Mesophilic Anaerobic Sludge Research Articles

Biomineralization of phosphorus during anaerobic treatment of distillery wastewaters

This study addresses the pressing environmental concerns associated with the rapidly growing distillery industry, which is a significant contributor to wastewater generation. By focusing on the treatment of distillery wastewater using anaerobic digestion, this research explores the potential to convert organic materials into biofuels (methane). Moreover, the study aims to recover both methane and phosphorus from distillery wastewater in a single anaerobic reactor, which represents a novel and unexplored approach. Laboratory-scale experiments were conducted using mesophilic and thermophilic upflow anaerobic sludge blanket reactors. A key aspect of the study involved the implementation of a unique strategy: the mixing of centrate and spent caustic wastewater streams. This approach was intended to enhance treatment performance, manipulate the microbial community structure, and thereby optimizing the overall treatment performance. The integration of the centrate and spent caustic streams yielded remarkable co-benefits, resulting in significant biomethane production and efficient phosphorus precipitation. The study demonstrated a phosphorus removal efficiency of ∼60 % throughout the 130–140 days operation period. The recovery of phosphorus via the reactor sludge offers exciting opportunities for its utilization as a fertilizer or as a raw material within the phosphorus refinery industry. The biomethane produced during the treatment exhibits significant energy potential, estimated at 0.5 GJ/(m3 distillery wastewater).

Application of Fuzzy Mamdani Model for Biogas Yield Prediction in Anaerobic Co-Digestion of Decomposable Wastes

Accurate prediction of biogas yield is crucial for optimizing waste-to-energy conversion systems in anaerobic co-digestion processes. In this study, a double input and single output (DISO) fuzzy mamdani model (FMM) was developed for the prediction of biogas yield in a pilot scale of 105-L mesophilic anaerobic sludge bio-digester. The input variables considered are the combination of cow dung and pig waste and the retention time (RT), while the output variable is the experimental biogas yield. Triangular Fuzzy Membership Functions (TFMF) were utilized to define the input and output datasets, and rules were derived from de-fuzzification. Comparative analysis between the FMM's predicted results and experimental values showcased its effectiveness in forecasting biogas yield during the anaerobic co-digestion of the hybrid wastes. Significantly, the FMM consistently produced results with low error values for the sample dataset, underscoring its accuracy even under stochastic conditions. This study emphasizes the FMM's ability to generate predictions with minimal deviations, offering superior results. As a prospect for future research, the implementation of hybrid algorithms may further enhance biogas yield prediction accuracy within waste-to-energy systems.

The role of microbial ecology in improving the performance of anaerobic digestion of sewage sludge.

The use of next-generation diagnostic tools to optimise the anaerobic digestion of municipal sewage sludge has the potential to increase renewable natural gas recovery, improve the reuse of biosolid fertilisers and help operators expand circular economies globally. This review aims to provide perspectives on the role of microbial ecology in improving digester performance in wastewater treatment plants, highlighting that a systems biology approach is fundamental for monitoring mesophilic anaerobic sewage sludge in continuously stirred reactor tanks. We further highlight the potential applications arising from investigations into sludge ecology. The principal limitation for improvements in methane recoveries or in process stability of anaerobic digestion, especially after pre-treatment or during co-digestion, are ecological knowledge gaps related to the front-end metabolism (hydrolysis and fermentation). Operational problems such as stable biological foaming are a key problem, for which ecological markers are a suitable approach. However, no biomarkers exist yet to assist in monitoring and management of clade-specific foaming potentials along with other risks, such as pollutants and pathogens. Fundamental ecological principles apply to anaerobic digestion, which presents opportunities to predict and manipulate reactor functions. The path ahead for mapping ecological markers on process endpoints and risk factors of anaerobic digestion will involve numerical ecology, an expanding field that employs metrics derived from alpha, beta, phylogenetic, taxonomic, and functional diversity, as well as from phenotypes or life strategies derived from genetic potentials. In contrast to addressing operational issues (as noted above), which are effectively addressed by whole population or individual biomarkers, broad improvement and optimisation of function will require enhancement of hydrolysis and acidogenic processes. This will require a discovery-based approach, which will involve integrative research involving the proteome and metabolome. This will utilise, but overcome current limitations of DNA-centric approaches, and likely have broad application outside the specific field of anaerobic digestion.

