Biomethane Production in an AnSBBR Treating Wastewater from Biohydrogen Process

An anaerobic sequencing batch reactor containing immobilized biomass (AnSBBR) was used to produce biomethane by treating the effluent from another AnSBBR used to produce biohydrogen from glucose- (AR-EPHG) and sucrose-based (AR-EPHS) wastewater. In addition, biomethane was also produced from sucrose-based synthetic wastewater (AR-S) in a single AnSBBR to compare the performance of biomethane production in two steps (acidogenic and methanogenic) in relation to a one-step operation. The system was operated at 30 °C and at a fixed stirring rate of 300 rpm. For AR-EPHS treatment, concentrations were 1,000, 2,000, 3,000, and 4,000 mg chemical oxygen demand?(COD)?L^?1 and cycle lengths were 6 and 8 h. The applied volumetric organic loads were 2.15, 4.74, 5.44, and 8.22 g COD L^?1 day^?1. For AR-EPHG treatment, concentration of 4,000 mg COD L^?1 and 4-h cycle length (7.21 g COD L^?1 day^?1) were used. For AR-S treatment, concentration was 4,000 mg COD L^?1 day^?1 and cycle lengths were 8 (7.04 g COD L^?1 day^?1) and 12 h (4.76 g COD L^?1 day^?1). The condition of 8.22 g COD L^?1 day^?1 (AR-EPHS) showed the best performance with respect to the following parameters: applied volumetric organic load of 7.56 g COD L^?1 day^?1, yield between produced methane and removed organic material of 0.016 mol CH₄?g COD^?1, CH₄ content in the produced biogas of 85 %, and molar methane productivity of 127.9 mol CH₄?m^?3 day^?1. In addition, a kinetic study of the process confirmed the trend that, depending on the biodegradability characteristics of the wastewaters used, the two-step treatment (acidogenic for biohydrogen production and methanogenic for biomethane production) has potential advantages over the single-step process.     

Single-cell Protein and Xylitol Production by a Novel Yeast Strain <Emphasis Type="Italic">Candida intermedia</Emphasis> FL023 from Lignocellulosic Hydrolysates and Xylose

Yeasts are good candidates to utilize the hydrolysates of lignocellulose, the most abundant bioresource, for bioproducts. This study aimed to evaluate the efficiencies of single-cell protein (SCP) and xylitol production by a novel yeast strain, Candida intermedia FL023, from lignocellulosic hydrolysates and xylose. This strain efficiently assimilated hexose, pentose, and cellubiose for cell mass production with the crude protein content of 484.2 g kg^?1 dry cell mass. SCP was produced by strain FL023 using corncob hydrolysate and urea as the carbon and nitrogen sources with the dry cell mass productivity 0.86 g L^?1 h^?1 and the yield of 0.40 g g^?1 sugar. SCP was also produced using NaOH-pretreated Miscanthus sinensis straw and corn steep liquor as the carbon and nitrogen sources through simultaneous saccharification and fermentation with the dry cell productivity of 0.23 g L^?1 h^?1 and yield of 0.17 g g^?1 straw. C. intermedia FL023 was tolerant to 0.5 g L^?1 furfural, acetic acid, and syringaldehyde in xylitol fermentation and produced 45.7 g L^?1 xylitol from xylose with the productivity of 0.38 g L^?1 h^?1 and the yield of 0.57 g g^?1 xylose. This study provides feasible methods for feed and food additive production from the abundant lignocellulosic bioresources.     

Succinic Acid Production from Corn Cob Hydrolysates by Genetically Engineered Corynebacterium glutamicum

Corynebacterium glutamicum wild type lacks the ability to utilize the xylose fractions of lignocellulosic hydrolysates. In the present work, we constructed a xylose metabolic pathway in C. glutamicum by heterologous expression of the xylA and xylB genes coming from Escherichia coli. Dilute-acid hydrolysates of corn cobs containing xylose and glucose were used as a substrate for succinic acid production by recombinant C. glutamicum NC-2. The results indicated that the available activated charcoal pretreatment in dilute-acid hydrolysates of corn cobs could be able to overcome the inhibitory effect in succinic acid production. Succinic acid was shown to be efficiently produced from corn cob hydrolysates (55 g l^?1 xylose and 4 g l^?1 glucose) under oxygen deprivation with addition of sodium carbonate. Succinic acid concentration reached 40.8 g l^?1 with a yield of 0.69 g g^?1 total sugars within 48 h. It was the first report of succinic acid production from corn cob hydrolysates by metabolically engineered C. glutamicum. This study suggested that dilute-acid hydrolysates of corn cobs may be an alternative substrate for the efficient production of succinic acid by C. glutamicum.     

