Acid hydrolysis of corn stover for biohydrogen production using Thermoanaerobacterium thermosaccharolyticum W16

Lignocellulosic biomass, if properly hydrolyzed, can be an ideal feedstock for fermentative hydrogen production. This work considered the pretreatment of corn stover (CS) using a dilute acid hydrolysis process and studied its fermentability for hydrogen production by the strain Thermoanaerobacterium thermosaccharolyticum W16. The effects of sulfuric acid concentration and reaction time in the hydrolysis stage of the process were determined based on a 2² central composite experimental design with respect to maximum hydrogen productivity. The optimal hydrolysis conditions to yield the maximum quantity of hydrogen by W16 were 1.69% sulfuric acid and 117 min reaction time. At these conditions, the hydrogen yield was shown to be 3305 ml H₂ L⁻¹ medium, which corresponds to 2.24 mol H₂ mol⁻¹ sugar. The present results indicate the potential of using T. thermosaccharolyticum W16 for high-yield conversion of CS hemicellulose into bio-H₂ integrated with acid hydrolysis.     

Bio-methanization of energy crops through mono-digestion for continuous production of renewable biogas

The aim of this laboratory-scale study was to investigate the long-term anaerobic fermentation of an extremely sour substrate, an energy crop, for continuous production of methane (CH₄) as a source of renewable energy. The sugar beet silage was used as the mono-substrate, which had a low pH of around 3.3–3.4, without the addition of manure. The mesophilic biogas digester was operated in a hydraulic retention time (HRT) range between 15 and 9.5 days, and an organic loading rate (OLR) range of between 6.33 and 10 g VS l⁻¹ d⁻¹. The highest specific gas production rate (spec. GPR) and CH₄ content were 0.67 l g VS⁻¹ d⁻¹ and 74%, respectively, obtained at an HRT of 9.5 days and OLR of 6.35 g VS l⁻¹ d⁻¹. The digester worked within the neutral pH range as well. Since this substrate lacked the availability of macro and micro nutrients, and the buffering capacity as well, external supplementation was definitely required to provide a stable and efficient operation, as provided using NH₄Cl and KHCO₃ in this case. The findings of this ongoing long-term fermentation of an extremely acidic biomass substrate without manure addition have reflected crucial information about how to appropriately maintain the operational and particularly the environmental parameters in an agricultural biogas plant.     

An anaerobic sequential batch reactor for enhanced continuous hydrogen production from fungal pretreated cornstalk hydrolysate

Anaerobic sequencing batch reactor (ASBR) process offers great potential for H₂ production from wastewaters. In this study, an ASBR was used at first time for enhanced continuous H₂ production from fungal pretreated cornstalk hydrolysate by Thermoanaerobacterium thermosaccharolyticum W16. The reactor was operated at different hydraulic retention times (HRTs) of 6, 12, 18, and 24 h by keeping the influent hydrolysate constant at 65 mmol sugars L⁻¹. Results showed that increasing the HRT from 6 to 12 h led to the H₂ production rate increased from 6.7 to the maximum of 9.6 mmol H₂ L⁻¹ h⁻¹ and the substrate conversion reached 90.3%, although the H₂ yield remained at the same level of 1.7 mol H₂ mol⁻¹ substrate. Taking into account both H₂ production and substrate utilization efficiencies, the optimum HRT for continuous H₂ production via an ASBR was determined at 12 h. Compared with other continuous H₂ production processes, ASBR yield higher H₂ production at relatively lower HRT. ASBR is shown to be another promising process for continuous fermentative H₂ production from lignocellulosic biomass.     

Optimal operational conditions for biohydrogen production from sugar refinery wastewater in an ASBR

The performance of biohydrogen production in an anaerobic sequencing batch reactor (ASBR) was evaluated with respect to variations in the key operational parameters – pH, hydraulic retention time HRT, and organic loading rate OLR using sugar refinery wastewater as substrate. Analysis of variance (ANOVA) indicated HRT had less significant influence on hydrogen content and yield in comparison to pH and OLR, whereas OLR has much impact on hydrogen production rate. Taxonomic analysis results showed that diverse bacterial species contributed to hydrogen production and the dominant species in the bioreactor were governed by all operational parameters. Even without pretreatment of the seed sludge, a high proportion of Clostridium spp. over the other bacterial species was observed at pH 5.5, and this is compatible with the high hydrogen productivity. Consequently, pH 5.5, HRT 10 h, and OLR 15 kg/m³ d were delineated as the optimal operational conditions for an ASBR fed with sugar refinery wastewater.     

