Kinetic exploration of intracellular nitrate storage in marine microalgae.

In this study, a recently developed model accounting for intracellular nitrate storage kinetics was thoroughly studied to understand and compare the storage capacity of Phaeodactylum tricornutum and Amphora coffeaeformis. In the first stage the identifiability of the biokinetic parameters was examined. Next, the kinetic model was calibrated for both microalgal species based on experimental observations during batch growth experiments. Two kinetic parameters were calibrated, namely the maximum specific growth rate [Formula: see text] and the nitrate storage rate ([Formula: see text]). A significant difference was observed for the nitrate storage rate between both species. For P. tricornutum, the nitrate storage rate was much higher ([Formula: see text] = 0.036 m3 g-1 DW d-1) compared to A. coffeaeformis ([Formula: see text] = 0.0004 m3 g-1 DW d-1). This suggests that P. tricornutum has a more efficient nitrate uptake ability and intracellular nitrate storage capacity and also indicates the need for determination of [Formula: see text] in order to quantify nitrate storage.

Laccase enzyme detoxifies hydrolysates and improves biogas production from hemp straw and miscanthus.

The impact of various phenolic compounds, vanillic acid, ferulic acid, p-coumaric acid and 4-hydroxybenzoic acid on anaerobic digestion of lignocellulosic biomass (hemp straw and miscanthus) was studied. Such phenolic compounds have been known to inhibit biogas production during anaerobic digestion. The different phenolic compounds were added in various concentrations: 0, 100, 500, 1000 and 2000mg/L. A difference in inhibition of biomethane production between the phenolic compounds was noted. Hydrolysis rate, during anaerobic digestion of miscanthus was inhibited up to 50% by vanillic acid, while vanillic acid had no influence on the initial rate of biogas production during the anaerobic digestion of hemp straw. Miscanthus has a higher lignin concentration (12-30g/100gDM) making it less accessible for degradation, and in combination with phenolic compounds released after harsh pretreatments, it can cause severe inhibition levels during the anaerobic digestion, lowering biogas production. To counter the inhibition, lignin degrading enzymes can be used to remove or degrade the inhibitory phenolic compounds. The interaction of laccase and versatile peroxidase individually with the different phenolic compounds was studied to have insight in the polymerization of inhibitory compounds or breakdown of lignocellulose. Hemp straw and miscanthus were incubated with 0, 100 and 500mg/L of the different phenolic compounds for 0, 6 and 24h and pretreated with the lignin degrading enzymes. A laccase pretreatment successfully detoxified the substrate, while versatile peroxidase however was inhibited by 100mg/L of each of the individual phenolic compounds. Finally a combination of enzymatic detoxification and subsequent biogas production showed that a decrease in phenolic compounds by laccase treatment can considerably lower the inhibition levels of the biogas production.

Electricity-assisted production of caproic acid from grass.

BACKGROUND: Medium chain carboxylic acids, such as caproic acid, are conventionally produced from food materials. Caproic acid can be produced through fermentation by the reverse ß-oxidation of lactic acid, generated from low value lignocellulosic biomass. In situ extraction of caproic acid can be achieved by membrane electrolysis coupled to the fermentation process, allowing recovery by phase separation. RESULTS: Grass was fermented to lactic acid in a leach-bed-type reactor, which was then further converted to caproic acid in a secondary fermenter. The lactic acid concentration was 9.36 ± 0.95 g L-1 over a 33-day semi-continuous operation, and converted to caproic acid at pH 5.5-6.2, with a concentration of 4.09 ± 0.54 g L-1 during stable production. The caproic acid product stream was extracted in its anionic form, concentrated and converted to caproic acid by membrane electrolysis, resulting in a >70 wt% purity solution. In a parallel test exploring the upper limits of production rate through cell retention, we achieved the highest reported caproic acid production rate to date from a lignocellulosic biomass (grass, via a coupled process), at 0.99 ± 0.02 g L-1 h-1. The fermenting microbiome (mainly consisting of Clostridium IV and Lactobacillus) was capable of producing a maximum caproic acid concentration of 10.92 ± 0.62 g L-1 at pH 5.5, at the border of maximum solubility of protonated caproic acid. CONCLUSIONS: Grass can be utilized as a substrate to produce caproic acid. The biological intermediary steps were enhanced by separating the steps to focus on the lactic acid intermediary. Notably, the pipeline was almost completely powered through electrical inputs, and thus could potentially be driven from sustainable energy without need for chemical input.Graphical abstractMicrobial and electrochemical production of lactic acid, caproic acid and decane from grass.

