Full-scale and laboratory-scale anaerobic treatment of citric acid production wastewater

This paper reviews the operation of a full-scale, fixed-bed digester treating a citric acid production wastewater with a COD: sulphate ratio of 3–4 : 1. Support matrix pieces were removed from the digester at intervals during the first 5 years of operation in order to quantify the vertical distribution of biomass within the digester. Detailed analysis of the digester biomass after 5 years of operation indicated that H2 and propionate-utilising SRB had outcompeted hydrogenophilic methanogens and propionate syntrophs. Acetoclastic methanogens were shown to play the dominant role in acetate conversion. Butyrate and ethanol-degrading syntrophs also remained active in the digester after 5 years of operation.Laboratory-scale hybrid reactor treatment at 55 °C of a diluted molasses influent, with and without sulphate supplementation, showed that the reactors could be operated with high stability at volumetric loading rates of 24 kgCOD.m-3.d-1 (12 h HRT). In the presence of sulphate (2 g/l-1; COD/sulphate ratio of 6 : 1), acetate conversion was severely inhibited, resulting in effluent acetate concentrations of up to 4000 mg.l-1.     

Toxic effects of thiol-reactive compounds on anaerobic biomass

The objective of this study was to characterize the toxic effects of three well known thiol-reactive electrophilic compounds, N-ethylmaleimide (NEM), pentachlorophenol (PCP) and 1-chloro-2,4-dinitrobenzene (CDNB) on anaerobic biotransformation process. The work was part of a larger investigation on potassium efflux as a possible response mechanism of anaerobic microorganisms to the presence of thiol-reactive organic compounds and the interference of such compounds on the reductive dehalogenation process. Using anaerobic toxicity assay (ATA) and granular anaerobic biomass from a full-scale upflow anaerobic sludge blanket (UASB) reactor, inhibitory concentrations of these compounds that reduced the microbial activity of granular biomass to 50% of a control (IC50) were determined to be 592, 0.97, and 450 mg/l for NEM, PCP, and CDNB, respectively. Toxicity of NEM was also tested on anaerobic biomass from a municipal wastewater treatment plant digester and slightly lower IC50 of 532 mg/l was obtained. The results presented here indicate that anaerobic biomass can acclimate to the three thiol-reactive compounds studied and recover from inhibition as long as the toxicant concentration is below a threshold level. That threshold concentration was found to be 500 mg/l for NEM on biomass from the municipal digester, 1 mg/l for PCP, and 500 mg/l for CDNB, both on granular biomass. Granular anaerobic biomass showed recovery even at NEM concentrations of 1000 mg/l.     

Acetoclastic methanogenic activity measurement by a titration bioassay.

A titration bioassay, designed to accurately determine the activity of acetoclastic methanogens, is described that also allows evaluation of inhibition due to potential toxicants on the active biomass. The instrument is made of a pH-stat connected to an anaerobic batch reactor. Acetate is blended and mixed with anaerobic sludge in the reactor where a 1:1 N2 and CO2 mixture is sparged at the beginning of each test. As the acetoclastic methanogens consume acetate, the pH increase, and the titration unit adds acetic acid and keeps the pH constant. The rate of titrant addition is directly proportional to the methanogenic activity. A very useful feature of the system is its potential to operate for long periods (days) at constant pH and substrate (acetate) concentration. The theoretical background and principle of operation are described as well as some of the practical problems encountered with the use of the instrument. Estimation of kinetic constants for an anaerobic culture according to the Michaelis-Menten model is presented. Examples of inhibition by inorganics (NaCl) and chlorinated solvents (chloroform) are also given.     

Evaluation of parameters for monitoring an anaerobic co-digestion process

The system investigated in this study is an anaerobic digester at a municipal wastewater treatment plant operating on sludge from the wastewater treatment, co-digested with carbohydrate-rich food-processing waste. The digester is run below maximum capacity to prevent overload. Process monitoring at present is not extensive, even for the measurement of on-line gas production rate and off-line pH. Much could be gained if a better program for monitoring and control was developed, so that the full capacity of the system could be utilised without the risk of overload. The only limit presently set for correct process operation is that the pH should be above 6.8. In the present investigation, the pH was compared with alkalinity, gas production rate, gas composition and the concentration of volatile fatty acids (VFA). Changes in organic load were monitored in the full-scale anaerobic digester and in laboratory-scale models of the plant. Gas-phase parameters showed a slow response to changes in load. The VFA concentrations were superior for indicating overload of the microbial system, but alkalinity and pH also proved to be good monitoring parameters. The possibility of using pH as a process indicator is, however, strongly dependent on the buffering capacity. In this study, a minor change in the amount of carbohydrates in the substrate had drastic effects on the buffering effect of the system. Received: 21 January 2000 / Received revision: 10 July 2000 / Accepted: 16 July 2000     

Importance of cobalt for individual trophic groups in an anaerobic methanol-degrading consortium.

