Effect of substrate feeding frequencies on the methane production and microbial communities of laboratory-scale anaerobic digestion reactors

Even though full-scale digesters have been designed based on laboratory-scale tests, the substrate feeding modes of laboratory-scale tests might be different from those of full-scale digesters. The effect of substrate feeding frequencies on the performance and microbial community of laboratory-scale anaerobic digestion reactors was investigated. Feeding frequencies of twice a day, once a day, and every two days were tested in three 2-L reactors with an organic loading rate of 0.5 g-glucose/L/day under mesophilic condition. According to the results of this study, all the reactors showed similar methane production rates and SCOD removal efficiencies after sufficient time of acclimation, but frequently feeding promoted more stable digestion. Although there was no significant difference in microbial diversities from pyrosequencing analyses, the changes of archaeal community composition were observed. The decrease in feeding frequency appeared to cause shifts from acetoclastic methanogens affiliated with Methanosaeta to H₂-utilizing methanogens. The increase of Methanosaeta at a frequently feeding might contribute to the stability of reactor operation. Since this study uses glucose as the substrate, there is still possibility of different results for more complex substrates, such as sludge, food waste, etc.     

Microbial diversity and dynamics during methane production from municipal solid waste

The objectives of this study were to characterize development of bacterial and archaeal populations during biodegradation of municipal solid waste (MSW) and to link specific methanogens to methane generation. Experiments were conducted in three 0.61-m-diameter by 0.90-m-tall laboratory reactors to simulate MSW bioreactor landfills. Pyrosequencing of 16S rRNA genes was used to characterize microbial communities in both leachate and solid waste. Microbial assemblages in effluent leachate were similar between reactors during peak methane generation. Specific groups within the Bacteroidetes and Thermatogae phyla were present in all samples and were particularly abundant during peak methane generation. Microbial communities were not similar in leachate and solid fractions assayed at the end of reactor operation; solid waste contained a more abundant bacterial community of cellulose-degrading organisms (e.g., Firmicutes). Specific methanogen populations were assessed using quantitative polymerase chain reaction. Methanomicrobiales, Methanosarcinaceae, and Methanobacteriales were the predominant methanogens in all reactors, with Methanomicrobiales consistently the most abundant. Methanogen growth phases coincided with accelerated methane production, and cumulative methane yield increased with increasing total methanogen abundance. The difference in methanogen populations and corresponding methane yield is attributed to different initial cellulose and hemicellulose contents of the MSW. Higher initial cellulose and hemicellulose contents supported growth of larger methanogen populations that resulted in higher methane yield.     

Utilisation of single added fatty acids by consortia of digester sludge in batch culture

Inocula derived from an anaerobic digester were used to study (i) their potential for methane production and (ii) the utilisation rates of different short chain fatty acids (SCFAs) by the microbial community in defined media with mono-carbon sources (formic-, acetetic-, propionic-, butyric acid) in batch culture. It could be demonstrated that the microbial reactor population could be transferred successfully to the lab, and its ability to build up methane was present even with deteriorating biogas plant performance. Therefore, this reduction in performance of the biogas plant was not due to a decrease in abundance, but due to an inactivity of the microbial community. Generally, the physico-chemical properties of the biogas plant seemed to favour hydrogenotrophic methanogens, as seen by the high metabolisation rates of formate compared with all other carbon sources. In contrast, acetoclastic methanogenesis could be shown to play a minor role in the methane production of the investigated biogas plant, although the origin of up to 66% of methane is generally suggested to be generated through acetoclastic pathway.     

Anaerobic biodegradation of cellulosic material: Batch experiments and modelling based on isotopic data and focusing on aceticlastic and non-aceticlastic methanogenesis