Simultaneous thermophilic hydrogen production and phenol removal from palm oil mill effluent by Thermoanaerobacterium-rich sludge

Thermoanaerobacterium-rich sludge was used for hydrogen production and phenol removal from palm oil mill effluent (POME) in the presence of phenol concentration of 100–1000 mg/L. Thermoanaerobacterium-rich sludge yielded the most hydrogen of 4.2 L H₂/L-POME with 65% phenol removal efficiency at 400 mg/L phenol. Butyric acid and acetic acid were the main metabolites. The effects of oil palm ash, NH₄NO₃ and iron concentration (Fe²⁺) on hydrogen production and phenol removal efficiency from POME by Thermoanaerobacterium-rich sludge was investigated using response surface methodology (RSM). The RSM results indicated that the presence of 0.2 g Fe²⁺/L, 0.3 g/L NH₄NO₃ and 20 g/L oil palm ash in POME could improved phenol removal efficiency, with predicted hydrogen production and phenol removal efficiency of 3.45 L H₂/L-POME and 93%, respectively. In a confirmation experiment under optimized conditions highly reproducible results were obtained, with hydrogen production and phenol removal efficiency of 3.43 ± 0.12 L H₂/L-POME and 92 ± 1.5%, respectively. Simultaneous hydrogen production and phenol removal efficiency in continuous stirred tank reactor at hydraulic retention time (HRT) of 1 and 2 days were 4.0 L H₂/L-POME with 85% and 4.2 L H₂/L-POME with 92%, respectively. Phenol degrading Thermoanaerobacterium-rich sludge comprised of Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacterium aciditolerans, Desulfotomaculum sp., Bacillus coagulans and Clostridium uzonii. Phenol degrading Thermoanaerobacterium-rich sludge has great potential to harvest hydrogen from phenol-containing wastewater.     

Effect of hydraulic retention time on biohydrogen production from palm oil mill effluent in anaerobic sequencing batch reactor

The feasibility of hydrogen generation from palm oil mill effluent (POME), a high strength wastewater with high solid content, was evaluated in an anaerobic sequencing batch reactor (ASBR) using enriched mixed microflora, under mesophilic digestion process at 37 °C. Four different hydraulic retention times (HRT), ranging from 96 h to 36 h at constant cycle length of 24 h and various organic loading rate (OLR) concentrations were tested to evaluate hydrogen productivity and operational stability of ASBR. The results showed higher system efficiency was achieved at HRT of 72 h with maximum hydrogen production rate of 6.7 LH₂/L/d and hydrogen yield of 0.34 LH₂/g COD_feeding, while in longer and shorter HRTs, hydrogen productivity decreased. Organic matter removal efficiency was affected by HRT; accordingly, total and soluble COD removal reached more than 37% and 50%, respectively. Solid retention time (SRT) of 4-19 days was achieved at these wide ranges of HRTs. Butyrate was found to be the dominant metabolite in all HRTs. Low concentration of volatile fatty acid (VFA) confirmed the state of stability and efficiency of sequential batch mode operation was achieved in ASBR. Results also suggest that ASBR has the potential to offer high digestion rate and good stability of operation for POME treatment.     

High rate low solids methane fermentation of sorghum, corn and cellulose

Sorghum, sorghum/alpha-cellulose mixture, and corn were anaerobically digested at 55°C at effluent solids contents of 8–12% total solids (TS), using trace nutrient supplementation. Volatile solids (VS) loading rates at much higher levels than conventional maxima were maintained without volatile fatty acid (VFA) accumulation. Semi-continuously fed digesters with organic loading rates (OLR) up to 12 gVS kg⁻¹ d⁻¹ produced methane at rates up to 3.3 L kg⁻¹d⁻¹. Continuous feeding of corn at an OLR of 18 gVS kg⁻¹ d⁻¹ resulted in a methane production rate of 5.4 L kg⁻¹d⁻¹. VS removal efficiencies at maximum OLRs were 60% (sorghum) and 67% (corn). At an OLR of 4 gVS kg⁻¹ d⁻¹ sorghum alone as a feedstock led to excess ammonia-N accumulation. Excess ammonia did not accumulate at sorghum loading rates of 8 and 12 gVS kg⁻¹ d⁻¹ nor with a sorghum/alpha-cellulose mix loaded at 8 gVS kg⁻¹ d⁻¹. Instantaneous gas production rates were directly related to feedstock cell soluble content, with peak instantaneous biogas production rates from corn (OLR of 8 gVS kg⁻¹ d⁻¹ approaching 25 L kg⁻¹ d⁻¹ following a three-day feeding.     

