Biological caproate production by Clostridium kluyveri from ethanol and acetate as carbon sources.

Caproate is a valuable industrial product and chemical precursor. In this study, batch tests were conducted to investigate the fermentative caproate production through chain elongation from acetate and ethanol. The effect of acetate/ethanol ratio and initial ethanol concentration on caproate production was examined. When substrate concentration was controlled at 100mM total carbon, hydrogen was used as an additional electron donor. The highest caproate concentration of 3.11g/L was obtained at an ethanol/acetate ratio of 7:3. No additional electron donor was needed upon an ethanol/acetate ratio ≥7:3. Caproate production increased with the increase of carbon source until ethanol concentration over 700mM, which inhibited the fermentation process. The highest caproate concentration of 8.42g/L was achieved from high ethanol strength wastewater with an ethanol/acetate ratio of 10:1 (550mM total carbon). Results obtained in this study can pave the way towards efficient chain elongation from ethanol-rich wastewater.

Integrated production of cellulosic bioethanol and succinic acid from industrial hemp in a biorefinery concept.

The aim of this study was to develop integrated biofuel (cellulosic bioethanol) and biochemical (succinic acid) production from industrial hemp (Cannabis sativa L.) in a biorefinery concept. Two types of pretreatments were studied (dilute-acid and alkaline oxidative method). High cellulose recovery (>95%) as well as significant hemicelluloses solubilization (49-59%) after acid-based method and lignin solubilization (35-41%) after alkaline H2O2 method were registered. Alkaline pretreatment showed to be superior over the acid-based method with respect to the rate of enzymatic hydrolysis and ethanol productivity. With respect to succinic acid production, the highest productivity was obtained after liquid fraction fermentation originated from steam treatment with 1.5% of acid. The mass balance calculations clearly showed that 149kg of EtOH and 115kg of succinic acid can be obtained per 1ton of dry hemp. Results obtained in this study clearly document the potential of industrial hemp for a biorefinery.

Effects of Benzalkonium Chloride, Proxel LV, P3 Hypochloran, Triton X-100 and DOWFAX 63N10 on anaerobic digestion processes.

In this study, the individual and synergistic toxicity of the following xenobiotics: Benzalkonium Chloride (BKC), Proxel LV (PRX), P3 Hypochloran (HPC), Triton X-100 (TRX), and DOWFAX 63N10 (DWF), on anaerobic digestion (AD) process, was assessed. The experiments were performed in batch and continuous (up-flow anaerobic sludge blanket, UASB) reactors with biochemical-industrial wastewater, as substrate. In batch experiments, half-maximal inhibitory concentrations (IC50) for the tested xenobiotics were found to be 13.1, 1003, 311.5 and 24.3 mg L(-1) for BKC, PRX, DWF and TRX, respectively while HPC did not affect the AD process. Furthermore, the xenobiotics mixture tested did not present any synergistic inhibitory effect on the AD process. In continuous experiments, BKC and xenobiotics' mixture induced even stronger (more than 85%) of inhibition, expressed as IC50, compared to the levels observed from the batch reactors. Oppositely, TRX showed no inhibition in continuous mode, while inhibition was detected at batch mode.

Thermochemical pretreatments for enhancing succinic acid production from industrial hemp (Cannabis sativa L.).

The aim of this study was to develop an efficient thermochemical method for treatment of industrial hemp biomass, in order to increase its bioconversion to succinic acid. Industrial hemp was subjected to various thermochemical pretreatments using 0-3% H2SO4, NaOH or H2O2 at 121-180°C prior to enzymatic hydrolysis. The influence of the different pretreatments on hydrolysis and succinic acid production by Actinobacillus succinogenes 130Z was investigated in batch mode, using anaerobic bottles and bioreactors. Enzymatic hydrolysis and fermentation of hemp material pretreated with 3% H2O2 resulted in the highest overall sugar yield (73.5%), maximum succinic acid titer (21.9 g L(-1)), as well as the highest succinic acid yield (83%). Results obtained clearly demonstrated the impact of different pretreatments on the bioconversion efficiency of industrial hemp into succinic acid.