Utilizing stable carbon isotope data to account for aceticlastic and non-aceticlastic pathways of methane generation, a model was created to describe laboratory batch anaerobic decomposition of cellulosic materials (office paper and cardboard). The total organic and inorganic carbon concentrations, methane production volume, and methane and CO₂ partial pressure values were used for the model calibration and validation. According to the fluorescent in situ hybridization observations, three groups of methanogens including strictly hydrogenotrophic methanogens, strictly aceticlastic methanogens (Methanosaeta sp.) and Methanosarcina sp., consuming both acetate and H₂/H₂CO₃ as well as acetate-oxidizing syntrophs, were considered. It was shown that temporary inhibition of aceticlastic methanogens by non-ionized volatile fatty acids or acidic pH was responsible for two-step methane production from office paper at 35 °C where during the first and second steps methane was generated mostly from H₂/H₂CO₃ and acetate, respectively. Water saturated and unsaturated cases were tested. According to the model, at the intermediate moisture (150%), much lower methane production occurred because of full-time inhibition of aceticlastic methanogens. At the lowest moisture, methane production was very low because most likely hydrolysis was seriously inhibited. Simulations showed that during cardboard and office paper biodegradation at 55 °C, non-aceticlastic syntrophic oxidation by acetate-oxidizing syntrophs and hydrogenotrophic methanogens were the dominant methanogenic pathways.     

Population dynamics during startup of thermophilic anaerobic digesters: The mixing factor

Two thermophilic digesters were inoculated with manure and started-up under mixed and stagnant conditions. The Archaea in the mixed digester (A) were dominated by hydrogenotrophic Methanobateriaceae (61%) with most of the methane being produced via syntrophic pathways. Methanosarcinales (35%) were the only acetoclastic methanogens present. Acetate dissipation seems to depend on balanced hydrogenotrophic-to-acetotrophic abundance, which in turn was statistically correlated to free ammonia levels. Relative abundance of bacterial community was associated with the loading rate. However, in the absence of mixing (digester B), the relationship between microbial composition and operating parameters was not discernible. This was attributed to the development of microenvironments where environmental conditions are significantly different from average measured parameters. The impact of microenvironments was accentuated by the use of a non-acclimated seed that lacks adequate propionate degraders. Failure to disperse the accumulated propionate, and other organics, created high concentration niches where competitive and inhibiting conditions developed and favored undesired genera, such as Halobacteria (65% in B). As a result, digester B experienced higher acid levels and lower allowable loading rate. Mixing was found necessary to dissipate potential inhibitors, and improve stability and loading capacity, particularly when a non-acclimated seed, often lacking balanced thermophilic microflora, is used.     

Comparison of different liquid anaerobic digestion effluents as inocula and nitrogen sources for solid-state batch anaerobic digestion of corn stover

Effluents from three liquid anaerobic digesters, fed with municipal sewage sludge, food waste, or dairy waste, were evaluated as inocula and nitrogen sources for solid-state batch anaerobic digestion of corn stover in mesophilic reactors. Three feedstock-to-effluent (F/E) ratios (i.e., 2, 4, and 6) were tested for each effluent. At an F/E ratio of 2, the reactor inoculated by dairy waste effluent achieved the highest methane yield of 238.5 L/kgVS_feed, while at an F/E ratio of 4, the reactor inoculated by food waste effluent achieved the highest methane yield of 199.6 L/kgVS_feed. The microbial population and chemical composition of the three effluents were substantially different. Food waste effluent had the largest population of acetoclastic methanogens, while dairy waste effluent had the largest populations of cellulolytic and xylanolytic bacteria. Dairy waste also had the highest C/N ratio of 8.5 and the highest alkalinity of 19.3 g CaCO₃/kg. The performance of solid-state batch anaerobic digestion reactors was closely related to the microbial status in the liquid anaerobic digestion effluents.     

Evolution of butyric acid and the methanogenic microbial population in a thermophilic dry anaerobic reactor

Knowledge of the relationship between the evolution of butyric acid, the main precursor of methane, and the methanogenic microbial population, quantified by fluorescent in situ hybridization (FISH), is important for understating and controlling the anaerobic digestion of solid waste. In this study, a statistical analysis has been made of this relationship in a laboratory-scale continuously stirred tank reactor (CSTR) without recycling of biomass. The CSTR has been operated at four organic loading rates (OLR) between 4.42 and 7.50 kg VS m^?3 day^?1, using the Organic Fraction of Municipal Solid Waste (OFMSW) as the substrate. During the start-up stage, the consumption of butyric acid is related to the population of H₂-utilising methanogens (R² = 0.774), whereas in the stabilization stage, it is related to that of acetoclastic methanogens (R² = 0.716). Therefore, it can be concluded that the methanogenic population dynamics are closely correlated with the concentration of volatile fatty acids, and in particular, with that of butyric acid.     