Microalgal bacterial flocs treating paper mill effluent: A sunlight-based approach for removing carbon, nitrogen, phosphorus, and calcium.

Treatment of upflow anaerobic sludge blanket (UASB) effluent from a paper mill in aerated activated sludge reactors involves high aeration costs. Moreover, this calcium-rich effluent leads to problematic scale formation. Therefore, a novel strategy for the aerobic treatment of paper mill UASB effluent in microalgal bacterial floc sequencing batch reactors (MaB-floc SBRs) is proposed, in which oxygen is provided via photosynthesis, and calcium is removed via bio-mineralization. Based on the results of batch experiments in the course of this study, a MaB-floc SBR was operated at an initial neutral pH. This SBR removed 58±21% organic carbon, 27±8% inorganic carbon, 77±5% nitrogen, 73±2% phosphorus, and 27±11% calcium. MaB-flocs contained 10±3% calcium, including biologically-influenced calcite crystals. The removal of calcium and inorganic carbon by MaB-flocs significantly decreased when inhibiting extracellular carbonic anhydrase (CA), an enzyme that catalyses the hydration and dehydration of CO2. This study demonstrates the potential of MaB-floc SBRs for the alternative treatment of calcium-rich paper mill effluent, and highlights the importance of extracellular CA in this treatment process.

Technical potential of microalgal bacterial floc raceway ponds treating food-industry effluents while producing microalgal bacterial biomass: An outdoor pilot-scale study.

To replace costly mechanical aeration by photosynthetical aeration, upflow anaerobic sludge blanket (UASB) effluent of food-industry was treated in an outdoor MaB-floc raceway pond. Photosynthetic aeration was sufficient for nitrification, but the raceway effluent quality was below current discharge limits, despite the high hydraulic retention time (HRT) of 35days. Hereafter, conventional activated sludge (CAS) effluent of food-industry was treated in this pond to recover phosphorus. The two-day HRT results in a more realistic pond area, but the phosphorus removal efficiency was low (20%). High biomass productivities were obtained, i.e. 31.3 and 24.9ton total suspended solids hapond(-1)year(-1) for UASB and CAS effluent, respectively. Bioflocculation enabled successful harvesting of CAS effluent-fed MaB-flocs by settling and filtering at 150-250µm to 22.7% total solids. To conclude, MaB-floc raceway ponds cannot be recommended as the sole treatment for these food-industry effluents, but huge potential lies in added-value biomass production.

Acetate accumulation enhances mixed culture fermentation of biomass to lactic acid.

Lactic acid is a high-in-demand chemical, which can be produced through fermentation of lignocellulosic feedstock. However, fermentation of complex substrate produces a mixture of products at efficiencies too low to justify a production process. We hypothesized that the background acetic acid concentration plays a critical role in lactic acid yield; therefore, its retention via selective extraction of lactic acid or its addition would improve overall lactic acid production and eliminate net production of acetic acid. To test this hypothesis, we added 10 g/L of acetate to fermentation broth to investigate its effect on products composition and concentration and bacterial community evolution using several substrate-inoculum combinations. With rumen fluid inoculum, lactate concentrations increased by 80 ± 12 % (cornstarch, p < 0.05) and 16.7 ± 0.4 % (extruded grass, p < 0.05) while with pure culture inoculum (Lactobacillus delbrueckii and genetically modified (GM) Escherichia coli), a 4 to 23 % increase was observed. Using rumen fluid inoculum, the bacterial community was enriched within 8 days to >69 % lactic acid bacteria (LAB), predominantly Lactobacillaceae. Higher acetate concentration promoted a more diverse LAB population, especially on non-inoculated bottles. In subsequent tests, acetate was added in a semi-continuous percolation system with grass as substrate. These tests confirmed our findings producing lactate at concentrations 26 ± 5 % (p < 0.05) higher than the control reactor over 20 days operation. Overall, our work shows that recirculating acetate has the potential to boost lactic acid production from waste biomass to levels more attractive for application.