Methanol is an important anaerobic substrate in industrial wastewater treatment and the natural environment. Previous studies indicate that cobalt greatly stimulates methane formation during anaerobic treatment of methanolic wastewaters. To evaluate the effect of cobalt in a mixed culture, a sludge with low background levels of cobalt was cultivated in an upflow anaerobic sludge blanket reactor. Specific inhibitors in batch assays were then utilized to study the effect of cobalt on the growth rate and activity of different microorganisms involved in the anaerobic degradation of methanol. Only methylotrophic methanogens and acetogens were stimulated by cobalt additions, while the other trophic groups utilizing downstream intermediates, H2-CO2 or acetate, were largely unaffected. The optimal concentration of cobalt for the growth and activity of methanol-utilizing methanogens and acetogens was 0.05 mg liter-1. The higher requirement of cobalt is presumably due to the previously reported production of unique corrinoid-containing enzymes (or coenzymes) by direct utilizers of methanol. This distinctly high requirement of cobalt by methylotrophs should be considered during methanolic wastewater treatment. Methylotroph methanogens presented a 60-fold-higher affinity for methanol than acetogens. This result in combination with the fact that acetogens grow slightly faster than methanogens under optimal cobalt conditions indicates that acetogens can outcompete methanogens only when reactor methanol and cobalt concentrations are high, provided enough inorganic carbon is available.     

Inhibitory effect of chlorinated guaiacols on methanogenic activity of anaerobic digester sludge

Of four chlorinated guaiacols, tetrachloroguaiacol at 62 M inhibited acetate methanogenesis, the strongest decreasing activity by 50%. 4,5,6-Trichloroguaiacol, 4,5-dichloroguaiacol, and 4-chloroguaiacol showed 50% inhibition at 0.13, 0.32, and 1.50 mM, respectively. Degradation test results of volatile fatty acids (acetic, propionic, and butyric acid) by anaerobic digester sludge (stored 5 weeks) indicated that syntrophic butyrate degraders of this sludge were more sensitive to tetrachloroguaiacol than acetoclastic methanogens and syntrophic propionate degraders.     

Performance of an UASB reactor treating synthetic wastewater at low-temperature using cold-adapted seed slurry

The present study is an attempt to investigate if a long-term acclimation of digester contents to low-temperatures would improve wastewater treatment at low-temperatures similar to mesophilic ranges. The feasibility of low-temperature (15 °C) anaerobic treatment of synthetic wastewater in an upflow anaerobic sludge blanket reactor was studied using inoculum from a cattle manure digester adapted to 15 °C. The effect of varying hydraulic retention time was studied by decreasing the retention time from 7 days to 1 day. Under a constant temperature of 15 °C with a hydraulic retention time of 1 day and a corresponding loading rate of 7.2 g-chemical oxygen demand (COD)/l/day, 90–95% removal efficiency was achieved. The methane production of 250 l/kg-COD removed at standard temperature pressure (STP) is a major highlight of the study complementing the high treatment efficiency achieved. Loading rates >5 g-COD/l/day was accompanied by increase in effluent volatile fatty acids (VFA) concentrations. Due to the presence of a high concentration of active granular sludge in the lower compartment of the reactor, 80% reduction of COD occurred within the granular bed of the reactor. Treatment of low strength wastewater for a short period showed 70–75% removal efficiencies with methane yield of 300 l/kg-COD removed. Specific methanogenic activity profiles of the anaerobic biomass revealed low-temperature (15 °C) optima, indicating selection of cold-active microorganisms during the acclimation process. The SMA assays also indicate the development of a putatively psychrophilic acetoclastic methanogenic community and biogas analysis showed 75% efficiency in energy recovery as methane.     

Bacteriological composition and structure of granular sludge adapted to different substrates.