Application of an early warning indicator and CaO to maximize the time–space-yield of an completely mixed waste digester using rape seed oil as co-substrate

In order to increase the organic loading rate (OLR) and hereby the performance of biogas plants an early warning indicator (EWI-VFA/Ca) was applied in a laboratory-scale biogas digester to control process stability and to steer additive dosing. As soon as the EWI-VFA/Ca indicated the change from stable to instable process conditions, calcium oxide was charged as a countermeasure to raise the pH and to bind long-chain fatty acids (LCFAs) by formation of aggregates. An interval of eight days between two increases of the OLR, which corresponded to 38% of the hydraulic residence time (HRT), was sufficient for process adaptation. An OLR increase by a factor of three within six weeks was successfully used for biogas production. The OLR was increased to 9.5 kg volatile solids (VS) m^?3 d^?1 with up to 87% of fat. The high loading rates affected neither the microbial community negatively nor the biogas production process. Despite the increase of the organic load to high rates, methane production yielded almost its optimum, amounting to 0.9 m³ (kg VS)^?1. Beneath several uncharacterized members of the phylum Firmicutes mostly belonging to the family Clostridiaceae, a Syntrophomonas-like organism was identified that is known to live in a syntrophic relationship to methanogenic archaea. Within the methanogenic group, microorganisms affiliated to Methanosarcina, Methanoculleus and Methanobacterium dominated the community.     

Increasing biogas production from sewage sludge anaerobic co-digestion process by adding crude glycerol from biodiesel industry

In an effort to convert waste streams to energy in a green process, glycerol from biodiesel manufacturing has been used to increase the gas production and methane content of biogas within a mesophilic anaerobic co-digestion process using primary sewage sludge. Glycerol was systematically added to the primary digester from 0% to 60% of the organic loading rate (OLR). The optimum glycerol loading range was from 25% to 60% OLR. This resulted in an 82–280% improvement in specific gas production. Following the feeding schedule described, the digesters remained balanced and healthy until inhibition was achieved at 70% glycerol OLR. This suggests that high glycerol loadings are possible if slow additions are upheld in order to allow the bacterial community to adjust properly. Waste water treatment plant operators with anaerobic digesters can use the data to increase loadings and boost biogas production to enhance energy conversion. This process provides a safe, environmentally friendly method to convert a typical waste stream to an energy stream of biogas.     

Ultrasound assisted biogas production from landfill leachate

The aim of this study is to increase biogas production and methane yield from landfill leachate in anaerobic batch reactors by using low frequency ultrasound as a pretreatment step. In the first part of the study, optimum conditions for solubilization of organic matter in leachate samples were investigated using various sonication durations at an ultrasound frequency of 20 kHz. The level of organic matter solubilization during ultrasonic pretreatment experiments was determined by calculating the ratio of soluble chemical oxygen demand (sCOD) to total chemical oxygen demand (tCOD). The sCOD/tCOD ratio was increased from 47% in raw leachate to 63% after 45 min sonication at 600 W/l. Non-parametric Friedman’s test indicated that ultrasonic pretreatment has a significant effect on sCOD parameter for leachate (p < 0.05). In the second part of the study, anaerobic batch reactors were operated for both ultrasonically pretreated and untreated landfill leachate samples in order to assess the effect of sonication on biogas and methane production rate. In anaerobic batch reactor feed with ultrasonically pretreated leachate, 40% more biogas was obtained compared to the control reactor. For statistical analysis, Mann–Whitney U test was performed to compare biogas and methane production rates for raw and pretreated leachate samples and it has been found that ultrasonic pretreatment significantly enhanced biogas and methane production rates from leachate (p < 0.05) in anaerobic batch reactors. The overall results showed that low frequency ultrasound pretreatment can be potentially used for wastewater management especially with integration of anaerobic processes.     

Effect of inoculum to substrate ratio (I/S) on municipal solid waste anaerobic degradation kinetics and potential

The goal of this study is to evaluate the impact of the inoculum to substrate ratio (I/S) on the anaerobic degradation potential of municipal solid waste (MSW). Reconstituted MSW samples were thus incubated under batch anaerobic conditions and inoculated with an increasing amount of inoculum originating from a mesophilic sludge digester. I/S tested values were 0 (no inoculum added), 0.015, 0.03, 0.06, 0.12, 0.25, 1, 2 and 4 (gVM_inoculum/gVM_waste). The results indicate that the apparent maximal rate of dissolved organic carbon accumulation is reached at I/S = 0.12. Under this level, the hydrolysis process is limited by the concentration of biomass and can thus be described as first order kinetics phenomena with respect to biomass for I/S ratios below 0.12. The maximum methane production rate and the minimal latency are reached at a ratio of 2. In addition to that, both methane signature and ARISA show a shift in the methanogenic populations from hydrogenotrophic to acetoclastic.     