Effective harvesting of the microalgae Chlorella protothecoides via bioflocculation with cationic starch.

In the present work, the flocculation efficiency of cationic starch (Greenfloc 120) was tested on the fresh water microalga Chlorella protothecoides under different conditions (pH and flocculant concentrations). Different concentrations of Greenfloc 120 (0, 2.5, 5, 10, 20, 40 mg L(-1)) were screened against different algal densities (0.44, 0.56 and 0.77 g L(-1)). Once the optimal flocculation concentration had been established (40 mg L(-1) for all different biomasses densities) a more detailed analysis was performed in order to investigate if different pH (4.0, 7.7, and 10.0) could increase the flocculation efficiency of cationic starch. Highest flocculation efficiency without addition of Greenfloc 120 was obtained at pH 10, while in the presence of flocculant, the efficiency increased for all the tested pH values, with a maximum of 98% for pH 7.7 and 10. Cationic starch confirmed to be as an easy to use, efficient and cost-effective flocculant for harvesting of microalgae.

Bioaugmentation as a solution to increase methane production from an ammonia-rich substrate.

Ammonia-rich substrates inhibit the anaerobic digestion (AD) process and constitute the main reason for low energy recovery in full-scale reactors. It is estimated that many full-scale AD reactors are operating in ammonia induced "inhibited steady-state" with significant losses of the potential biogas production yield. To date there are not any reliable methods to alleviate the ammonia toxicity effect or to efficiently digest ammonia-rich waste. In the current study, bioaugmentation as a possible method to alleviate ammonia toxicity effect in a mesophilic continuously stirred-tank reactor (CSTR) operating under "inhibited steady state" was tested. A fast growing hydrogenotrophic methanogen (i.e., Methanoculleus bourgensis MS2(T)) was bioaugmented in the CSTR reactor at high ammonia levels (5 g NH4(+)-N L(-1)). A second CSTR reactor was used as control with no bioaugmentation. The results derived from this study clearly demonstrated a 31.3% increase in methane production yield in the CSTR reactor, at steady-state, after bioaugmentation. Additionally, high-throughput 16S rRNA gene sequencing analysis showed a 5-fold increase in relative abundance of Methanoculleus spp. after bioaugmentation. On the contrary to all methods used today to alleviate ammonia toxicity effect, the tested bioaugmentation process performed without interrupting the continuous operation of the reactor and without replacing the ammonia-rich feedstock.

An environmentally-friendly fluorescent method for quantification of lipid contents in yeast.

This study aimed at developing an efficient, fast and environmentally-friendly method to quantify neutral lipid contents in yeast. After optimising the fluorescence instrument parameters and influence of organic solvent concentrations, a new method to quantify neutral lipids in yeast based on fluorescence was demonstrated. Isopropanol and Nile red in concentrations of 5% (final volume%) and 500 µg/L, respectively, were added to washed cells suspended in potassium chloride phosphate buffered saline (PBSKCl). Fluorescence was measured after 10 min in the dark. Glyceryltrioleate was used as model lipid and the calibration curve showed linearity (R(2)=0.994) between 0.50 and 25 mg/L. Compared with traditional gravimetric analysis, the developed method is much faster and uses less organic solvents. Lipid contents determined by fluorescence and gravimetry were the same for some strains, but for other strains the lipid contents determined by fluorescence were less. This new method will therefore be suitable for fast screening purposes.

Bioaugmentation with an acetate-oxidising consortium as a tool to tackle ammonia inhibition of anaerobic digestion.

