Maximizing Hydrogen Production and Substrate Consumption by Escherichia coli WDHL in Cheese Whey Fermentation

Fermentative hydrogen production is strongly affected by pH. In order to maximize hydrogen production and substrate consumption in Escherichia coli ΔhycA, ΔlacI (WDHL) cheese whey fermentation, the influence of pH control at values of 5.5, 6, and 6.5 was studied in batch stirred-tank bioreactors. From the conditions evaluated, pH 6.5 was the best condition, at which the highest cumulative hydrogen production and yield (1.78 mol H₂/mol lactose) were obtained. Moreover, at this pH, all carbohydrates from the cheese whey were consumed, and a mix of ethanol and organic acids, mainly lactate, were produced from glucose, whereas galactose yielded acetate, ethanol, and succinate. Operating the reactor at pH 5.5 resulted in the highest maximum specific production rate, but smaller hydrogen yield because only glucose was metabolized and galactose was accumulated. At pH 6, not all cheese whey carbohydrates were consumed, and it was not favorable for hydrogen production. Lactose consumption and growth kinetics were not affected by the pH. The results show the importance of controlling pH to maximize hydrogen production and substrate consumption using cheese whey as substrate.     

Effect of nutrient supplements addition on ethanol production from cheese whey usingCandida pseudotropicalis under batch condition

Candida pseudotropicalis ATCC 8619 was selected among nine strains of lactose fermenting yeast for the production of ethanol from cheese whey. The effects of three nutrients (ammonium sulfate (NH₄)₂SO₄, dipotassium hydrogen phosphate K₂HPO₄, yeast extract, and a combination of them) on the ethanol yield from cheese whey were investigated. The results indicated that no addition of nutrient supplement is necessary to achieve complete lactose utilization during the cheese whey ethanol fermentation. However, addition of a small concentration (0.005% w/v) of these supplements reduced the lag period and the total fermentation time and increased the specific growth rate of the yeast. Higher concentrations (0.01 and 0.015% w/v) of ammonium sulfate and dipotassium hydrogen phosphate inhibited the cell growth and reduced lactose consumption. The highest ethanol (21.17 g/L) was achieved using yeast extract at a concentration of 0.01% w/v, given a conversion efficiency of 98.3%. No indication of alcohol inhibition was observed in this study.     

Succinic Acid Production from Cheese Whey using Actinobacillus succinogenes 130 Z

Actinobacillus succinogenes 130 Z was used to produce succinic acid from cheese whey in this study. At the presence of external CO₂ supply, the effects of initial cheese whey concentration, pH, and inoculum size on the succinic acid production were studied. The by-product formation during the fermentation process was also analyzed. The highest succinic acid yield of 0.57 was obtained at initial cheese whey concentration of 50 g/L, while the highest succinic acid productivity of 0.58 g h⁻¹ L⁻¹ was obtained at initial cheese whey concentration of 100 g/L. Increase in pH and inoculum size caused higher succinic acid yield and productivity. At the preferred fermentation condition of pH 6.8, inoculum size of 5% and initial cheese whey concentration of 50 g/L, succinic acid yield of 0.57, and productivity of 0.44 g h⁻¹ L⁻¹ were obtained. Acetic acid and formic acid were the main by-products throughout the fermentation run of 48 h. It is feasible to produce succinic acid using lactose from cheese whey as carbon resource by A. succinogenes 130 Z.     

Production of lactic acid from cheese whey by batch and repeated batch cultures of Lactobacillus sp. RKY2

The fermentative production of lactic acid from cheese whey and corn steep liquor (CSL) as cheap raw materials was investigated by using Lactobacillus sp. RKY2 in order to develop a cost-effective fermentation medium. Lactic acid yields based on consumed lactose were obtained at more than 0.98 g/g from the medium containing whey lactose. Lactic acid productivities and yields obtained from whey lactose medium were slightly higher than those obtained from pure lactose medium. The lactic acid productivity gradually decreased with increase in substrate concentration owing to substrate and product inhibitions. The fermentation efficiencies were improved by the addition of more CSL to the medium. Moreover, through the cell-recycle repeated batch fermentation, lactic acid productivity was maximized to 6.34 g/L/h, which was 6.2 times higher than that of the batch fermentation.     