Analysis of methanogenic activity in a thermophilic-dry anaerobic reactor: use of fluorescent in situ hybridization

Methanogenic activity in a thermophilic-dry anaerobic reactor was determined by comparing the amount of methane generated for each of the organic loading rates with the size of the total and specific methanogenic population, as determined by fluorescent in situ hybridization. A high correlation was evident between the total methanogenic activity and retention time [-0.6988Ln(x)+2.667] (R(2) 0.8866). The total methanogenic activity increased from 0.04x10(-8) mLCH(4) cell(-1)day(-1) to 0.38x10(-8) mLCH(4) cell(-1)day(-1) while the retention time decreased, augmenting the organic loading rates. The specific methanogenic activities of H(2)-utilizing methanogens and acetate-utilizing methanogens increased until they stabilised at 0.64x10(-8) mLCH(4) cell(-1)day(-1) and 0.33x10(-8) mLCH(4) cell(-1)day(-1), respectively. The methanogenic activity of H(2)-utilizing methanogens was higher than acetate-utilizing methanogens, indicating that maintaining a low partial pressure of hydrogen does not inhibit the acetoclastic methanogenesis or the anaerobic process.     

Co-digestion of cattle manure with food waste and sludge to increase biogas production

Anaerobic co-digestion strategies are needed to enhance biogas production, especially when treating certain residues such as cattle/pig manure. This paper presents a study of co-digestion of cattle manure with food waste and sewage sludge. With the aim of maximising biogas yields, a series of experiments were carried out under mesophilic and thermophilic conditions using continuously stirred-tank reactors, operating at different hydraulic residence times. Pretreatment with ultrasound was also applied to compare the results with those obtained with non-pretreated waste. Specific methane production decreases when increasing the OLR and decreasing HRT. The maximum value obtained was 603 LCH(4)/kg VS(feed) for the co-digestion of a mixture of 70% manure, 20% food waste and 10% sewage sludge (total solid concentration around 4%) at 36°C, for an OLR of 1.2g VS/Lday. Increasing the OLR to 1.5g VS/Lday led to a decrease of around 20-28% in SMP. Lower methane yields were obtained when operating at 55°C. The increase in methane production when applying ultrasound to the feed mixtures does not compensate for the energy spent in this pretreatment.     

Managing Excessive Methanogenesis During ERD/ISCR Remedial Action

Excessive production of methane has been observed at some remediation sites following the addition of organic hydrogen donors such as (emulsified) oils/lecithin, sugars, and conventional carbon + zero‐valent iron (ZVI) amendments. This is due to the fact that methanogens are commonly the most ubiquitous indigenous microbes in anoxic aquifer settings, and, under enriched environmental conditions, methanogens replicate every one to two hours (whereas Dehalococcoides spp., e.g., double in 24–48 hr). Hence, methanogens often bloom and dominate the microbial ecosystem following the addition of remedial amendments, thereby liberating large amounts of methane gas. There are at least three important consequences of this response:

1. By utilizing hydrogen, the methanogens compete with dechlorinating microbes, thus making inefficient use of the remedial amendment (just 20 ppm methane in groundwater represents an approximate 30 percent “waste” of added fermentable substrate (i.e., hydrogen donor)—this is a common and tangible detriment);
2. Methanogens can methylate heavy metals and their rapid growth consumes alkalinity, while generating acidity, thereby facilitating multiple potential mechanisms for creating secondary contaminant issues (i.e., arsenic plumes); and
3. Elevated methane concentrations can exceed current and pending regulations of <10 to <28 ppm methane in groundwater and/or 0.5 percent by volume methane in soil gas (e.g., 10 percent of the lower explosive limit) and/or indoor air (methane is flammable between 5 percent and 15 percent by volume) and this will induce migration of contaminant vapors potentially causing indoor air issues.