Responses of mesophilic anaerobic sludge microbiota to thermophilic conditions: Implications for start-up and operation of thermophilic THP-AD systems

Anaerobic digestion (AD) has been widely employed for wastewater and organic waste treatment, in which methanogenesis is highly driven by close microbial interactions among intricate microbial communities. However, the ecological processes underpinning the community assembly that support methanogenesis in such engineered ecosystems remain largely unknown, especially when exposed to challenging circumstances (e.g., high temperature, ammonium content). Here, eight AD bioreactors were seeded with four different inocula (two from full-scale mesophilic AD systems and the other two from lab-scale mesophilic AD systems), and were operated under thermophilic conditions (55 °C) for treating thermal hydrolysis process (THP) pre-treated waste activated sludge to investigate how mesophilic community responds to thermophilic conditions during the long-term cultivation. Results showed that the inocula collected from the full-scale systems were more resilient than that from the lab-scale systems, which may be primarily attributed to indigenous robust methanogens. As a result, the former efficiently generated methane which was predominantly contributed by Methanothermobacter and Methanosarcina (healthy AD ecosystem), while methanogenic activity was remarkably prohibited in the latter (dysfunctional AD ecosystem). Thermophilic environment was a strong selection force, resulting in the convergence of microbial communities in both the healthy and dysfunctional AD ecosystems. Deterministic processes predominated the community assembly regardless of AD ecosystem function, but stronger influences of stochastic processes were observed in dysfunctional AD ecosystems, which was likely attributable from the stronger effect of immigrants from the feedstock. As indicated by molecular ecological network analysis, the microbial network structures in the healthy AD ecosystems were more stable than those in the dysfunctional AD ecosystems. Although keystone taxa were different among the bioreactors, most of which played vital roles in organic hydrolysis/fermentation. To sum up, this study greatly improved our understanding of the relationships between microbiological traits and AD ecosystem function under thermophilic conditions, which could provide useful information to guide thermophilic AD (e.g., THP-AD) start-up and health diagnosis during operation.

The Start-Up of Continuous Biohydrogen Production from Cheese Whey: Comparison of Inoculum Pretreatment Methods and Reactors with Moving and Fixed Polyurethane Carriers

This article presents the results of the start-up of continuous production of biohydrogen from cheese whey (CW) in an anaerobic filter (AF) and anaerobic fluidized bed (AFB) with a polyurethane carrier. Heat and acid pretreatments were used for the inactivation of hydrogen-scavengers in the inoculum (mesophilic and thermophilic anaerobic sludge). Acid pretreatment was effective for thermophilic anaerobic sludge to suppress methanogenic activity, and heat treatment was effective for mesophilic anaerobic sludge. Maximum specific yields of hydrogen, namely 178 mL/g chemical oxygen demand (COD) and 149 mL/g COD for AFB and AF, respectively, were obtained at the hydraulic retention time (HRT) of 4.5 days and organic load rate (OLR) of 6.61 kg COD/(m3 day). At the same time, the maximum hydrogen production rates of 1.28 and 1.9 NL/(L day) for AF and AFB, respectively, were obtained at the HRT of 2.02 days and OLR of 14.88 kg COD/(m3 day). At the phylum level, the dominant taxa were Firmicutes (65% in AF and 60% in AFB), and at the genus level, Lactobacillus (40% in AF and 43% in AFB) and Bifidobacterium (24% in AF and 30% in AFB).

Sulfur Reduction at Hyperthermoacidophilic Conditions with Mesophilic Anaerobic Sludge as the Inoculum.

Sulfur reduction at hyperthermoacidophilic conditions represents a promising opportunity for metal sulfide precipitation from hot acidic metallurgical streams, avoiding costly cooling down. The suitability of mesophilic anaerobic sludges as the inoculum for sulfur-reducing bioreactors operated at high temperature and low pH was explored. We examined sludges from full-scale anaerobic reactors for sulfur-reducing activity at pH 2.0–3.5 and 70 or 80 °C, with H2 as an electron donor. At pH 3.5 in batch experiments, sulfidogenesis started within 4 days, reaching up to 100–200 mg·L–1 of dissolved sulfide produced after 19–24 days, depending on the origin of the sludge. Sulfidogenesis resumed after removing H2S by flushing with nitrogen gas, indicating that sulfide was limiting the conversion. The best performing sludge was used to inoculate a 4 L gas-lift reactor fed with H2 as the electron donor, CO2 as the carbon source, and elemental sulfur as the electron acceptor. The reactor was operated in semibatch mode at a pH 3.5 and 80 °C, and stable sulfide production rates of 60–80 mg·L–1·d–1 were achieved for a period of 24 days, without formation of methane or acetate. Our results reveal the potential of mesophilic anaerobic sludges as seed material for sulfur-reducing bioprocesses operated at hyperthermoacidophilic conditions. The process needs further optimization of the volumetric sulfide production rate to gain relevance for practice.