Co-Fermentation of Cheese Whey and Crude Glycerol in EGSB Reactor as a Strategy to Enhance Continuous Hydrogen and Propionic Acid Production

This study evaluated the production of hydrogen and propionic acid in an expanded granular sludge bed (EGSB) reactor by co-fermentation of cheese whey (CW) and crude glycerol (CG). The reactor was operated at hydraulic retention time (HRT) of 8 h by changing the CW/CG ratio from 5:1 to 5:2, 5:3, 5:4, and 5:5. At the ratio of 5:5, HRT was reduced from 8 to 0.5 h. The maximum hydrogen yield of 0.120 mmol H₂ g COD^?1 was observed at the CW/CG ratio of 5:1. Increasing the CG concentration repressed hydrogen production in favor of propionic acid, with a maximum yield of 6.19 mmol HPr g COD^?1 at the CW/CG ratio of 5:3. Moreover, by reducing HRT of 8 to 0.5 h, the hydrogen production rate was increased to a maximum value of 42.5 mL H₂ h^?1 L^?1at HRT of 0.5 h. The major metabolites were propionate, 1,3-propanediol, acetate, butyrate, and lactate.     

Effect of Light/Dark Regimens on Hydrogen Production by <Emphasis Type="Italic">Tetraselmis subcordiformis</Emphasis> Coupled with an Alkaline Fuel Cell System

To improve the photoproduction of hydrogen (H₂) by a green algae-based system, the effect of light/dark regimens on H₂ photoproduction regulated by carbonyl cyanide m-chlorophenylhydrazone (CCCP) was investigated. A fuel cell was integrated into a photobioreactor to allow online monitoring of the H₂ evolution rate and decrease potential H₂ feedback inhibition by consuming the generated H₂ in situ. During the first 15 h of H₂ evolution, the system was subjected to dark treatment after initial light illumination (L/D = 6/9 h, 9/6 h, and 12/3 h). After the dark period, all systems were again exposed to light illumination until H₂ evolution stopped. Two peaks were observed in the H₂ evolution rate under all three light/dark regimens. Additionally, a high H₂ yield of 126 ± 10 mL L^?1 was achieved using a light/dark regimen of L 9 h/D 6 h/L until H₂ production ceased, which was 1.6 times higher than that obtained under continuous illumination. H₂ production was accompanied by some physiological and morphological changes in the cells. The results indicated that light/dark regimens improved the duration and yield of H₂ photoproduction by the CCCP-regulated process of Tetraselmis subcordiformis.     

Lactic acid production from cheese whey by immobilized bacteria

The performance of immobilized Bifidobacterium longum in sodium alginate beads and on a spiral-sheet bioreactor for the production of lactic acid from cheese whey was evaluated. Lactose utilization and lactic acid yield of B. longum were compared with those of Lactobacillus helveticus. B. longum immobilized in sodium alginate beads showed better performance in lactose utilization and lactic acid yield than L. helveticus. In the spiral-sheet bioreactor, a lactose conversion ratio of 79% and lactic acid yield of 0.84 g of lactic acid/g of lactose utilized were obtained during the first run with the immobilized L. helveticus. A lactose conversion ratio of 69% and lactic acid yield of 0.51 g of lactic acid/g of lactose utilized were obtained during the first run with immobilized B. longum in the spiral-sheet bioreactor. In producing lactic acid L. helveticus performed better when using the Spiral Sheet Bioreactor and B. longum showed better performance with gel bead immobilization. Because B. longum is a very promising new bacterium for lactic acid production from cheese whey, its optimum fermentation conditions such as pH and metabolic pathway need to be studied further. The ultrafiltration tests have shown that 94% of the cell and cheese whey proteins were retained by membranes with a mol wt cutoff of 5 and 20 KDa.     