Considering the recent guidelines for indoor air published by the US Environmental Protection Agency, it is increasingly important to prevent excessive methanogenesis associated with remedial actions. From a regulatory perspective, public safety issues are paramount; from a property re‐use or real estate (brownfield) developers’ perspective, project delays are costly and can jeopardize an entire program. The use of antimethanogenic compounds as inhibitors of protein biosynthesis and the activity of enzyme systems unique to Archaea (i.e., methanogens) during in situ remedial action can improve contaminant removal while offering safer, more efficacious treatment, simply by impeding the methanogenic bacteria's ability to proliferate and out compete desired bacterial communities (e.g., Dehalococcoides spp.). ©2016 Wiley Periodicals, Inc.     

Investigation into the effect of high concentrations of volatile fatty acids in anaerobic digestion on methanogenic communities

A study was conducted to determine whether differences in the levels of volatile fatty acids (VFAs) in anaerobic digester plants could result in variations in the indigenous methanogenic communities. Two digesters (one operated under mesophilic conditions, the other under thermophilic conditions) were monitored, and sampled at points where VFA levels were high, as well as when VFA levels were low. Physical and chemical parameters were measured, and the methanogenic diversity was screened using the phylogenetic microarray ANAEROCHIP. In addition, real-time PCR was used to quantify the presence of the different methanogenic genera in the sludge samples. Array results indicated that the archaeal communities in the different reactors were stable, and that changes in the VFA levels of the anaerobic digesters did not greatly alter the dominating methanogenic organisms. In contrast, the two digesters were found to harbour different dominating methanogenic communities, which appeared to remain stable over time. Real-time PCR results were inline with those of microarray analysis indicating only minimal changes in methanogen numbers during periods of high VFAs, however, revealed a greater diversity in methanogens than found with the array.     

Physical modelling of the composting environment: a review. Part 2: Simulation performance

This paper reviews previously published heat balance data for experimental and full-scale composting reactors, and then presents an evaluation of the simulation performance of laboratory and pilot-scale reactors, using both quantitative and qualitative temperature profile characteristics. The review indicates that laboratory-scale reactors have typically demonstrated markedly different heat balance behaviour in comparison to full-scale systems, with ventilative heat losses of 36-67%, and 70-95% of the total flux, respectively. Similarly, conductive/convective/radiative (CCR) heat losses from laboratory reactors have been reported at 33-62% of the total flux, whereas CCR losses from full-scale composting systems have ranged from 3% to 15% of the total. Full-scale windrow temperature-time profiles from the literature were characterised by the present authors. Areas bounded by the curve and a 40 degrees C baseline (A(40)) exceeded 624 degrees C. days, areas bounded by the curve and a 55 degrees C baseline (A(55)) exceeded 60 degrees C days, and times at 40 and 55 degrees C were >46 days and >24 days, respectively, over periods of 50-74 days. For forced aeration systems at full scale, values of A(40) exceeded 224 degrees C days, values of A(55) exceeded 26 degrees C days, and times at 40 and 55 degrees C were >14 days and >10 days, respectively, over periods of 15-35 days. Values of these four parameters for laboratory-scale reactors were typically considerably lower than for the full-scale systems, although temperature shape characteristics were often similar to those in full-scale profiles. Evaluation of laboratory-, pilot- and full-scale profiles from systems treating the same substrate showed that a laboratory-scale reactor and two pilot-scale reactors operated at comparatively high aeration rates poorly simulated full-scale temperature profiles. However, the curves from two moderately insulated, self-heating, pilot-scale reactors operated at relatively low aeration rates appeared to closely replicate full-scale temperature profiles. The importance of controlling aeration rates and CCR losses is discussed and further work suggested in order to investigate the links between simulation of the composting environment and process performance.     

A side-by-side comparison of two systems of sequencing coupled reactors for anaerobic digestion of the organic fraction of municipal solid waste.