Mixing strategies – Activated carbon nexus: Rapid start-up of thermophilic anaerobic digestion with the mesophilic anaerobic sludge as inoculum

This study evaluated the mixing – activate carbon nexus in anaerobic digestion with the aim of accelerating start-up of thermophilic anaerobic co-digestion of food waste and chicken manure using mesophilic anaerobic sludge as inoculum. Results showed that the methane yield in the continuous stirred reactor is 71.3% higher than that of intermittent agitated reactor, and the addition of activated carbon can further improve the yield of methane by 18.2%. Continuous mixing mode followed by intermittent mixing was proved to be an alternative strategy to accelerate start-up of thermophilic anaerobic digestion. The optimum mixing time of 120 s/hour were obtained using computational fluid dynamics modeling. Analysis of genomic annotation metabolism indicated that the addition of activated carbon enhanced the dominant metabolism pathways of amino acid, methane and energy. Results of enzymes gene expression suggested that carbohydrates esterases, glycoside hydrolases and glycosyl transferases were dominant, respectively.

Activity of preserved anaerobic sludge

There is a need for a broad study addressing different preservation conditions of anaerobic sludge and its activity after a prolonged storage. This study compared four different preservation methods of mesophilic anaerobic sludge for a period of up to 12 months: storage at 23 ± 2 °C, +4 °C, ‒20 °C, and freeze-dried. Anaerobic sludge was sampled from upper and bottom ports of an up flow anaerobic sludge blanket (UASB) reactor fed with microalgae and sodium acetate at organic loading rate of 5.4 gCOD/L·d. Specific methanogenic activity (SMA) tests were performed on the sludge samples after 2.5, 6, and 12 months of storage. Results demonstrated a statistically significant decrease in the SMA of the bottom port preserved sludge, but not of the upper port sludge, regardless of the method used for preservation. A varying susceptibility to the storage of the two types of the anaerobic sludge can be explained by the content of the methanogenic microorganisms, with bottom port sludge having a higher amount of the methane producing species. Interestingly, lyophilized samples were able to produce similar amounts of biogas when compared to the other three storage conditions, with the only difference of having a longer re-activation period.

Mesophilic, thermophilic and hyperthermophilic acidogenic fermentation of food waste in batch: Effect of inoculum source

Distinctions in hydrolysis and acidogenesis were examined for a series of anaerobic batch reactors inoculated with three different anaerobic mixed cultures (mesophilic, thermophilic and hyperthermophilic anaerobic sludge) and operated at the temperature of inoculum’s origin and additionally at 70 °C. Hyperthermophilic temperatures led to increased hydrolysis rates during the start-up stage but a rapid drop in pH limited the overall hydrolysis efficiency, indicating the importance of pH control to sustain the high reaction rates at higher temperatures. No significant difference (P > 0.05) was observed among hydrolysis efficiencies obtained for different reactors which ranged between 27 ± 3% and 40 ± 14%. The highest fermentation yield of 0.44 g COD of fermentation products/g VSS-CODadded was obtained under thermophilic conditions, followed by mesophilic (0.33 g COD ferm. prod./g VSS-CODadded) and hyperthermophilic conditions (0.05–0.08 g COD ferm. prod./g VSS-CODadded). Fermentative performance was better at mesophilic and thermophilic conditions as indicated by improved production of volatile fatty acids (VFA). VFAs accounted for 60–71% of the solubilised matter at thermophilic and mesophilic conditions. Acetic acid formed the primary VFA (70%) at mesophilic temperatures, while butyric acid was the major VFA at thermophilic (60%) conditions. Hyperthermophilic conditions led to increased production of lactic acid, which comprised up to 32% of the solubilised matter. Overall, the results indicate that different operating temperatures may not significantly affect the substrate degradation efficiency but clearly influence the biotransformation pathways.