Utilization of Cheese Whey Using Synergistic Immobilization of β-Galactosidase and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Cells in Dual Matrices

Whey is a byproduct of the dairy industry, which has prospects of using as a source for production of various valuable compounds. The lactose present in whey is considered as an environmental pollutant and its utilization for enzyme and fuel production, may be effective for whey bioremediation. The dairy yeast Kluyveromyces marxianus have the ability to utilize lactose sharply as the major carbon source for the production of the enzyme. Five strains were tested for the production of the β-galactosidase using whey. The maximum β-galactosidase activity of 1.74 IU/mg dry weight was achieved in whey using K. marxianus MTCC 1389. The biocatalyst was further immobilized on chitosan macroparticles and exhibited excellent functional activity at 35 °C. Almost 89 % lactose hydrolysis was attained for concentrated whey (100 g/L) and retained 89 % catalytic activity after 15 cycles of reuse. Finally, β-galactosidase was immobilized on chitosan and Saccharomyces cerevisiae on calcium alginate, and both were used together for the production of ethanol from concentrated whey. Maximal ethanol titer of 28.9 g/L was achieved during fermentation at 35 °C. The conclusions generated by employing two different matrices will be beneficial for the future modeling using engineered S. cerevisiae in scale-up studies.     

Green Synthesized Iron Oxide Nanoparticles Effect on Fermentative Hydrogen Production by Clostridium acetobutylicum

A green synthesis of iron oxide nanoparticles (FeNPs) was developed using Murraya koenigii leaf extract as reducing and stabilizing agent. UV–vis spectra show that the absorption band centred at a wavelength of 277 nm which corresponds to the surface plasmon resonances of synthesized FeNPs. Fourier transform infrared spectroscopy spectrum exhibits that the characteristic band at 580 cm^?1 is assigned to Fe–O of γ-Fe₂O₃. Transmission electron microscopy image confirms that the spherical with irregular shaped aggregates and average size of nanoparticles was found to be ～59 nm. The effect of synthesized FeNPs on fermentative hydrogen production was evaluated from glucose by Clostridium acetobutylicum NCIM 2337. The hydrogen yield in control experiment was obtained as 1.74?±?0.08 mol H₂/mol glucose whereas the highest hydrogen yield in FeNPs supplemented experiment was achieved as 2.33?±?0.09 mol H₂/mol glucose at 175 mg/L of FeNPs. In addition, the hydrogen content and hydrogen production rate were also increased from 34?±?0.8 to 52?±?0.8 % and 23 to 25.3 mL/h, respectively. The effect of FeNPs was compared with supplementation of FeSO₄ on fermentative process. The supplementation of FeNPs enhanced the hydrogen production in comparison with control and FeSO₄. The supplementation of FeNPs led to the change of the metabolic pathway towards high hydrogen production due to the enhancement of ferredoxin activity. The fermentation type was shifted from butyrate to acetate/butyrate fermentation type at the addition of FeNPs.     

Biohydrogen Production from Cheese Whey Wastewater in a Two-Step Anaerobic Process