Enhanced Production of Poly-γ-glutamic Acid by <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> TISTR 1010 with Environmental Controls

Bacillus licheniformis TISTR 1010 was used for glutamic acid-independent production of poly-γ-glutamic acid (γ-PGA). A fed-batch production strategy was developed involving feedings of glucose, citric acid, and ammonium chloride at specified stages of the fermentation. With the dissolved oxygen concentration controlled at ≥50% of air saturation and the pH controlled at ~7.4, the fed-batch operation at 37 °C afforded a peak γ-PGA concentration of 39.9 ± 0.3 g L^?1 with a productivity of 0.926 ± 0.006 g L^?1 h^?1. The observed productivity was nearly threefold greater than previously reported for glutamic acid-independent production using the strain TISTR 1010. The molecular weight of γ-PGA was in the approximate range of 60 to 135 kDa.     

Screening of <Emphasis Type="Italic">Isochrysis</Emphasis> Strains and Utilization of a Two-Stage Outdoor Cultivation Strategy for Algal Biomass and Lipid Production

Isochrysis is a genus of marine algae without cell wall and capable of accumulating lipids. In this study, the lipid production potential of Isochrysis was assessed by comparing 15 Isochrysis strains with respect to their growth rate, lipid production, and fatty acid profiles. Three best strains were selected (lipid productivity, 103.0~121.7 mg L^?1 day^?1) and their lipid-producing capacities were further examined under different controlled parameters, e.g., growth phase, medium nutrient, and light intensity in laboratory cultures. Furthermore, the three Isochrysis strains were monitored in outdoor panel photobioreactors with various initial cell densities and optical paths, and the strain CS177 demonstrated the superior potential for outdoor cultivation. A two-stage semi-continuous strategy for CS177 was subsequently developed, where high productivities of biomass (1.1 g L^?1 day^?1) and lipid (0.35 g L^?1 day^?1) were achieved. This is a comprehensive study to evaluate the lipid-producing capability of Isochrysis strains under both indoor and outdoor conditions. Results of the present work lay a solid foundation for the physiological and biochemical responses of Isochrysis to various conditions, shedding light on the future utilization of this cell wall-lacking marine alga for biofuel production.     

High Level Production of β-Galactosidase Exhibiting Excellent Milk-Lactose Degradation Ability from Aspergillus oryzae by Codon and Fermentation Optimization

A β-galactosidase gene from Aspergillus oryzae was engineered utilizing codon usage optimization to be constitutively and highly expressed in the Pichia pastoris SMD1168H strain in a high-cell-density fermentation. After fermentation for 96 h in a 50-L fermentor using glucose and glycerol as combined carbon sources, the recombinant enzyme in the culture supernatant had an activity of 4,239.07 U mL^?1 with o-nitrophenyl-β-d-galactopyranoside as the substrate, and produced a total of extracellular protein content of 7.267 g L^?1 in which the target protein (6.24 g L^?1) occupied approximately 86 %. The recombinant β-galactosidase exhibited an excellent lactose hydrolysis ability. With 1,000 U of the enzyme in 100 mL milk, 92.44 % lactose was degraded within 24 h at 60 °C, and the enzyme could also accomplish the hydrolysis at low temperatures of 37, 25, and 10 °C. Thus, this engineered strain had significantly higher fermentation level of A. oryzae lactase than that before optimization and the β-galactosidase may have a good application potential in whey and milk industries.     

<Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> application as a prospective approach for biosynthesis of pyruvic acid from glycerol

With the problems related to chemical methods of pyruvic acid (PA) synthesis, a fast-growing interest has been observed in research aiming at reducing the production cost of PA by applying biotechnological methods. This study aimed to investigate the potential applicability of Yarrowia lipolytica Wratislavia 1.31 yeast strain for valorisation of pure and crude glycerol through the production of industrially desired PA. Conditions required for the effective PA biosynthesis, i.e., pH value, thiamine concentration, agitation, and substrate concentration, were examined in batch and fed-batch cultivation modes. The efficient production of PA occurred under the limitation of thiamine (1 µg L^?1) and was stimulated by moderate pH (4.5) and agitation (800 rev min^?1) of the culture. Under optimal conditions, Y. lipolytica Wratislavia 1.31 was able to produce 85.2 g L^?1 of PA with volumetric productivity of 0.90 g L^?1 h^?1. The yield of PA biosynthesis reached a high level of 1.03 g g^?1. Obtained results confirmed the aptitude of Y. lipolytica yeast to produce high amounts of PA from simple glycerol-containing media. Presented process was very promising and might be considered as an attractive alternative for currently used chemical methods of PA synthesis.     

Isolation of Novel Microalgae from Acid Mine Drainage and Its Potential Application for Biodiesel Production

Microalgae were selected and isolated from acid mine drainage in order to find microalgae species which could be cultivated in low pH condition. In the present investigation, 30 microalgae were isolated from ten locations of acid mine drainage in South Korea. Four microalgae were selected based on their growth rate, morphology, and identified as strains of KGE1, KGE3, KGE4, and KGE7. The dry biomass of microalgae species ranged between 1 and 2 g L^?1 after 21 days of cultivation. The growth kinetics of microalgae was well described by logistic growth model. Among these, KGE7 has the highest biomass production (2.05?±?0.35 g L^?1), lipid productivity (0.82?±?0.14 g L^?1), and C16–C18 fatty acid contents (97.6 %). These results suggest that Scenedesmus sp. KGE 7 can be utilized for biodiesel production based on its high biomass and lipid productivity.     

Production of <Emphasis Type="Italic">R</Emphasis>-Mandelic Acid Using Nitrilase from Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Cells Immobilized with Tris(Hydroxymethyl)Phosphine

Recombinant Escherichia coli cells harboring nitrilase from Alcaligenes faecalis were immobilized using tris(hydroxymethyl)phosphine (THP) as the coupling agent. The optimal pH and temperature of the THP-immobilized cells were determined at pH 8.0 and 55 °C. The half-lives of THP-immobilized cells measured at 35, 40, and 50 °C were 1800, 965, and 163 h, respectively. The concentration of R-mandelic acid (R-MA) reached 358 mM after merely 1-h conversion by the immobilized cells with 500 mM R,S-mandelonitrile (R,S-MN), affording the highest productivity of 1307 g L^?1 day^?1 and the space-time productivity of 143.2 mmol L^?1 h^?1 g^?1. The immobilized cells with granular shape were successfully recycled for 60 batches using 100 mM R,S-MN as substrate at 40 °C with 64% of relative activity, suggesting that the immobilized E. coli cells obtained in this study are promising for the production of R-MA.     

Bioflocculant Exopolysaccharide Production by Azotobacter indicus Using Flower Extract of Madhuca latifolia L

Efficacy of Azotobacter indicus ATCC 9540 strain for production exopolysaccharide (EPS) bioflocculant was investigated. Mahua flower extract (Madhuca latifolia L), a natural substrate at the concentration of 20 g L^?1, gave maximum recovery of EPS followed by sucrose and mannitol as compared to other carbon sources after 172 h. Yeast extract was found to be the most effective nitrogen source as compared to beef extract, sodium nitrate, ammonium sulfate, casein hydrolysate, and urea for the production of EPS. EPS production was increased in presence of nitrogen (5.51 g L^?1) as compared to nitrogen-free medium (3.51 g L^?1), and fermentation time was also reduced by 28 h. Maximum EPS production (6.10 g L^?1) was found in the presence of 20 g L^?1 flower extract and 0.5 g L^?1 yeast extract containing Ashby’s media with 180 rpm at 30 °C at 144 h, under controlled conditions in 2.5 L fermenter using optimized medium. The isolated EPS showed cation-dependent flocculating activity. Concentration of EPS played an important role in bioflocculating activity which increased in a concentration-dependent manner up to a certain limit, with the maximum flocculation of 72% at 500 mg L^?1 concentration but remained almost static after this concentration. Extracted polymer was characterized by different chemical tests, FT-IR spectroscopy, and TLC which showed presence of uronic acids, O-acetyl groups, and Orcinol with suggestive indication of alginate like polymer. This study suggests that use of M. latifolia L. flowers can be a potential alternative bioresource for production of exopolysaccharide.     