Improvement of methanogenic activity of anaerobic digestion using poly(l-lactic acid) with enhanced chemical hydrolyzability based on physicochemical parameters

Because packing bags and disposable items of poly (l-lactic acid) (PLLA) waste are discharged together with other organic waste including garbage, anaerobic co-digestion of PLLA and other organic waste is required. However, because of low hydrolyzability of PLLA products, the chemical hydrolyzability must be improved for PLLA treatment during anaerobic digestion. This study aimed to assess weight-average molecular weight (Mw) and crystallinity (Xc), to determine the chemical hydrolyzability of PLLA, for PLLA treatment during anaerobic digestion. Moreover, the possibility of anaerobic co-digestion of the PLLA after improvement of chemical hydrolyzability and other organic waste was also discussed. Detectable methanogenic activity of the mesophilic and thermophilic anaerobic sludges of PLLA occurred in the Mw range of 6,800 to 16,500, and 6,800 and 38,000, respectively. The methanogenic activity of mesophilic and thermophilic anaerobic sludge was higher with PLLA with a high crystallinity (Xc = 39.9–46.1%) than with nearly amorphous PLLA (Xc = 0.3–3.5%). The maximum methanogenic activity of anaerobic sludge using PLLA with an Xc of approximately 40–45% and with a Mw of 10,300 and 16,500 for mesophilic and thermophilic anaerobic sludge were 0.013 gCOD·gVS−1·d−1 and 0.13 gCOD·gVS−1·d−1, respectively. A survey on the possibility of anaerobic co-digestion of PLLA after improvement in chemical hydrolyzability based on Mw and Xc and organic wastes revealed that thermophilic conditions at 55 °C are more advantageous than mesophilic conditions at 37 °C.

Effect of elevated nitrate and sulfate concentrations on selenate removal by mesophilic anaerobic granular sludge bed reactors

The molar ratio of NO3−and SO42−to SeO42−is a key factor in SeO42−removal. A UASB reactor showed no inhibition of SeO42−at high NO3−and SO42−concentrations achieving 100% NO3−, 30% SO42−and 80% total Se removal efficiencies.

Optimization of the Acid Stage of a Two-Stage Anaerobic Digestion Process to Treat Urban Wastewater Sludge

AbstractThe efficiency of a two-stage mesophilic anaerobic sludge digestion process was studied in comparison with a conventional process to treat urban sludge in relation to the hydraulic retentio...

The impact of furfural concentrations and substrate-to-biomass ratios on biological hydrogen production from synthetic lignocellulosic hydrolysate using mesophilic anaerobic digester sludge

This study evaluated the impact of furfural (a furan derivative) on hydrogen production rates and yields at initial substrate-to-microorganism ratios (S°/X°) of 4, 2, 1, and 0.5gCOD/gVSS and furfural concentrations of 4, 2, 1, and 0.5g/L. Fermentation studies were carried out in batches using synthetic lignocellulosic hydrolysate as substrate and mesophilic anaerobic digester sludge as seed. Contrary to other literature studies where furfural was inhibitory, this study showed that furfural concentrations of up to 1g/L enhanced hydrogen production with yields as high as 19% from the control (batch without furfural). Plots of hydrogen yields against gfurfural/gsugars and hydrogen yields versus gfurfural/gbiomass showed negative linear correlation indicating that these parameters influence biohydrogen production. Regression analysis indicated that gfurfural/gsugarsinitial exerted a greater effect on the degree of inhibition of hydrogen production than gfurfural/gVSSfinal.

Co-digestion of press liquids of source-sorted municipal organic waste in anaerobic sludge treatment of municipal wastewater treatment plants.

This paper describes a semi-continuous laboratory-scale investigation of a potential co-substrate for mesophilic anaerobic sludge digestion in a municipal wastewater treatment plant. A feed liquid produced from source-sorted municipal organic waste by pretreatment with a screw press was subjected to the investigation. Quantities produced in press trials as well as the composition of the feed liquid are presented. Mass balances for N, P and chemical oxygen demand are given in order to verify the methane production of the feed liquid in co-digestion with sewage sludge at mesophilic conditions. Hydraulic retention time of the reactors were 14.7 to 16 d and organic loading rates were 1.5 to 2.7 kg volatile solids (VS) per cubic metre per day. The pretreatment by screw press is compared to the production of feed liquids with pulper-based pretreatment processes. While the addition of the feed liquid increased methane production by about 345 ml CH(4)/g VS(in), total solids of the feed liquid were reduced to about 63%. With respect to co-digestion at municipal wastewater treatment plants, several risks associated with the investigated feed liquid are outlined.