Cheese whey-based biohydrogen production was seen in batch experiments via dark fermentation by free and immobilized Enterobacter aerogenes MTCC 2822 followed by photofermentation of VFAs (mainly acetic and butyric acid) in the spent medium by Rhodopseudomonas BHU 01 strain. E. aerogenes free cells grown on cheese whey diluted to 10 g lactose/L, had maximum lactose consumption (～79%), high production of acetic acid (1,900 mg/L), butyric acid (537.2 mg/L) and H(2) yield (2.04 mol/mol lactose; rate,1.09 mmol/L/h). The immobilized cells improved lactose consumption (84%), production of acetic acid (2,100 mg/L), butyric acid (718 mg/L) and also H(2) yield (3.50 mol/mol lactose; rate, 1.91 mmol/L/h). E. aerogenes spent medium (10 g lactose/L) when subjected to photofermentation by free Rhodopseudomonas BHU 01 cells, the H(2) yield reached 1.63 mol/mol acetic acid (rate, 0.49 mmol/L/h). By contrast, immobilized Rhodopseudomonas cells improved H(2) yield to 2.69 mol/mol acetic acid (rate, 1.87 mmol/L/h). The cumulative H(2) yield for free and immobilized bacterial cells was 3.40 and 5.88 mol/mol lactose, respectively. Bacterial cells entrapped in alginate, had a sluggish start of H(2) production but outperformed the free cells subsequently. Also, the concomitant COD reduction for free cells (29.5%) could be raised to 36.08% by immobilized cells. The data suggest that two-step fermentative H(2) production from cheese whey involving immobilized bacterial cells, offers greater substrate to- hydrogen conversion efficiency, and the effective removal of organic load from the wastewater in the long-term.     

Semicontinuous production of lactic acid from cheese whey using integrated membrane reactor

Semicontinuous production of lactic acid from cheese whey using free cells of Bifidobacterium longum with and without nanofiltration was studied. For the semicontinuous fermentation without membrane separation, the lactic acid productivity of the second and third runs is much lower than the first run. The semicontinuous fermentation with nanoseparation was run semicontinuously for 72 h with lactic acid to be harvested every 24 h using a nanofiltration membrane unit. The cells and unutilized lactose were kept in the reactor and mixed with newly added cheese whey in the subsequent runs. Slight increase in the lactic acid productivity was observed in the second and third runs during the semicontinuous fermentation with nanofiltration. It can be concluded that nanoseparation could improve the lactic acid productivity of the semicontinuous fermentation process.     

Synthesis,characterization, and catalytic properties of a cobalt(II) complex supported by an amine-bis(phenolate) ligand

A cobalt(II) complex [L′CoPy] 1 was prepared by the reaction of dimethylaminoethylamino-N,N-bis(2,4-dibromo)phenol (H₂L′) with CoCl₂. Electrochemical studies indicate that this complex is among the most efficient homogeneous catalysts for water reduction, with a turnover frequency of 917.7 mol of hydrogen per mole of catalyst per hour at an overpotential of 636.7 mV (pH 7.0). Additionally, under photoirradiation with blue light (λ _max = 469 nm), complex 1 in combination with [Ru(bpy)₃]Cl₂ and ascorbic acid (pH 4.0 in aqueous solution) also produces hydrogen with a turnover number of 4.9 × 10⁵ mol of H₂ per mol of catalyst.     

Additional Paper Waste in Pulping Sludge for Biohydrogen Production by Heat-Shocked Sludge

Dark anaerobic fermentation is an interesting alternative method for producing biohydrogen (H₂) as a renewable fuel because of its low cost and various usable organic substrates. Pulping sludge from wastewater treatment containing plentiful cellulosic substrate could be feasibly utilized for H₂ production by dark fermentation. The objective of this study was to investigate the optimal proportion of pulping sludge to paper waste, the optimal initial pH, and the optimal ratio of carbon and nitrogen (C/N) for H₂ production by anaerobic seed sludge pretreated with heat. The pulping sludge was pretreated with NaOH solution at high temperature and further hydrolyzed with crude cellulase. Pretreatment of the pulping sludge with 3% NaOH solution under autoclave at 121 °C for 2 h, hydrolysis with 5 FPU crude cellulase at 50 °C, and pH 4.8 for 24 h provided the highest reducing sugar production yield (229.68 ± 2.09 mg/g_TVS). An initial pH of 6 and a C/N ratio of 40 were optimal conditions for H₂ production. Moreover, the supplement of paper waste in the pulping sludge enhanced the cumulative H₂ production yield. The continuous hydrogen production was further conducted in a glass reactor with nylon pieces as supporting media and the maximum hydrogen production yield was 151.70 ml/g_TVS.     