Isolation of anoxygenic photosynthetic bacteria from Songkhla Lake for use in a two-staged biohydrogen production process from palm oil mill effluent

We are developing a process to produce biohydrogen from palm oil mill effluent. Part of this process will involve photohydrogen production from volatile fatty acids under low light conditions. We sought to isolate suitable bacteria for this purpose from Songkhla Lake in Southern Thailand. Enrichment for phototrophic bacteria from 34 samples was conducted providing acetate as a major carbon source and applying culturing conditions of anaerobic-low light (3000 lux) at 30 °C. Among the independent isolates from these enrichments 19 evolved hydrogen with productivities between 4 and 326 ml l⁻¹ d⁻¹. Isolate TN1 was the most efficient producer at a rate of 1.85 mol H₂ mol acetate⁻¹ with a light conversion efficiency of 1.07%. The maximum hydrogen production rate for TN1 was determined to be 43 ml l⁻¹ h⁻¹. Environmentally desirable features of photohydrogen production by TN1 included the absence of pH change in the cultures and no detectable residual CO₂.     

Biohydrogen production from pig slurry in a CSTR reactor system with mixed cultures under hyper-thermophilic temperature (70 °C)

A continuous stirred tank reactor (CSTR) (750 cm³ working volume) was operated with pig slurry under hyper-thermophilic (70 °C) temperature for hydrogen production. The hydraulic retention time (HRT) was 24 h and the organic loading rate was 24.9 g d⁻¹ of volatile solid (VS). The inoculum used in the hyper-thermophilic reactor was sludge obtained from a mesophilic methanogenic reactor. The continuous feeding with active biomass (inoculum) from the mesophilic methanogenic reactor was necessary in order to achieve hydrogen production. The hyper-thermophilic reactor started to produce hydrogen after a short adapted period of 4 days. During the steady state period the mean hydrogen yield was 3.65 cm³ g⁻¹ of volatile solid added. The high operation temperature of the reactor enhanced the hydrolytic activity in pig slurry and increased the volatile fatty acids (VFA) production. The short HRT (24 h) and the hyper-thermophilic temperature applied in the reactor were enough to prevent methanogenesis. No pre-treatment methods or other control methods for preventing methanogenesis were necessary. Hyper-thermophilic hydrogen production was demonstrated for the first time in a CSTR system, fed with pig slurry, using mixed culture. The results indicate that this system is a promising one for biohydrogen production from pig slurry.     

Effects of the reduction of the hydraulic retention time to 1.5 days at constant organic loading in CSTR,ASBR, and fixed-bed reactors – Performance and methanogenic community composition

The hydraulic retention time (HRT) is one of the key parameters in biogas processes and often it is postulated that a minimum HRT of 10–25 days is obligatory in continuous stirred tank reactors (CSTR) to prevent a washout of slow growing methanogens. In this study the effects of the reduction of the HRT from 6 to 1.5 days on performance and methanogenic community composition in different systems with and without immobilization operated with simulated thin stillage (STS) at mesophilic conditions and constant organic loading rates (OLR) of 10 g L⁻¹d⁻¹ of volatile solids were investigated. With the reduction of the HRT process instability was first observed in the anaerobic sequencing batch reactor (ASBR) (at HRT of 3 days) followed by the CSTR (at HRT of 2 days). The fixed bed reactor (FBR) was stable until the end of the experiment, but the reduction of the HRT to 1.5 days caused a decrease of the specific biogas production to about 450 L kg⁻¹ of VS compared to about 600 L kg⁻¹ of VS at HRTs of 4–5 days. Methanoculleus and Methanosarcina were the dominant genera under stable process conditions in the CSTR and the ASBR and members of Methanosaeta and Methanospirillum were only present at HRT of 4 days and lower. In the effluent of the FBR Methanosarcina spp. were not detected and Methanosaeta spp. were more abundant then in the other reactors.     