Survival of Antibiotic Resistant Bacteria and Horizontal Gene Transfer Control Antibiotic Resistance Gene Content in Anaerobic Digesters.

Understanding fate of antibiotic resistant bacteria (ARB) vs. their antibiotic resistance genes (ARGs) during wastewater sludge treatment is critical in order to reduce the spread of antibiotic resistance through process optimization. Here, we spiked high concentrations of tetracycline-resistant bacteria, isolated from mesophilic (Iso M1-1—a Pseudomonas sp.) and thermophilic (Iso T10—a Bacillus sp.) anaerobic digested sludge, into batch digesters and monitored their fate by plate counts and quantitative polymerase chain reaction (QPCR) of their corresponding tetracycline ARGs. In batch studies, spiked ARB plate counts returned to baseline (thermophilic) or 1-log above baseline (mesophilic) while levels of the ARG present in the spiked isolate [tet(G)] remained high in mesophilic batch reactors. To compare results under semi-continuous flow conditions with natural influent variation, tet(O), tet(W), and sul1 ARGs, along with the intI1 integrase gene, were monitored over a 9-month period in the raw feed sludge and effluent sludge of lab-scale thermophilic and mesophilic anaerobic digesters. sul1 and intI1 in mesophilic and thermophilic digesters correlated positively (Spearman rho = 0.457–0.829, P < 0.05) with the raw feed sludge. There was no correlation in tet(O) or tet(W) ratios in raw sludge and mesophilic digested sludge or thermophilic digested sludge (Spearman rho = 0.130–0.486, P = 0.075–0.612). However, in the thermophilic digester, the tet(O) and tet(W) ratios remained consistently low over the entire monitoring period. We conclude that the influent sludge microbial composition can influence the ARG content of a digester, apparently as a result of differential survival or death of ARBs or horizontal gene transfer of genes between raw sludge ARBs and the digester microbial community. Notably, mesophilic digestion was more susceptible to ARG intrusion than thermophilic digestion, which may be attributed to a higher rate of ARB survival and/or horizontal gene transfer between raw sludge bacteria and the digester microbial community.

Sewage sludge addition modifies soil microbial communities and plant performance depending on the sludge stabilization process

Despite the widespread use of sewage sludge as an organic amendment to improve soil stability and plant productivity, relatively little is known about how the different sludge stabilization processes affect the microbial composition and diversity of the sludge and the soil microbial populations as well as plant performance. In this study, the effects caused by addition of thermophilic aerobic (ATAD) and mesophilic anaerobic (MAD) sludge and inorganic fertilization on soil microbial community structure and diversity was assessed by pyrosequencing of 16S and 18S rRNA genes. Melon (Cucumis melo L., cv. Giotto) was used as model crop and its performance (growth and physiological state) was monitored together with changes in soil chemical parameters. Our results showed that the stabilization process of sewage sludge determined the feasibility of the final by-product as an organic amendment by altering in different manner the soil environment and modifying the soil microbial community structure and functioning. Changes in soil microbial community were related more to changes in the soil chemical environment rather than to the introduction of sludge-borne microorganisms. We also have shown that changes in a single physicochemical parameter (electrical conductivity) due to sludge application are associated with a pronounced shift in microbial community structure and activity as well as in plant performance. Along these lines, we showed that the application of ATAD sludge into soil resulted in less pronounced changes in its chemistry and microbial community structure, while enhancing soil microbial activity and plant performance. This study shows, therefore, that ATAD sludge could be applied as an excellent alternative to MAD sludge or inorganic fertilization.

Xylose substrate as the only nutrient in the operation of microbial fuel cells

Microbial fuel cells are bioelectrochemical devices converting chemical energy of organic compounds (i.e. biomass) into electrical energy by catalytic reactions of microbes under anaerobic conditions. The operation of two-chamber microbial fuel cells by Shewanella putrefaciens and mesophilic anaerobic sludge was studied comparatively, using xylose and glucose as solo substrates during the experiments. It was found that higher electric power was generated by the multicultural system than by the monoculture.