Production of lactic acid from cheese whey by batch and repeated batch cultures of Lactobacillus sp. RKY2

The fermentative production of lactic acid from cheese whey and corn steep liquor (CSL) as cheap raw materials was investigated by using Lactobacillus sp. RKY2 in order to develop a cost-effective fermentation medium. Lactic acid yields based on consumed lactose were obtained at more than 0.98 g/g from the medium containing whey lactose. Lactic acid productivities and yields obtained from whey lactose medium were slightly higher than those obtained from pure lactose medium. The lactic acid productivity gradually decreased with increase in substrate concentration owing to substrate and product inhibitions. The fermentation efficiencies were improved by the addition of more CSL to the medium. Moreover, through the cell-recycle repeated batch fermentation, lactic acid productivity was maximized to 6.34 g/L/h, which was 6.2 times higher than that of the batch fermentation.     

Biohydrogen Production Based on the Evaluation of Kinetic Parameters of a Mixed Microbial Culture Using Glucose and Fruit–Vegetable Waste as Feedstocks

Hydrogen (H₂) production from the organic fraction of solid waste such as fruit and vegetable waste (FVW) is a novel and feasible energy technology. Continuous application of this process would allow for the simultaneous treatment of organic residues and energy production. In this study, batch experiments were conducted using glucose as substrate, and data of H₂ production obtained were successfully adjusted by a logistic model. The kinetic parameters (μ _max?=?0.101 h^?1, K _s?=?2.56 g/L) of an H₂-producing microbial culture determined by the Monod and Haldane–Andrews growth models were used to establish the continuous culture conditions. This strategy led to a productive steady state in continuous culture. Once the steady state was reached in the continuous reactor, a maximum H₂ production of 700 mL was attained. The feasibility of producing H₂ from the FVW obtained from a local market in Mexico City was also evaluated using batch conditions. The effect of the initial FVW concentration on the H₂ production and waste organic material degradation was determined. The highest H₂ production rate (1.7 mmol/day), the highest cumulative H₂ volume (310 mL), and 25 % chemical oxygen demand (COD) removal were obtained with an initial substrate (FVW) concentration of 37 g COD/L. The lowest H₂ production rates were obtained with relatively low initial substrate concentrations of 5 and 11 g COD/L. The H₂ production rates with FVW were also characterized by the logistic model. Similar cumulative H₂ production was obtained when glucose and FVW were used as substrates.     

Direct Fermentation of Gelatinized Cassava Starch to Acetone,Butanol, and Ethanol Using Clostridium acetobutylicum Mutant Obtained by Atmospheric and Room Temperature Plasma

The mutant strain designated as ART18, obtained from the wild-type strain Clostridium acetobutylicum PW12 treated by atmospheric and room temperature plasma, showed higher solvent tolerance and butanol production than that of the wild-type strain. The production of butanol was 11.3?±?0.5 g/L, 31 % higher than that of the wild-type strain when it was used for acetone, butanol, and ethanol fermentation in P2 medium. Furthermore, the effects of cassava flour concentration, pH regulators, and vitamins on the ABE production were also investigated. The highest butanol production of 15.8?±?0.8 g/L and butanol yield (0.31 g/g) were achieved after the above factors were optimized. When acetone, butanol, and ethanol fermentation by ART18 was carried out in a 15-L bioreactor, the butanol production, the productivity of butanol, and the total solvent were 16.3?±?0.9, 0.19, and 0.28 g/L^/h, respectively. These results indicate that ART18 is a promising industrial producer in ABE fermentation.     