Start-up study of biohydrogen production from palm oil mill effluent in a lab-scale up-flow anaerobic sludge blanket fixed-film reactor

A start-up study of lab-scale up-flow anaerobic sludge blanket fixed-film reactor (UASFF) was conducted to produce biohydrogen from palm oil mill effluent (POME). The reactor was fed with POME at different hydraulic retention time (HRT) and organic loading rate (OLR) to obtain the optimum fermentation time for maximum hydrogen yield (HY). The results showed the HY, volumetric hydrogen production rate (VHPR), and COD removal of 0.5–1.1 L H₂/g COD_consumed, 1.98–4.1 L H₂ L^?1 day^?1, and 33.4–38.5%, respectively. The characteristic study on POME particles was analyzed by particle size distribution (PSD), Scanning electron microscopy (SEM), and Energy-dispersive X-ray spectroscopy (EDX). The microbial Shannon and Simpson diversity indices and Principal Component Analysis assessed the alpha and beta diversity, respectively. The results indicated the change of bacterial community diversity over the operation, in which Clostridium sensu stricto 1 and Lactobacillus species were contributed to hydrogen fermentation.     

Pilot-scale of biohythane production from palm oil mill effluent by two-stage thermophilic anaerobic fermentation

The pilot-scale of two-stage thermophilic (55 °C) for biohythane production from palm oil mill effluent (POME) was operated at hydraulic retention time (HRT) of 2 days and organic loading rate (OLR) of 27.5 gCOD/L⋅d) for first stage and HRT of 10 days and OLR of 5.5 gCOD/L⋅d for second stage. Biohythane production rate was 1.93 L-gas/L⋅d with biogas containing 11% H₂, 37% CO₂, and 52% CH₄. Recirculation of methane effluent mixed with POME at a ratio of 1:1 can control pH in the first stage at an optimal range of 5.0–6.5. Microbial community in hydrogen stage dominated by Thermoanaerobacterium sp., while methane stage dominated by Methanosarcina sp. The H₂/CH₄ ratio of biohythane was 0.13–0.18 which suitable for vehicle fuel. Biohythane production from POME could be promising cleaner biofuel with flexible and controllable H₂/CH₄ ratio.     

The use of acidified palm oil mill effluent for thermophilic biomethane production by changing the hydraulic retention time in anaerobic sequencing batch reactor

The feasibility of thermophilic biomethane production from acidified palm oil mill effluent (POME) was assessed in a 5 L anaerobic sequencing batch reactor (ASBR). The effects of various hydraulic retention time (HRT) (10-1 d) on methane production performance and the stability of ASBR in treating acidified POME were evaluated herein. It was found that the highest methane productivity of 5.65 L CH₄/L/d could be attained at HRT of 2 d. However, the removal of chemical oxygen demand (COD) and volatile fatty acid (VFA) at this HRT is rather low (65-62%) hence making it inefficient to operate at HRT 2 d since most of the contaminants remained in the liquid streams. Thus the most recommended HRT was 3 d with maximum methane productivity of 3.96 L CH₄/L/d with corresponding methane yield of 260.3 L CH₄/kgCOD_removed. The COD removal efficiency at 3 d HRT was 71%, and the VFA consumption was more than 80%. The correlation of total VFA: total alkalinity (TVFA: TA) at HRT of 3 d was found to be 0.1. This recommended HRT of 3 is equally shorter than any previously reported application of POME as a substrate for thermophilic biomethane.     

Biological hydrogen production from sweet sorghum syrup by mixed cultures using an anaerobic sequencing batch reactor (ASBR)