Influence of biodegradation in thermophilic anaerobic aqueous conditions on crystallization of poly(butylene succinate)

This work investigates the biodegradation of poly(butylene succinate) (PBS) in an environment of thermophilic (55 °C) and mesophilic (37 °C) anaerobic sludge. The thermophilic anaerobic sludge was prepared in a laboratory from mesophilic sludge sourced from a municipal waste water treatment plant and placed under thermophilic conditions. It was confirmed that PBS failed to decompose under mesophilic anaerobic conditions (2 wt. %), while the degree of biodegradation achieved under thermophilic conditions reached 24.8 wt. %. Abiotic hydrolysis of PBS in an environment of phosphate buffer (pH = 7) at 55 °C reached 17.8%, leading to the conclusion that hydrolytic enzymes present in thermophilic anaerobic sludge are involved in the biodegradation process as well as abiotic hydrolysis. Nonisothermal crystallization kinetics of PBS before and after biodegradation was investigated by differential scanning calorimetry (DSC). After biodegradation, a large shift in melting temperature Tm was observed, while shift of crystallization temperature Tc was even more pronounced. Lower Tm means smaller crystals. Crystallization kinetics was approximately 50% slower, which could be explained by lower nucleation density. Scanning electron microscopy (SEM) revealed a beautiful spherulitic structure after biodegradation at elevated temperature. During biodegradation, microorganisms assimilated mostly in the amorphous part of the polymer, and elevated temperature helped healing of crystal imperfections. By X-ray diffraction (XRD), it was found that biodegradation does not have a large effect on crystal form of PBS.

Recovery strategies of inhibition for mesophilic anaerobic sludge treating the de-oiled grease trap waste

De-oiled grease trap waste (GTW) could be a feasible substrate for anaerobic digestion. However, due to high concentration of lipid, the long-chain fatty acid (LCFA), as an intermediate in the anaerobic lipid degradation, easily inhibits the anaerobic digestion, resulting in long recovery period. Four recovery strategies: bentonite addition, water dilution, mixing with low LCFA substrate and mixing with inocula, with control test as comparison, were employed in order to accelerate the recovery process of mesophilic anaerobic sludge. Adding bentonite, water dilution with 80% mixing ratio and mixing with active inocula achieved shortening of the recovery process. The whole recovery time took approximately 3.5 months for the control test, as calculated from the beginning to the end of the lag time after de-oiled GTW was re-added as F/I ratio of 0.4. At least 10 g l−1 of bentonite addition was necessary for the fast recovery of the inhibited sludge, with the whole recovery time one month shorter than the control test. 1.5 months could be saved for the whole recovery process when the strategy of water dilution with 80% mixing ratio was considered. The more inocula were mixed with the inhibited sludge, the more the recovery period was lessened. For the strategy of mixing with inocula with the mixing ratio 80%, only 20 days were needed for the whole recovery process.

Bioelectrochemical treatment of municipal waste liquor in microbial fuel cells for energy valorization

Microbial fuel cells (MFCs) are recognized as promising applications to produce bioelectricity by utilizing various waste materials. In this study, dual-chamber microbial fuel cells were employed for energy valorization of an untested substrate, the liquid fraction of pressed municipal solid waste (LPW). This by-product is potentially applicable as a substrate in MFCs because of its high organic matter content. In the course of the experiments, the anodic biofilm response and energy production efficiency have been investigated by experimental design approach, taking substrate and fresh inoculum – mesophilic anaerobic sludge (MAS) – addition into account as factors. It was observed that reinoculation could result in a negative effect on the energy production, especially at low substrate (LPW) dosing levels. However, when the LPW to fresh MAS ratio in the anode chamber exceeded a particular value, the biofilm-associated electrical utilization dominated against the degradation in the bulk phase. Furthermore, the results indicated that the highest energy yields (8–9 J g−1 ΔCOD d−1) could be attained at the lowest input COD concentrations. The maximal and average COD removal efficiencies were 94% and 87%, respectively, which indicate the excellent biodegradability of LPW. As for COD removal rate, 1.2–1.9 kg COD m−3 d−1 could be reached.