In situ Removal of Hydrogen Sulfide During Biogas Fermentation at Microaerobic Condition

In this paper, rice straw was used as a raw material to produce biogas by anaerobic batch fermentation at 35 °C (mesophilic) or 55 °C (thermophilic). The hydrogen sulfide in biogas can be converted to S⁰ or sulfate and removed in-situ under micro-oxygen environment. Trace oxygen was conducted to the anaerobic fermentation tank in amount of 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, or 10.0 times stoichiometric equivalence, respectively, and the control experiment without oxygen addition was carried out. The results showed that the initial H₂S concentrations of biogas are about 3235?±?185 mg/m³ (mesophilic) or 3394?±?126 mg/m³ (thermophilic), respectively. The desulfurization efficiency is 72.3 % (mesophilic) or 65.6 % (thermophilic), respectively, with oxygen addition by stoichiometric relation. When the oxygen feeded in amount of 2～4 times, theoretical quantity demanded the removal efficiency of hydrogen sulfide could be over 92 %, and the oxygen residue in biogas could be maintained less than 0.5 %, which fit the requirement of biogas used as vehicle fuel or combined to the grid. Though further more oxygen addition could promote the removal efficiency of hydrogen sulfide (about 93.6 %), the oxygen residue in biogas would be higher than the application limit concentration (0.5 %). Whether mesophilic or thermophilic fermentation with the extra addition of oxygen, there were no obvious changes in the gas production and methane concentration. In conclusion, in-situ desulfurization can be achieved in the anaerobic methane fermentation system under micro-oxygen environment. In addition, air could be used as a substitute oxygen resource on the situation without strict demand for the methane content of biogas.     

Ascorbic Acid/Hydrogen Peroxide–Initiated Graft Copolymerization of MMA onto Bombyx mori Silk Fibroin

Graft copolymerization of methyl methacrylate (MMA) onto Bombyx mori silk fibroin was investigated using ascorbic acid and hydrogen peroxide as redox system. The effect of different reaction parameters such as reaction time, temperature, and initiator and monomer concentrations on graft yield were determined. The optimum grafting conditions were found to be temperature = 65°C, time = 120 min, monomer concentration = 2.82 × 10^?1 mol/L, ascorbic acid concentration = 2.83 × 10^?2 mol/L, and H₂O₂ concentration = 1.58 × 10^?1 mol/L. The structural properties of the fibers were studied with the help of FT-IR, scanning electron microscopy (SEM), X-ray diffraction, and TGA techniques.     

Thermal Drying of <Emphasis Type="BoldItalic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="BoldItalic">bulgaricus</Emphasis> and its Efficient Use as Starter for Whey Fermentation and Unsalted Cheese Making

Lactobacillus bulgaricus grown on whey was dried by a simple thermal drying method at the range 35–55°C and its efficiency for lactic acid fermentation of whey was evaluated. Drying of cells in whey suspension in the examined temperature range did not affect significantly their viability (82–87% survival), indicating a protective effect of whey as both growth and drying medium. The kinetics of fermentation of whey and mixtures of whey/molasses using the dried culture were comparable to those of non-dried cells, and only low pH had a detrimental effect on the fermentation ability of the dried cells. Furthermore, dried L. bulgaricus, free or immobilized on casein coagulates, was used as starter for the production of unsalted hard-type cheese. The effects of the amount of starter culture and the immobilization technique, the evolution of microbial counts, and the sensory properties of the produced cheeses were evaluated during ripening at various temperatures.     

Separate and Concentrate Lactic Acid Using Combination of Nanofiltration and Reverse Osmosis Membranes

The processes of lactic acid production include two key stages, which are (a) fermentation and (b) product recovery. In this study, free cell of Bifidobacterium longum was used to produce lactic acid from cheese whey. The produced lactic acid was then separated and purified from the fermentation broth using combination of nanofiltration and reverse osmosis membranes. Nanofiltration membrane with a molecular weight cutoff of 100–400 Da was used to separate lactic acid from lactose and cells in the cheese whey fermentation broth in the first step. The obtained permeate from the above nanofiltration is mainly composed of lactic acid and water, which was then concentrated with a reverse osmosis membrane in the second step. Among the tested nanofiltration membranes, HL membrane from GE Osmonics has the highest lactose retention (97 ± 1%). In the reverse osmosis process, the ADF membrane could retain 100% of lactic acid to obtain permeate with water only. The effect of membrane and pressure on permeate flux and retention of lactose/lactic acid was also reported in this paper.