Effect of enzymatic pretreatment of various lignocellulosic substrates on production of phenolic compounds and biomethane potential.

Pretreatment of lignocellulosic biomass is necessary to enhance the hydrolysis, which is the rate-limiting step in biogas production. Laccase and versatile peroxidase are enzymes known to degrade lignin. Therefore, the impact of enzymatic pretreatment was studied on a variety of biomass. A significant higher release in total phenolic compounds (TPC) was observed, never reaching the inhibiting values for anaerobic digestion. The initial concentration of TPC was higher in the substrates containing more lignin, miscanthus and willow. The anaerobic digestion of these two substrates resulted in a significant lower biomethane production (68.8-141.7 Nl/kg VS). Other substrates, corn stover, flax, wheat straw and hemp reached higher biomethane potential values (BMP), between 241 and 288 Nl/kg VS. Ensilaged maize reached 449 Nl/kg VS, due to the ensilation process, which can be seen as a biological and acid pretreatment. A significant relation (R(2) = 0.89) was found between lignin content and BMP.

Low temperature calcium hydroxide treatment enhances anaerobic methane production from (extruded) biomass.

Ca(OH)2 treatment was applied to enhance methane yield. Different alkali concentration, incubation temperature and duration were evaluated for their effect on methane production and COD conversion efficiency from (non-)extruded biomass during mesophilic anaerobic digestion at lab-scale. An optimum Ca(OH)2 pretreatment for grass is found at 7.5% lime loading at 10°C for 20h (37.3% surplus), while mild (50°C) and high temperatures perform sub-optimal. Ca(OH)2 post-treatment after fast extrusion gives an additional surplus compared to extruded material of 15.2% (grass), 11.2% (maize straw) and 8.2% (sprout stem) regarding methane production. COD conversion improves accordingly, with additional improvements of 10.3% (grass), 9.0% (maize straw) and 6.8% (sprout stem) by Ca(OH)2 post-treatment. Therefore, Ca(OH)2 pretreatment and post-treatment at low temperature generate an additional effect regarding methane production and COD conversion efficiency. Fast extrusion gives a higher energy efficiency ratio compared to slow extrusion.

Impact of enzymatic pretreatment on corn stover degradation and biogas production.

Corn stover is an agricultural residue consisting of lignocellulose, cellulose and hemicellulose polymers, sheeted in a lignin barrier. Corn stover can be used as feedstock for biogas production. Previous studies have shown biological pretreatment of lignocellulose materials can increase digestibility of the substrate improving hydrolysis, the rate-limiting step in biogas production. The impact of pretreating with different enzymes (laccase, manganese peroxidase and versatile peroxidase) and different incubation times, (0, 6 and 24 h) was studied. The effect on the matrix and biomethane production was determined. Pretreatments did not yield high concentrations of phenolic compounds, inhibitors of biogas production. The laccase enzyme showed an increase in biomethane production of 25% after 24 h of incubation. Pretreatment with peroxidase enzymes increased biomethane production with 17% after 6 h of incubation. As such it can be concluded that by introducing the different enzymes at different stages during pretreatment an increased biomethane production can be obtained.

Effect of liquid hot water pre-treatment on sugarcane press mud methane yield.