Ammonia is the major inhibitor of anaerobic digestion (AD) process in biogas plants. In the current study, the bioaugmentation of the ammonia tolerant SAO co-culture (i.e. Clostridium ultunense spp. nov. in association with Methanoculleus spp. strain MAB1) in a mesophilic up-flow anaerobic sludge blanket (UASB) reactor subjected to high ammonia loads was tested. The co-cultivation in fed-batch reactors of a fast-growing hydrogenotrophic methanogen (i.e. Methanoculleus bourgensis MS2(T)) with the SAO co-culture was also investigated. Results demonstrated that bioaugmentation of SAO co-culture in a UASB reactor was not possible most likely due to the slow maximum growth rate (µmax=0.007 h(-1)) of the culture caused by the methanogenic partner. The addition of M. bourgensis to SAO led to 42% higher growth rate (µmax=0.01 h(-1)) in fed-batch reactors. This indicates that methanogens were the slowest partners of the SAO co-culture and therefore were the limiting factor during bioaugmentation in the UASB reactor.

Microwave and thermal pretreatment as methods for increasing the biogas potential of secondary sludge from municipal wastewater treatment plants.

In the present study, the sludge was pretreated with microwave irradiation and low-temperature thermal method, both conducted under the same temperature range (30-100°C). Microwave pretreatment was found to be superior over the thermal treatment with respect to sludge solubilization and biogas production. Taking into account the specific energy demand of solubilization, the sludge pre-treated at 60-70°C by microwaves of 900 W was chosen for further experiments in continuous mode, which was more energetically sustainable compared to lower value (700 W) and thermal treatment. Continuous biogas reactor experiments indicated that pre-treated sludge (microwave irradiation: 900 W, temperature: 60-70°C) gave 35% more methane, compared to untreated sludge. Moreover, the results of this study clearly demonstrated that microwave pretreated sludge showed better degree of sanitation.

Extreme thermophilic ethanol production from rapeseed straw: using the newly isolated Thermoanaerobacter pentosaceus and combining it with Saccharomyces cerevisiae in a two-step process.

The newly isolated extreme thermophile Thermoanaerobacter pentosaceus was used for ethanol production from alkaline-peroxide pretreated rapeseed straw (PRS). Both the liquid and solid fractions of PRS were used. T. pentosaceus was able to metabolize the typical process inhibitors present in lignocellulosic hydrolysate, namely 5-hydroxymethyl furfural (HMF) and furfural, up to concentrations of 1 and 0.5 g L(-1) , respectively. Above these levels, xylose consumption was inhibited up to 70% (at 3.4 g-furfural L(-1) ) and 75% (at 3.4 g-HMF L(-1) ). T. pentosaceus was able to grow and produce ethanol directly from the liquid fraction of PRS, without any dilution or need for additives. However, when the hydrolysate was used undiluted the ethanol yield was only 37% compared to yield of the control, in which pure sugars in synthetic medium were used. The decrease of ethanol yield was attributed to the high amounts of salts resulting from the alkaline-peroxide pretreatment. Finally, a two-stage ethanol production process from PRS using Saccharomyces cerevisiae (utilization of hexoses in the first step) and T. pentosaceus (utilization of pentoses in the second step) was developed. Results showed that the two strains together could achieve up to 85% of the theoretical ethanol yield based on the sugar composition of the rapeseed straw, which was 14% and 50% higher compared to the yield with the yeast or the bacteria alone, respectively.

Effect of ammonium and acetate on methanogenic pathway and methanogenic community composition.

Methanogenesis from acetate (aceticlastic methanogenesis or syntrophic acetate oxidation (SAO) coupled with hydrogenotrophic methanogenesis) is the most important step for the biogas process. The major environmental factors influencing methanogenesis are volatile fatty acids, ammonia, pH, and temperature. In our study, the effect of acetate and ammonia concentration on the methanogenic pathway from acetate and on the methanogenic communities was elucidated in two experiments: one where inocula were gradually exposed to increasing concentrations of acetate or ammonia, and another with direct exposure to different ammonia concentrations. The methanogenic pathway was determined by following the production of (14) CH(4) and (14) CO(2) from acetate labeled in the methyl group (C-2). Microbial communities' composition was determined by fluorescence in situ hybridization. Upon acclimatization to acetate and ammonia, thermophilic cultures clearly shifted their acetate bioconversion pathway from SAO with subsequent hydrogenotrophic methanogenesis (mediated by Methanobacteriales spp. and/or Methanomicrobiales spp.) to aceticlastic methanogenesis (mediated by Methanosarcinaceae spp.). On the contrary, acclimatization process resulted in no pathway shift with the mesophilic acclimatized culture. When nonacclimatized thermophilic culture was exposed to high ammonia levels (7 g NH4 +-N L(-1)), aceticlastic Methanosarcinaceae spp. was found to be the dominant methanogen.