The objective of this work was to compare the performance of two laboratory-scale, mesophilic systems aiming at the anaerobic digestion of the organic fraction of municipal solid wastes (OFMSW). The first system consisted of two coupled reactors packed with OFMSW (PBR1.1-PBR1.2) and the second system consisted of an upflow anaerobic sludge bed reactor (UASB) coupled to a packed reactor (UASB2.1-PBR2.2). For the start-up phase, both reactors PBR 1.1 and the UASB 2.1 (also called leading reactors) were inoculated with a mixture of non-anaerobic inocula and worked with leachate and effluent full recirculation, respectively. Once a full methanogenic regime was achieved in the leading reactors, their effluents were fed to the fresh-packed reactors PBR1.2 and PBR2.2, respectively. The leading PBR 1.1 reached its full methanogenic regime after 118 days (Tm, time to achieve methanogenesis) whereas the other leading UASB 2.1 reactor reached its full methanogenesis regime after only 34 days. After coupling the leading reactors to the corresponding packed reactors, it was found that both coupled anaerobic systems showed similar performances regarding the degradation of the OFMSW. Removal efficiencies of volatile solids and cellulose and the methane pseudo-yield were 85.95%, 80.88% and 0.109 NL CH4 g(-1) VS(fed) in the PBR-PBR system; and 88.75%, 82.61% and 0.115 NL CH4 g(-1) VS(fed0 in the UASB-PBR system [NL, normalized litre (273 degrees K, 1 ata basis)]. Yet, the second system UASB-PBR system showed a faster overall start-up.     

Dry anaerobic digestion in batch mode: Design and operation of a laboratory-scale,completely mixed reactor

A laboratory-scale (40 l) reactor was designed to investigate dry anaerobic digestion. The reactor is equipped with an intermittent paddle mixer, enabling complete mixing in the reactor. Three consecutive batch dry digestion tests of municipal solid waste were performed under mesophilic conditions and compared to operation results obtained on a pilot-scale (21 m³) with the same feedstock. Biogas and methane production at the end of the tests were similar (around 200 m³ CH₄STP/tVS), and the dynamics of methane production and VFA accumulation concurred. However, the maximal levels of VFA transitory accumulation varied between reactors and between runs in a same reactor. Ammonia levels were similar in both reactors. These results show that the new reactor accurately imitates the conditions found in larger ones. Adaptation of micro-organisms to the waste and operating conditions was also pointed out along the consecutive batches.     

Effects of various fatty acid amendments on a microbial digester community in batch culture

Since biogas production is becoming increasingly important the understanding of anaerobic digestion processes is fundamental. However, large-scale digesters often lack online sensor equipment to monitor key parameters. Furthermore the possibility to selectively change fermenting parameter settings in order to investigate methane output or microbial changes is limited. In the present study we examined the possibility to investigate the microbial community of a large scale (750,000 L) digester within a laboratory small-scale approach. We studied the short-term response of the downscaled communities on various fatty acids and its effects on gas production and compared it with data from the original digester sludge. Even high loads of formic acid led to distinct methane formation, whereas high concentrations of other acids (acetic, butyric, propionic acid) caused a marked inhibition of methanogenesis coupled with an increase in hydrogen concentration. Molecular microbial techniques (DGGE/quantitative real-time-PCR) were used to monitor the microbial community changes which were related to data from GC and HPLC analysis. DGGE band patterns showed that the same microorganisms which were already dominant in the original digester re-established again in the lab-scale experiment. Very few microorganisms dominated the whole fermenting process and species diversity was not easily influenced by moderate varying fatty acid amendments - Methanoculleus thermophilus being the most abundant species throughout the variants. MCR-copy number determined via quantitative real-time-PCR - turned out to be a reliable parameter for quantification of methanogens, even in a very complex matrix like fermenter sludge. Generally the downscaled batch approach was shown to be appropriate to investigate microbial communities from large-scale digesters.     

Long-term anaerobic digestion of food waste stabilized by trace elements

The purpose of this study was to examine if long-term anaerobic digestion of food waste in a semi-continuous single-stage reactor could be stabilized by supplementing trace elements. Contrary to the failure of anaerobic digestion of food waste alone, stable anaerobic digestion of food waste was achieved for 368 days by supplementing trace elements. Under the conditions of OLR (organic loading rates) of 2.19-6.64 g VS (volatile solid)/L day and 20-30 days of HRT (hydraulic retention time), a high methane yield (352-450 mL CH(4)/g VS(added)) was obtained, and no significant accumulation of volatile fatty acids was observed. The subsequent investigation on effects of individual trace elements (Co, Fe, Mo and Ni) showed that iron was essential for maintaining stable methane production. These results proved that the food waste used in this study was deficient in trace elements.