Continuous biological hydrogen production from sweet sorghum syrup by mixed cultures was investigated by using anaerobic sequencing batch reactor (ASBR). The ASBR was conducted based on the optimum condition obtained from batch experiment i.e. 25 g/L of total sugar concentration, 1.45 g/L of FeSO₄ and pH of 5.0. Feasibility of continuous hydrogen fermentation in ASBR operation at room temperature (30 ± 3 °C) with different hydraulic retention time (HRT) of 96, 48, 24 and 12 hr and cycle periods consisting of filling (20 min), settling (20 min), and decanting (20 min) phases was analyzed. Results showed that hydrogen content decreased with a reduction in HRT i.e. from 42.93% (96 hr HRT) to 21.06% (12 hr HRT). Decrease in HRT resulted in a decrease of solvents produced which was from 10.77 to 2.67 mg/L for acetone and 78.25 mg/L to zero for butanol at HRT of 96 hr-12 hr, respectively. HRT of 24 hr was the optimum condition for ASBR operation indicated by the maximum hydrogen yield of 0.68 mol H₂/mol hexose. The microbial determination in DGGE analysis indicated that the well-known hydrogen producers Clostridia species were dominant in the reacting step. The presence of Sporolactobacillus sp. which could excrete the bacteriocins causing the adverse effect on hydrogen-producing bacteria might responsible for the low hydrogen content obtained.     

Comparison of the effectivities of two-phase and single-phase anaerobic sequencing batch reactors during dairy wastewater treatment

The performances of anaerobic sequencing batch reactors fed with two different substrates were studied. The substrates were raw acid whey and acid whey fermented with Kluyveromyces lactis in order to investigate the suitability of ethanol for biogas production. The organic loading rates (OLRs) during the experiment ranged from 1.6 to 12.8 g COD dm⁻³ d⁻¹ and the corresponding decreasing hydraulic retention times from 40 to 5 days for both reactor systems. The efficiency of each system depended on the OLR: the highest COD removal rate was observed at the lowest OLR applied (about 100% in both systems), and at maximum OLR the COD removal efficiency was 68% for the reactors fed with the raw whey and 80% for those fed with the pre-fermented whey. Under the same high OLR conditions the methane yield was 0.122 dm⁻³ CH₄ g⁻¹ COD_degraded for the anaerobic digesters fed with the untreated whey, and 0.197 dm⁻³ CH₄ g⁻¹ COD_degraded for those fed with the pre-fermented whey. The digesters functioned without pH control. At the maximum OLR the pH in the reactors fed with the raw acid whey was 5.1, while in those fed with the pre-fermented whey it was 7.15.The results demonstrate that the use of the pre-fermented acid whey as substrate for anaerobic digestion without pH control is feasible, especially at high OLR levels. This substrate is preferable to the raw acid whey, because of the ethanol formed as a non-acidic fermentation product of the yeast.     

Structural, thermal and electrochemical properties of layered perovskite SmBaCo2O5+d, a potential cathode material for intermediate-temperature solid oxide fuel cells

The synthesis, conductivity properties, area specific resistance (ASR) and thermal expansion behaviour of the layered perovskite SmBaCo₂O_5+d (SBCO) are investigated for use as a cathode material for intermediate-temperature solid oxide fuel cells (IT-SOFCs). The SBCO is prepared and shows the expected orthorhombic pattern. The electrical conductivity of SBCO exhibits a metal–insulator transition at about 200 °C. The maximum conductivity is 570 S cm⁻¹ at 200 °C and its value is higher than 170 S cm⁻¹ over the whole temperature range investigated. Under variable oxygen partial pressure SBCO is found to be a p-type conductor. The ASR of a composite cathode (50 wt% SBCO and 50 wt% Ce_0.9Gd_0.1O_2−d, SBCO:50) on a Ce_0.9Gd_0.1O_2−d (CGO91) electrolyte is 0.05 Ω cm² at 700 °C. An abrupt increase in thermal expansion is observed in the vicinity of 320 °C and is ascribed to the generation of oxygen vacancies. The coefficients of thermal expansion (CTE) of SBCO is 19.7 and 20.0 × 10⁻⁶ K⁻¹ at 600 and 700 °C, respectively. By contrast, CTE values for SBCO:50 are 12.3, 12.5 and 12.7 × 10⁻⁶ K⁻¹ at 500, 600 and 700 °C, that is, very similar to the value of the CGO91 electrolyte.     