Sugarcane press mud was pretreated by liquid hot water (LHW) at different temperatures (140-210 °C) and pre-treatment times (5-20 min) in order to assess the effects on the chemical oxygen demand (COD) solubilisation, inhibitors formation and methane yield. The experimental results showed that a high degree of biomass solubilisation was possible using LHW. Higher methane yields were obtained at lower severities (log(Ro) = 2.17-2.77) with (i) mild temperatures (140-150 °C) and long contact times (12.5 min, 20 min) or (ii) mild temperatures (175 °C) with short contact time (2 min). The highest increase in methane yield (up to 63%) compared to the untreated press mud was found at 150 °C for 20 min. At temperatures of 200 °C and 210 °C, low methane efficiency was attributed to the possible formation of refractory compounds through the Maillard reaction.

Treatment of industrial wastewaters by microalgal bacterial flocs in sequencing batch reactors.

Microalgal bacterial flocs in sequencing batch reactors (MaB-floc SBRs) represent a novel approach to wastewater treatment. In this approach, mechanical aeration is replaced by photosynthetic aeration and MaB-floc settling separates the treated wastewater from the produced biomass. However, its technical potential for industrial wastewaters needs to be shown. Therefore, wastewaters of aquaculture, manure treatment, food-processing and chemical industry were treated in MaB-floc SBRs. This treatment resulted in significantly different nutrient removal rates and effluent qualities among wastewaters. A high MaB-floc production was obtained for all wastewaters, ranging from 0.14 to 0.26g total suspended solids Lreactor(-1)day(-1). A major advantage of MaB-flocs is the harvesting via a filter press with a large pore size of 200µm, resulting in MaB-floc recoveries of 79-99% and cakes containing 12-21% dry matter. These results may contribute to evolving MaB-floc SBRs as a valuable remediation strategy, especially for aquaculture and food-processing wastewaters.

Up-scaling aquaculture wastewater treatment by microalgal bacterial flocs: from lab reactors to an outdoor raceway pond.

Sequencing batch reactors with microalgal bacterial flocs (MaB-floc SBRs) are a novel approach for photosynthetic aerated wastewater treatment based on bioflocculation. To assess their technical potential for aquaculture wastewater treatment in Northwest Europe, MaB-floc SBRs were up-scaled from indoor photobioreactors of 4 L over 40 and 400 L to a 12 m(3) outdoor raceway pond. Scale-up decreased the nutrient removal efficiencies with a factor 1-3 and the volumetric biomass productivities with a factor 10-13. Effluents met current discharge norms, except for nitrite and nitrate. Flue gas sparging was needed to decrease the effluent pH. Outdoor MaB-flocs showed enhanced settling properties and an increased ash and chlorophyll a content. Bioflocculation enabled successful harvesting by gravity settling and dewatering by filtering at 150-250 µm. Optimisation of nitrogen removal and biomass valorisation are future challenges towards industrial implementation of MaB-floc SBRs for aquaculture wastewater treatment.

Thermo-chemical pre-treatment to solubilize and improve anaerobic biodegradability of press mud.

Different pre-treatment severities by thermo-alkaline conditions (100°C, Ca(OH)2) on press mud were evaluated for different pre-treatment time and lime loading. COD solubilization and the methane yield enhancement were assessed. The biochemical methane potential was determined in batch assays under mesophilic conditions (37±1°C). The best pre-treatment resulted in a surplus of 72% of methane yield, adding 10g Ca(OH)2 100g(-1)TS(-1) for 1h. Pre-treatment also increased the COD solubilization, but the optimal severity for COD solubilization as determined by response surface methodology did not ensure the highest methane production. Inhibitory effects on anaerobic digestion were noticed when the severity was increased. These results demonstrate the relevance of thermo-alkaline pre-treatment severity in terms of both lime loading and pre-treatment time to obtain optimal anaerobic biodegradability of lignocellulosic biomass from press mud.

Flue gas compounds and microalgae: (bio-)chemical interactions leading to biotechnological opportunities.