Multivariate Analysis for the Classification Differentiation of Brazilian Sugarcane Spirits by Analysis of Organic and Inorganic Compounds

Abstract

Brazilian sugarcane spirits were analyzed to elucidate similarities and dissimilarities by principal component analysis. Nine aldehydes, six alcohols, and six metal cations were identified and quantified. Isobutanol (LD 202.9 µg L^?1), butiraldehyde (0.08–0.5 µg L^?1), ethanol (39–47% v/v), and copper (371–6068 µg L^?1) showed marked similarities, but the concentration levels of n-butanol (1.6–7.3 µg L^?1), sec-butanol (LD 89 µg L^?1), formaldehyde (0.1–0.74 µg L^?1), valeraldehyde (0.04–0.31 µg L^?1), iron (8.6–139.1 µg L^?1), and magnesium (LD 1149 µg L^?1) exhibited differences from samples.     

Microbial Fed-batch Production of 1,3-Propanediol Using Raw Glycerol with Suspended and Immobilized Klebsiella pneumoniae

The production of 1,3-propanediol (1,3-PD) was investigated with Klebsiella pneumoniae DSM 4799 using raw glycerol without purification obtained from a biodiesel production process. Fed-batch cultures with suspended cells revealed that 1,3-PD production was more effective when utilizing raw glycerol than pure glycerol (productivity after 47 h of fermentation, 0.84 g?L^?1?h^?1 versus 1.51 g?L^?1?h^?1 with pure and raw glycerol, respectively). In addition, more than 80 g/L of 1,3-PD was produced using raw glycerol; this is the highest 1,3-PD concentration reported thus far for K. pneumoniae using raw glycerol. Repeated fed-batch fermentation with cell immobilization in a fixed-bed reactor was performed to enhance 1,3-PD production. Production of 1,3-PD increased with the cycle number (1.06 g?L^?1?h^?1 versus 1.61 g?L^?1?h^?1 at the first and fourth cycle, respectively) due to successful cell immobilization. During 46 cycles of fed-batch fermentation taking place over 1,460 h, a stable and reproducible 1,3-PD production performance was observed with both pure and raw glycerol. Based on our results, repeated fed batch with immobilized cells is an efficient fermentor configuration, and raw glycerol can be utilized to produce 1,3-PD without inhibitory effects caused by accumulated impurities.     

Effects of Organic Loading, Influent Concentration, and Feed Time on Biohydrogen Production in a Mechanically Stirred AnSBBR Treating Sucrose-Based Wastewater

An anaerobic sequencing batch biofilm reactor (AnSBBR—total volume 7.5 L; liquid volume 3.6 L; treated volume per cycle 1.5 L) treated sucrose-based wastewater to produce biohydrogen (at 30 °C). Different applied volumetric organic loads (AVOL of 9.0, 12.0, 13.5, 18.0, and 27.0 kg COD m^?3 day^?1), which were varied according to the influent concentration (3,600 and 5,400 mg COD L^?1) and cycle length (4, 3, and 2 h), have been used to assess the following parameters: productivity and yield of biohydrogen per applied and removed load, reactor stability, and efficiency. The removed organic matter (COD) remained stable and close to 18 % and carbohydrates (sucrose) uptake rate remained between 83 and 97 % during operation. The decrease in removal performance of the reactor with increasing AVOL, by increasing the influent concentration (at constant cycle length) and decreasing the cycle lengths (at constant influent concentrations), resulted in lower conversion efficiencies. Under all conditions, when organic load increased there was a predominance of acetic, propionic, and butyric acid as well as ethanol. The highest concentration of biohydrogen in the biogas (24–25 %) was achieved at conditions with AVOL of 12.0 and 13.5 kg COD m^?3 day^?1, the highest daily production rate (0.139 mol H₂?day^?1) was achieved at AVOL of 18.0 kg COD m^?3 day^?1, and the highest production yields per removed and applied load were 2.83 and 3.04 mol H₂?kg SUC^?1, respectively, at AVOL of 13.5 kg COD m^?3 day^?1. The results indicated that the best productivity tends to occur at higher organic loads, as this parameter involves the “biochemical generation” of biogas, and the best yield tends to occur at lower and/or intermediate organic loads, as this parameter involves “biochemical consumption” of the substrate.     

