping in time averaged rates of 0.15 ml min⁻¹. Oxygen needle electrodes positioned above the burrow openings revealed that muscular undulation of the worm body pumps anoxic water out of the burrow. On the other hand, microscope observations of the animal showed that ventilation of oxygen-rich water in the burrow occurs by ciliary action. The volume of water irrigated by M. viridis appears to vary linearly within the first 24 h incubation, with rates ranging from 0.003 to 0.01 ml min⁻¹. From those rates we could estimate that the time averaged rate of cilia ventilation should be about 0.16 ml min⁻¹. Since the cilia pumping into the burrow occurs in periods of 24 ± 12 min and at 50-70% of the measured time, considerable amounts of water from deeper sediments may percolate upwards to the sediment surface. This water is rich in reduced compounds and nutrients and may have important associated ecological implications in the ecosystem (e.g. affecting redox conditions, organic matter degradation, benthic recruitment and primary production).     

Helicoverpa armigera (Lepidoptera: Noctuidae) larvae that survive sublethal doses of nucleopolyhedrovirus exhibit high metabolic rates

To determine the effect of sublethal doses of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearSNPV) on the metabolic rate of H. armigera, the respiration rates of third instar H. armigera larvae inoculated with sublethal doses of HearSNPV were evaluated. Respiration rates, measured as the rate of CO₂ production (VCO₂), were recorded daily using closed-system respirometry. By 4 days post-inoculation (dpi), the metabolic rates of LD₂₅ or LD₇₅ survivors were significantly higher than that of uninoculated controls. When dose data were pooled, the VCO₂ values of larvae that survived inoculation (0.0288 ml h⁻¹), the uninoculated controls (0.0250 ml h⁻¹), and the larvae that did not survive inoculation (0.0199 ml h⁻¹) differed significantly from one another. At 4 dpi, the VCO₂ of the uninoculated controls were significantly lower than the VCO₂ of inoculation survivors, but significantly higher than the VCO₂ of inoculation non-survivors. Inoculation survivors may have had high metabolic rates due to a combination of viral replication, organ damage, and an energy-intensive induced cellular immune response. The high 4 dpi metabolic rate of inoculation survivors may reflect an effective immune response and may be seen as the metabolic signature of larvae that are in the process of surviving inoculation with HearSNPV.     

Preliminary investigation on the production of fuels and bio-char from Chlamydomonas reinhardtii biomass residue after bio-hydrogen production

The aim of this work was to investigate the potential conversion of Chlamydomonas reinhardtii biomass harvested after hydrogen production. The spent algal biomass was converted into nitrogen-rich bio-char, biodiesel and pyrolysis oil (bio-oil). The yield of lipids (algal oil), obtained by solvent extraction, was 15 ± 2% w/w_dry-biomass. This oil was converted into biodiesel with a 8.7 ± 1% w/w_dry-biomass yield. The extraction residue was pyrolysed in a fixed bed reactor at 350 °C obtaining bio-char as the principal fraction (44 ± 1% w/w_dry-biomass) and 28 ± 2% w/w_dry-biomass of bio-oil. Pyrolysis fractions were characterized by elemental analysis, while the chemical composition of bio-oil was fully characterized by GC-MS, using various derivatization techniques. Energy outputs resulting from this approach were distributed in hydrogen (40%), biodiesel (12%) and pyrolysis fractions (48%), whereas bio-char was the largest fraction in terms of mass.