Flue gases are a resource yet to be fully utilised in microalgal biotechnology, not only to moderate the anthropogenic effects on our climate, but also to steer microalgal resource management towards innovative applications of microalgal biomass compounds. These gases, both untreated and treated into current discharge standards, contain CO2, N2, H2O, O2, NOx, SOx, CxHy, CO, particulate matter, halogen acids and heavy metals. To better steer and engineer flue gas-fed microalgal cultures, all these compounds need to be considered. Therefore, here, we review (i) the chemical composition and treatment technologies of flue gas, (ii) the uptake pathways and removal of the different compounds in microalgae reactors, and (iii) the tolerance and effects on microalgae of all flue gas compounds. By emphasising the interactions between microalgae and flue gas compounds, we envisage new pathways for microalgal biomass valorisation such as enzyme production for environmental technology, novel biogas production and biosequestration of minerals. Furthermore, we highlight fundamental and applied research niches that merit further investigation.

Bioflocculation of microalgae and bacteria combined with flue gas to improve sewage treatment.

Although microalgae are promising for a cradle-to-cradle design approach of sewage treatment, their application is hampered by high harvesting costs and low C:N ratios of sewage. Therefore, the potential of microalgal bacterial flocs (MaB-flocs) was investigated for the secondary treatment of sewage supplemented with different flue gas flow rates (FGFRs) from a coal power plant. Effluent (N, P, turbidity and pH) and off gas discharge levels (NO(x), SO(x)) met the European discharge limits with a hydraulic retention time of only 0.67 days and an FGFR of 0.6Lh(-1) (0.0025 vvm). This FGFR provided sufficient carbon and resulted in removal efficiencies of 48 ± 7% CO(2), 87 ± 5% NO(x) and 99 ± 1% SO(2). MaB-flocs settled fast reaching up to a density of 19 g VSSL(-1). High biomass productivities (0.18 gL(-1)day(-1)) were obtained under a low light intensity. This successful reactor performance indicates the large potential for the industrial application of MaB-flocs for flue gas sparged sewage treatment.

Microalgal bacterial floc properties are improved by a balanced inorganic/organic carbon ratio.

Microalgal bacterial floc (MaB-floc) reactors have been suggested as a more sustainable secondary wastewater treatment. We investigated whether MaB-flocs could be used as tertiary treatment. Tertiary influent has a high inorganic/organic carbon ratio, depending on the efficiency of the secondary treatment. In this study, the effect of this inorganic/organic carbon ratio on the MaB-flocs performance was determined, using three sequencing batch photobioreactors. The MaB-flocs were fed with synthetic wastewater containing 84, 42, and 0 mg L(-1) C-KHCO(3) supplemented with 0, 42, 84 mg L(-1) C-sucrose, respectively, representing inorganic versus organic carbon. Bicarbonate significantly decreased the autotrophic index of the MaB-flocs and resulted in poorly settling flocs. Moreover, sole bicarbonate addition led to a high pH of 9.5 and significant lower nitrogen removal efficiencies. Sucrose without bicarbonate resulted in good settling MaB-flocs, high nitrogen removal efficiencies and neutral pH levels. Despite the lower chlorophyll a content of the biomass and the lower in situ oxygen concentration, 92-96% of the soluble COD-sucrose was removed. This study shows that the inorganic/organic carbon ratio of the wastewater is of major importance and that organic carbon is requisite to guarantee a good performance of the MaB-flocs for wastewater treatment.

Calibration and statistical analysis of a simplified model for the anaerobic digestion of solid waste.

Modelling is increasingly used for optimizing environmental treatment processes such as anaerobic digestion. It allows problems such as instability of the process to be solved by predicting various scenarios. The anaerobic digestion model No. 1 (ADM1) is accepted worldwide as the standard model for the description of anaerobic digestion. However, it is sophisticated and complex, so it is not user friendly. Therefore, a mathematical method was developed that allows the calculation of the reactor pH, as well as the biogas flow rate (Q) and composition (expressed as the CO2 partial pressure, pCO2), based on a small number of widely available analyses such as chemical oxygen demand and total organic carbon. Furthermore, the ADM1 model was originally designed for anaerobic digestion of wastewater. In this work, the ADM1 model is evaluated for the first time for application in the modelling of solid waste digestion. This evaluation was performed in two steps. First, a list of experimentally available lab-scale data (pH and Q) was grouped according to the composition and origin of the treated solid waste (e.g. manure or vegetable waste). For each group the developed model for the calculation of pH, Q and pCO2 was calibrated with this lab-scale data. After calibration, the model was validated with additional experimental results. It could be demonstrated statistically that the model was able to predict the experimental results, although the confidence region was rather large.