Life cycle assessment of biofuel production from brown seaweed in Nordic conditions.

The use of algae for biofuel production is expected to play an important role in securing energy supply in the next decades. A consequential life cycle assessment (LCA) and an energy analysis of seaweed-based biofuel production were carried out in Nordic conditions to document and improve the sustainability of the process. Two scenarios were analyzed for the brown seaweed (Laminaria digitata), namely, biogas production (scenario 1) and bioethanol+biogas production (scenario 2). Potential environmental impact categories under investigation were Global Warming, Acidification and Terrestrial Eutrophication. The production of seaweed was identified to be the most energy intensive step. Scenario 1 showed better performance compared to scenario 2 for all impact categories, partly because of the energy intensive bioethanol separation process and the consequently lower overall efficiency of the system. For improved environmental performance, focus should be on optimization of seaweed production, bioethanol distillation, and management of digestate on land.

Thermoanaerobacter pentosaceus sp. nov., an anaerobic, extremely thermophilic, high ethanol-yielding bacterium isolated from household waste.

An extremely thermophilic, xylanolytic, spore-forming and strictly anaerobic bacterium, strain DTU01(T), was isolated from a continuously stirred tank reactor fed with xylose and household waste. Cells stained Gram-negative and were rod-shaped (0.5-2 µm in length). Spores were terminal with a diameter of approximately 0.5 µm. Optimal growth occurred at 70 °C and pH 7, with a maximum growth rate of 0.1 h(-1). DNA G+C content was 34.2 mol%. Strain DTU01(T) could ferment arabinose, cellobiose, fructose, galactose, glucose, lactose, mannitol, mannose, melibiose, pectin, starch, sucrose, xylan, yeast extract and xylose, but not cellulose, Avicel, inositol, inulin, glycerol, rhamnose, acetate, lactate, ethanol, butanol or peptone. Ethanol was the major fermentation product and a maximum yield of 1.39 mol ethanol per mol xylose was achieved when sulfite was added to the cultivation medium. Thiosulfate, but not sulfate, nitrate or nitrite, could be used as electron acceptor. On the basis of 16S rRNA gene sequence similarity, strain DTU01(T) was shown to be closely related to Thermoanaerobacter mathranii A3(T), Thermoanaerobacter italicus Ab9(T) and Thermoanaerobacter thermocopriae JT3-3(T), with 98-99 % similarity. Despite this, the physiological and phylogenetic differences (DNA G+C content, substrate utilization, electron acceptors, phylogenetic distance and isolation site) allow for the proposal of strain DTU01(T) as a representative of a novel species within the genus Thermoanaerobacter, for which the name Thermoanaerobacter pentosaceus sp. nov. is proposed, with the type strain DTU01(T) ( = DSM 25963(T) = KCTC 4529(T) = VKM B-2752(T) = CECT 8142(T)).

Biohydrogen production from arabinose and glucose using extreme thermophilic anaerobic mixed cultures.