La1−xSrxMO3 (M = Mn, Fe) perovskites as materials for thermochemical hydrogen production in conventional and membrane reactors

La_1−xSr_xMO₃ (M = Mn, Fe) perovskites are investigated as potential redox materials for the thermochemical production of hydrogen. Thermogravimetric oxidation/reduction experiments indicated that the materials are able to lose and uptake oxygen reversibly from their lattice up to 5.5 wt.% for La_1−xSr_xMnO₃ with x = 1 and up to 1.7 wt.% for La_1−xSr_xFeO₃ with x = 0. Pulse reaction experiments indicated that the materials can be used as redox catalysts in a redox process where water is dissociated giving rise to the production of pure hydrogen during the oxidation step. The oxidation and reduction steps can be combined in a membrane reactor constructed from dense perovskite membranes towards a continuous and isothermal operation. The system is also able to operate on partial pressure-based desorption without the need of a carbon-containing reductant, so that a process towards hydrogen production, based only on renewable hydrogen source such as water, can be established. At steady state and 900 ^°C, 25 ± 7 cm³ (STP) H₂ m⁻² min⁻¹ is produced in purified state.     

Effect of chitosan on reactor performance and population of specific methanogens in a modified CSTR treating raw POME

In this study, chitosan was periodically added to a CSTR treating raw POME in order to retain more working microorganisms at high OLRs. The data indicated that the CSTR with chitosan addition can be operated with reactor stability at an OLR of 26.5 kg m⁻³ d⁻¹ which is approximately 7.5 kg m⁻³ d⁻¹ higher than that for the control CSTR without chitosan addition. In the control CSTR, the biogas production did not increase with increased OLR, and the overall process was limited by slow methanogenic rates. For the CSTR with chitosan addition, the biogas production was 9.39 m³ m⁻³ d⁻¹ with a methane volume fraction of 68% at an OLR of 26.5 kg m⁻³ d⁻¹. Corresponding to this increase in methane production, it was found that Methanosarcinales numbers were significantly higher (P < 0.05) in the CSTR with chitosan addition than in the control CSTR.     

Isolation and evaluation of a high H2-producing lab isolate from cow dung

Hydrogen producing bacterial strain was isolated from Indian cow dung and identified of the bacterial family Enterobacteriaceae. This lab isolate was differentiated from Citrobacter Y-19 at molecular level by using RAPD, PCR based technique, and OPO-03₄₆₀ and OPO-17₈₀₀ RAPD marker for this specific strain (lab isolate) was identified. Fermentative studies were investigated for important parameters, starting with pH of the culture, temperature, inoculum age and inoculum volume, initial substrate concentration and different substrates. Among different substrates, dextrose and sucrose were the preferred substrates for hydrogen production. The optimal starting pH of the culture was found to be 5.0. The H₂ production increased with increase in temperature up to 30 °C. The maximum value of H₂ production was recorded when inoculum volume was 12.5% of the culture broth and inoculum age was 14 h. Under batch fermentation conditions, the maximum hydrogen production rate and yield were 355.2 ml l⁻¹ h⁻¹ and 2.1 mol/mol glucose (conversion 35%), respectively. These results indicate that this lab isolate is an ideal hydrogen producer.     

Hydrogen–methane production from swine manure: Effect of pretreatment and VFAs accumulation on gas yield

A two-phase anaerobic process to produce hydrogen and methane from swine manure was investigated, using pretreated sludge with heat, acid and alkali treatment as inoculum. The relative order of pretreatment methods of H₂ productivity effectiveness and CH₄ productivity effectiveness produced by the residua of the first phase was heat treatment > alkali treatment > acid treatment. When the inoculum sludge was heat-treated at 80°C for 30 min, the H₂ and CH₄ production rate was the highest of 36.6, 201.7 ml (g TS)_added⁻¹. There were significant correlations between biogas production and accumulation of acetic acid and butyric acids. When propionic acid and total VFA concentrations reached about 2850 mg L⁻¹ and 10.0 g L⁻¹, respectively, the average H₂ production rate and H₂ content decreased from 7.6 ml d⁻¹(g VS)_added⁻¹ and 55.3% to 1.4 ml d⁻¹(g VS)_added⁻¹ and 43.2%, respectively. The activity of methanogenic bacteria was inhibited to a significant extent when the total VFA concentration was above 10.0 g L⁻¹, but this inhibitory effect weakened when the VFA concentration fell to 6200–8500 mg L⁻¹. Correspondingly, average CH₄ production rate increased from 4.0 ml d⁻¹(g TS)_added⁻¹ to 12.5 ml d⁻¹(g TS)_added⁻¹. Propionic acid was degraded rapidly only when acetic and butyric acid concentrations dropped to 2500 mg L⁻¹ and 1000 mg L⁻¹, respectively.