Quantifying community dynamics of nitrifiers in functionally stable reactors.

A sequential batch reactor (SBR) and a membrane bioreactor (MBR) were inoculated with the same sludge from a municipal wastewater treatment plant, supplemented with ammonium, and operated in parallel for 84 days. It was investigated whether the functional stability of the nitrification process corresponded with a static ammonia-oxidizing bacterial (AOB) community. The SBR provided complete nitrification during nearly the whole experimental run, whereas the MBR showed a buildup of 0 to 2 mg nitrite-N liter(-1) from day 45 until day 84. Based on the denaturing gradient gel electrophoresis profiles, two novel approaches were introduced to characterize and quantify the community dynamics and interspecies abundance ratios: (i) the rate of change [Delta(t)((week))] parameter and (ii) the Pareto-Lorenz curve distribution pattern. During the whole sampling period, it was observed that neither of the reactor types maintained a static microbial community and that the SBR evolved more gradually than the MBR, particularly with respect to AOB (i.e., average weekly community changes of 12.6% +/- 5.2% for the SBR and 24.6% +/- 14.3% for the MBR). Based on the Pareto-Lorenz curves, it was observed that only a small group of AOB species played a numerically dominant role in the nitritation of both reactors, and this was true especially for the MBR. The remaining less dominant species were speculated to constitute a reserve of AOB which can proliferate to replace the dominant species. The value of these parameters in terms of tools to assist the operation of activated-sludge systems is discussed.

Poly-beta-hydroxybutyrate-accumulating bacteria protect gnotobiotic Artemia franciscana from pathogenic Vibrio campbellii.

A poly-beta-hydroxybutyrate (PHB)-accumulating enrichment culture was obtained using activated sludge from a polyphosphate-accumulating reactor as inoculum. PHB accumulated by the enrichment culture significantly enhanced the survival of Artemia nauplii, infected with the virulent pathogen Vibrio campbellii LMG 21363. A strain was isolated from the enrichment culture, based on its ability to accumulate PHB, and 16S rRNA gene sequencing of the isolate revealed 99% sequence similarity to Brachymonas denitrificans AS-P1. The isolate, named PHB2, showed good PHB-accumulating activity (up to 32% of the cell dry weight). PHB accumulated by isolate PHB2 was able to protect Artemia completely from the V. campbellii strain. Our data indicate that PHB-accumulating bacteria, such as B. denitrificans PHB2, could be used as an an effective and economically interesting alternative strategy to control infections in aquaculture.

Reactivation of aerobic and anaerobic ammonium oxidizers in OLAND biomass after long-term storage.

The biomass of an oxygen-limited autotrophic nitrification/denitrification (OLAND) biofilm reactor was preserved in various ways to find a storage method for both aerobic and anaerobic ammonium-oxidizing bacteria (AerAOB and AnAOB). Storage occurred at -20 degrees C with and without glycerol as cryoprotectant and at 4 and 20 degrees C with and without nitrate as redox buffer. After 2 and 5 months, reactivation of AerAOB and AnAOB was achieved with the biomass stored at 4 degrees C with and without nitrate and at 20 degrees C with nitrate. Moreover, the presence of the AerAOB and AnAOB was confirmed with fluorescent in situ hybridization (FISH). Preservation in a nitrate environment resulted in a lag phase for the AnAOB reactivation. The supplied nitrate was denitrified during storage, and a real-time polymerase chain reaction with nitrifying and denitrifying genes allowed to estimate that at least 1.0 to 6.0% of the OLAND biofilm consisted of denitrifiers. It was concluded that reactivation after long-term storage is possible and that preservation at 4 degrees C without nitrate addition is the recommended storage technique. The possibility to store OLAND biomass will facilitate research on AnAOB and can overcome larger-scale start-up and inhibition problems of novel nitrogen processes involving AnAOB.