BACKGROUND: Second generation hydrogen fermentation technologies using organic agricultural and forestry wastes are emerging. The efficient microbial fermentation of hexoses and pentoses resulting from the pretreatment of lingocellulosic materials is essential for the success of these processes. RESULTS: Conversion of arabinose and glucose to hydrogen, by extreme thermophilic, anaerobic, mixed cultures was studied in continuous (70°C, pH 5.5) and batch (70°C, pH 5.5 and pH 7) assays. Two expanded granular sludge bed (EGSB) reactors, Rarab and Rgluc, were continuously fed with arabinose and glucose, respectively. No significant differences in reactor performance were observed for arabinose and glucose organic loading rates (OLR) ranging from 4.3 to 7.1 kgCOD m-3 d-1. However, for an OLR of 14.2 kgCOD m-3 d-1, hydrogen production rate and hydrogen yield were higher in Rarab than in Rgluc (average hydrogen production rate of 3.2 and 2.0 LH2 L-1 d-1 and hydrogen yield of 1.10 and 0.75 molH2 mol-1substrate for Rarab and Rgluc, respectively). Lower hydrogen production in Rgluc was associated with higher lactate production. Denaturing gradient gel electrophoresis (DGGE) results revealed no significant difference on the bacterial community composition between operational periods and between the reactors. Increased hydrogen production was observed in batch experiments when hydrogen partial pressure was kept low, both with arabinose and glucose as substrate. Sugars were completely consumed and hydrogen production stimulated (62% higher) when pH 7 was used instead of pH 5.5. CONCLUSIONS: Continuous hydrogen production rate from arabinose was significantly higher than from glucose, when higher organic loading rate was used. The effect of hydrogen partial pressure on hydrogen production from glucose in batch mode was related to the extent of sugar utilization and not to the efficiency of substrate conversion to hydrogen. Furthermore, at pH 7.0, sugars uptake, hydrogen production and yield were higher than at pH 5.5, with both arabinose and glucose as substrates.

Effect of continuous oleate addition on microbial communities involved in anaerobic digestion process.

In the present study, the microbial diversity in anaerobic reactors, continuously exposed to oleate, added to a manure reactor influent, was investigated. Relative changes in archaeal community were less remarkable in comparison to changes in bacterial community indicating that dominant archaeal composition remained relatively stable. Majority of the analyzed bacterial amplicons were phylogenetically affiliated with uncultured bacteria belonging to Firmicutes, Bacteroidetes, Proteobacteria and Thermotogae phyla. Bacterial community changes in response to oleate addition resulted in a less diverse bacterial consortium related to functional specialization of the species towards oleate degradation. For the archaeal domain, the sequences were affiliated within Euryarchaeota phylum with three major groups (Methanosarcina, Methanosaeta and Methanobacterium genera). Results obtained in this study deliver a comprehensive picture on oleate degrading microbial communities in high organic strength wastewater. The findings might be utilized for development of strategies for biogas production from lipid-riched wastes.

Effect of xylose and nutrients concentration on ethanol production by a newly isolated extreme thermophilic bacterium.

An extreme thermophilic ethanol-producing strain was isolated from an ethanol high-yielding mixed culture, originally isolated from a hydrogen producing reactor operated at 70 degrees C. Ethanol yields were assessed with increasing concentrations of xylose, up to 20 g/l. The ability of the strain to grow without nutrient addition (yeast extract, peptone and vitamins) was also assessed. The maximum ethanol yield achieved was 1.28 mol ethanol/mol xylose consumed (77% of the theoretical yield), at 2 g/l of initial xylose concentration. The isolate was able to grow and produce ethanol as the main fermentation product under most of the conditions tested, including in media lacking vitamins, peptone and yeast extract. The results indicate that this new organism is a promising candidate for the development of a second generation bio-ethanol production process.

Long-term effect of inoculum pretreatment on fermentative hydrogen production by repeated batch cultivations: homoacetogenesis and methanogenesis as competitors to hydrogen production.