Enzymatic Hydrolysis and Fermentation of Pretreated Cashew Apple Bagasse with Alkali and Diluted Sulfuric Acid for Bioethanol Production

The aim of this work was to optimize the enzymatic hydrolysis of the cellulose fraction of cashew apple bagasse (CAB) after diluted acid (CAB-H) and alkali pretreatment (CAB-OH), and to evaluate its fermentation to ethanol using Saccharomyces cerevisiae. Glucose conversion of 82?±?2 mg/g CAB-H and 730?±?20 mg/g CAB-OH was obtained when 2% (w/v) of solid and 30 FPU/g bagasse was used during hydrolysis at 45 °C, 2-fold higher than when using 15 FPU/g bagasse, 44?±?2 mg/g CAB-H, and 450?±?50 mg/g CAB-OH, respectively. Ethanol concentration and productivity, achieved after 6 h of fermentation, were 20.0?±?0.2 g L^?1 and 3.33 g L^?1 h^?1, respectively, when using CAB-OH hydrolyzate (initial glucose concentration of 52.4 g L^?1). For CAB-H hydrolyzate (initial glucose concentration of 17.4 g L^?1), ethanol concentration and productivity were 8.2?±?0.1 g L^?1 and 2.7 g L^?1 h^?1 in 3 h, respectively. Hydrolyzates fermentation resulted in an ethanol yield of 0.38 and 0.47 g/g glucose with pretreated CAB-OH and CAB-H, respectively. Ethanol concentration and productivity, obtained using CAB-OH hydrolyzate, were close to the values obtained in the conventional ethanol fermentation of cashew apple juice or sugar cane juice.     

Cultivation of Chlorella vulgaris in Dairy Wastewater Pretreated by UV Irradiation and Sodium Hypochlorite

There is potential in the utilization of microalgae for the purification of wastewater as well as recycling the resource in the wastewater to produce biodiesel. The large-scale cultivation of microalgae requires pretreatment of the wastewater to eliminate bacteria and protozoa. This procedure is costly and complex. In this study, two methods of pretreatment, UV irradiation, and sodium hypochlorite (NaClO), in various doses and concentrations, were tested in the dairy wastewater. Combining the efficiency of biodiesel production, we proposed to treat the dairy wastewater with NaClO in the concentration of 30 ppm. In this condition, The highest biomass productivity and lipid productivity of Chlorella vulgaris reached 0.450 g L^?1 day^?1 and 51 mg L^?1 day^?1 after a 4-day cultivation in the dairy wastewater, respectively.     

Mixed Food Waste as Renewable Feedstock in Succinic Acid Fermentation

Mixed food waste, which was directly collected from restaurants without pretreatments, was used as a valuable feedstock in succinic acid (SA) fermentation in the present study. Commercial enzymes and crude enzymes produced from Aspergillus awamori and Aspergillus oryzae were separately used in hydrolysis of food waste, and their resultant hydrolysates were evaluated. For hydrolysis using the fungal mixture comprising A. awamori and A. oryzae, a nutrient-complete food waste hydrolysate was generated, which contained 31.9 g L^?1 glucose and 280 mg L^?1 free amino nitrogen. Approximately 80–90 % of the solid food waste was also diminished. In a 2.5 L fermentor, 29.9 g L^?1 SA was produced with an overall yield of 0.224 g g^?1 substrate using food waste hydrolysate and recombinant Escherichia coli. This is comparable to many similar studies using various wastes or by-products as substrates. Results of this study demonstrated the enormous potential of food waste as renewable resource in the production of bio-based chemicals and materials via microbial bioconversion.     