Long-term effects of inoculum pretreatments (heat, acid, loading-shock) on hydrogen production from glucose under different temperatures (37 °C, 55 °C) and initial pH (7 and 5.5) were studied by repeated batch cultivations. Results obtained showed that it was necessary to investigate the long-term effect of inoculum pretreatment on hydrogen production since pretreatments may just temporarily inhibit the hydrogen consuming processes. After long-term cultivation, pretreated inocula did not enhance hydrogen production compared to untreated inocula under mesophilic conditions (initial pH 7 and pH 5.5) and thermophilic conditions (initial pH 7). However, pretreatment could inhibit lactate production and lead to higher hydrogen yield under thermophilic conditions at initial pH 5.5. The results further demonstrated that inoculum pretreatment could not permanently inhibit either methanogenesis or homoacetogenesis, and methanogenesis and homoacetogenesis could only be inhibited by proper control of fermentation pH and temperature. Methanogenic activity could be inhibited at pH lower than 6, both under mesophilic and thermophilic conditions, while homoacetogenic activity could only be inhibited under thermophilic condition at initial pH 5.5. Microbial community analysis showed that pretreatment did not affect the dominant bacteria. The dominant bacteria were Clostridium butyricum related organisms under mesophilic condition (initial pH 7 and 5.5), Thermoanaerobacterium sp. related organisms under thermophilic condition (initial pH 7), and Thermoanaerobacterium thermosaccharolyticum related organisms under thermophilic condition (initial pH 5.5). Results from this study clearly indicated that the long-term effects of inoculum pretreatments on hydrogen production, methanogenesis, homoacetogenesis and dominant bacteria were dependent on fermentation temperature and pH.

Biomethanation and its potential.

Biomethanation is a process by which organic material is microbiologically converted under anaerobic conditions to biogas. Three main physiological groups of microorganisms are involved: fermenting bacteria, organic acid oxidizing bacteria, and methanogenic archaea. Microorganisms degrade organic matter via cascades of biochemical conversions to methane and carbon dioxide. Syntrophic relationships between hydrogen producers (acetogens) and hydrogen scavengers (homoacetogens, hydrogenotrophic methanogens, etc.) are critical to the process. Determination of practical and theoretical methane potential is very important for design for optimal process design, configuration, and effective evaluation of economic feasibility. A wide variety of process applications for biomethanation of wastewaters, slurries, and solid waste have been developed. They utilize different reactor types (fully mixed, plug-flow, biofilm, UASB, etc.) and process conditions (retention times, loading rates, temperatures, etc.) in order to maximize the energy output from the waste and also to decrease retention time and enhance process stability. Biomethanation has strong potential for the production of energy from organic residues and wastes. It will help to reduce the use of fossil fuels and thus reduce CO(2) emission.

Enhanced bioenergy recovery from rapeseed plant in a biorefinery concept.

The present study investigated the utilization of the whole rapeseed plant (seed and straw) for multi-biofuels production in a biorefinery concept. Results showed that bioethanol production from straw was technically feasible with ethanol yield of 0.15 g ethanol/g dry straw after combined alkaline peroxide and stream pretreatment. The byproducts (rapeseed cake, glycerol, hydrolysate and stillage) were evaluated for hydrogen and methane production. In batch experiments, the energy yields from each feedstock for, either methane production alone or for both hydrogen and methane, were similar. However, results from continuous experiments demonstrated that the two-stage hydrogen and methane fermentation process could work stably at organic loading rate up to 4.5 gVS/(Ld), while the single-stage methane production process failed. The energy recovery efficiency from rapeseed plant increased from 20% in the conventional biodiesel process to 60% in the biorefinery concept, by utilization of the whole rapeseed plant for biodiesel, bioethanol, biohydrogen and methane production.

Engineered heat treated methanogenic granules: a promising biotechnological approach for extreme thermophilic biohydrogen production.

In the present study, two granular systems were compared in terms of hydrogen production rate, stability and bacterial diversity under extreme thermophilic conditions (70 degrees C). Two EGSB reactors were individually inoculated with heat treated methanogenic granules (HTG) and HTG amended with enrichment culture with high capacity of hydrogen production (engineered heat treated methanogenic granules - EHTG), respectively. The reactor inoculated with EHTG (R(EHTG)) attained a maximum production rate of 2.7l H(2)l(-1)day(-1) in steady state. In comparison, the R(HTG) containing the HTG granules was very unstable, with low hydrogen productions and only two peaks of hydrogen (0.8 and 1.5l H(2)l(-1)day(-1)). The presence of active hydrogen producers in the R(EHTG) system during the reactor start-up resulted in the development of an efficient H(2)-producing bacterial community. The results showed that "engineered inocula" where known hydrogen producers are co-inoculated with HTG is an efficient way to start up biohydrogen-producing reactors.