Mineral Supplementation Increases Erythrose Reductase Activity in Erythritol Biosynthesis from Glycerol by Yarrowia lipolytica

The aim of this study was to examine the impact of divalent copper, iron, manganese, and zinc ions on the production of erythritol from glycerol by Yarrowia lipolytica and their effect on the activity of erythrose reductase. No inhibitory effect of the examined minerals on yeast growth was observed in the study. Supplementation with MnSO₄·7H₂O (25 mg l^?1) increased erythritol production by Y. lipolytica by 14.5 %. In the bioreactor culture with manganese ion addition, 47.1 g l^?1 of erythritol was produced from 100.0 g l^?1 of glycerol, which corresponded to volumetric productivity of 0.87 g l^?1 h^?1. The addition of Mn²⁺ enhanced the intracellular activity of erythrose reductase up to 24.9 U g^?1 of dry weight of biomass (DW), hence, about 1.3 times more than in the control.     

Occurrence of cytostatic compounds in hospital effluents and wastewaters,determined by liquid chromatography coupled to high-resolution mass spectrometry

The occurrence of 26 commonly used cytostatic compounds in wastewaters was evaluated using an automated solid-phase extraction (SPE) method with liquid chromatography–high-resolution mass spectrometry (LC–HRMS). Detection was optimized using Oasis HLB SPE cartridges at pH 2. Two hospital effluents and their two receiving wastewater treatment plants were sampled over five days. In hospital effluents, eight cytostatics were detected at levels up to 86.2 μg L^?1 for ifosfamide, 4.72 μg L^?1 for cyclophosphamide, and 0.73 μg L^?1 for irinotecan, the three most relevant compounds identified. Cyclophosphamide and megestrol acetate were found in wastewaters at concentrations up to 0.22 μg L^?1 for the latter. The predicted environmental concentrations (PEC) in sewage effluents of ifosfamide (2.4–4.3 ng L^?1), capecitabine (11.5–14.2 ng L^?1), and irinotecan (0.4–0.6 ng L^?1), calculated from consumption data in each hospital, published excretion values for the target compounds, and wastewater elimination rates, were in agreement with experimental values.     

Enhancing Carbohydrate Productivity of <Emphasis Type="Italic">Chlorella</Emphasis> sp. AE10 in Semi-continuous Cultivation and Unraveling the Mechanism by Flow Cytometry

Accumulated carbohydrate in microalgae is promising feedstock for bioethanol fermentation. Selection of suitable cultivation conditions in semi-continuous cultivation is critical to achieve a high carbohydrate productivity. In the current study, the effects of macro-nutrient (nitrogen, phosphorus, and sulfur) limitations and light intensity were evaluated for the carbohydrate accumulations of Chlorella sp. AE10 under 10% CO₂ conditions. It was shown that nitrogen limitation and high light intensity were effective for improving carbohydrate productivity. The average carbohydrate and biomass productivity in semi-continuous cultivation with 1/4 N medium and 1000 μmol photons m^?2 s^?1 was 0.673 and 0.93 g L^?1 day^?1, respectively. Sulfur and phosphorus limitations could improve the carbohydrate content but they could not enhance the carbohydrate productivity. The cell cycle progression and chlorophyll a were investigated using flow cytometry (FCM). The results showed that macro-nutrient limitation and high light intensity indeed influenced cell cycle progression and led to the formation of polyploid cells along with the carbohydrate accumulation in a certain range. FCM was rapid and accurate method to investigate the operation conditions why 1/4 N, 2 days as a cycle, and high light intensity were optimal ones. In addition, the remaining high level of photosynthesis activity was also important for achieving a high carbohydrate productivity. Dynamic tracking of carbohydrate accumulation is helpful for establishment of a semi-continuous cultivation for enhancing carbohydrate productivity in microalgae.     

Mercury speciation by a high performance liquid chromatography—atomic fluorescence spectrometry hyphenated system with photo-induced chemical vapour generation reagent in the mobile phase

Speciation of mercury was accomplished by using a simple interface with photo-induced chemical vapour generation in a high performance liquid chromatography—atomic fluorescence spectrometry (HPLC-AFS) hyphenated system. Acetic acid and 2-mercaptoethanol in the mobile phase were used as photochemical reagent. The operating parameters were optimized to give limits of detection of 0.53 µg L^?1, 0.22 µg L^?1, 0.18 µg L^?1 and 0.25 µg L^?1 for inorganic mercury, methylmercury, ethylmercury and phenylmercury, respectively. The method was validated with the certified reference material DORM-2 and applied to the analysis of seafood samples. The HPLC-AFS hyphenated system is simple, environmentally friendly, and represents an attractive alternative to the conventional peroxothiosulfate-borohydride method.