The bacteriological composition and ultrastructure of mesophilic granular methanogenic sludge from a large-scale Upflow Anaerobic Sludge Blanket reactor treating wastewater from a sugar plant and of sludge granules adapted to ethanol and propionate were studied by counting different bacterial groups and by immunocytochemical methods. Propionate-grown granular sludge consisted of two types of clusters, those of a rod-shaped bacterium immunologically related to Methanothrix soehngenii and those consisting of two different types of bacteria with a specific spatial orientation. One of these bacteria reacted with antiserum against Methanobrevibacter arboriphilus AZ, whereas the other is most likely a propionate-oxidizing bacterium immunologically unrelated to Syntrophobacter wolinii. Sludge granules obtained from the large-scale Upflow Anaerobic Sludge Blanket reactor and granules cultivated on ethanol did not show the typical spatial orientation of bacteria. Examination of the bacterial composition of the three types of granules by light and electron microscopy, the most-probable-number method, and by isolations showed that M. arboriphilus and M. soehngenii were the most abundant hydrogenotrophic and acetoclastic methanogens in propionate-grown sludge. Methanospirillum hungatei and Methanosarcina barkeri predominated in ethanol-grown granules, whereas many morphotypes of methanogens were abundant in granules from the full-scale reactor.     

Bacteriological composition and structure of granular sludge adapted to different substrates

The bacteriological composition and ultrastructure of mesophilic granular methanogenic sludge from a large-scale Upflow Anaerobic Sludge Blanket reactor treating wastewater from a sugar plant and of sludge granules adapted to ethanol and propionate were studied by counting different bacterial groups and by immunocytochemical methods. Propionate-grown granular sludge consisted of two types of clusters, those of a rod-shaped bacterium immunologically related to Methanothrix soehngenii and those consisting of two different types of bacteria with a specific spatial orientation. One of these bacteria reacted with antiserum against Methanobrevibacter arboriphilus AZ, whereas the other is most likely a propionate-oxidizing bacterium immunologically unrelated to Syntrophobacter wolinii. Sludge granules obtained from the large-scale Upflow Anaerobic Sludge Blanket reactor and granules cultivated on ethanol did not show the typical spatial orientation of bacteria. Examination of the bacterial composition of the three types of granules by light and electron microscopy, the most-probable-number method, and by isolations showed that M. arboriphilus and M. soehngenii were the most abundant hydrogenotrophic and acetoclastic methanogens in propionate-grown sludge. Methanospirillum hungatei and Methanosarcina barkeri predominated in ethanol-grown granules, whereas many morphotypes of methanogens were abundant in granules from the full-scale reactor.     

Concurrent microscopic observations and activity measurements of cellulose hydrolyzing and methanogenic populations during the batch anaerobic digestion of crystalline cellulose

This study compares process data with microscopic observations from an anaerobic digestion of organic particles. As the first part of the study, this article presents detailed observations of microbial biofilm architecture and structure in a 1.25-L batch digester where all particles are of an equal age. Microcrystalline cellulose was used as the sole carbon and energy source. The digestions were inoculated with either leachate from a 220-L anaerobic municipal solid waste digester or strained rumen contents from a fistulated cow. The hydrolysis rate, when normalized by the amount of cellulose remaining in the reactor, was found to reach a constant value 1 day after inoculation with rumen fluid, and 3 days after inoculating with digester leachate. A constant value of a mass specific hydrolysis rate is argued to represent full colonization of the cellulose surface and first-order kinetics only apply after this point. Additionally, the first-order hydrolysis rate constant, once surfaces were saturated with biofilm, was found to be two times higher with a rumen inoculum, compared to a digester leachate inoculum. Images generated by fluorescence in situ hybridization (FISH) probing and confocal laser scanning microscopy show that the microbial communities involved in the anaerobic biodegradation process exist entirely within the biofilm. For the reactor conditions used in these experiments, the predominant methanogens exist in ball-shaped colonies within the biofilm.     

Endurance of methanogenic archaea in anaerobic bioreactors treating oleate-based wastewater

Methanogenic archaea are reported as very sensitive to lipids and long chain fatty acids (LCFA). Therefore, in conventional anaerobic processes, methane recovery during LCFA-rich wastewater treatment is usually low. By applying a start-up strategy, based on a sequence of step feeding and reaction cycles, an oleate-rich wastewater was efficiently treated at an organic loading rate of 21 kg COD m^?3 day^?1 (50 % as oleate), showing a methane recovery of 72 %. In the present work, the archaeal community developed in that reactor is investigated using a 16S rRNA gene approach. This is the first time that methanogens present in a bioreactor converting efficiently high loads of LCFA to methane are monitored. Denaturing gradient gel electrophoresis profiling showed that major changes on the archaeal community took place during the bioreactor start-up, where phases of continuous feeding were alternated with batch phases. After the start-up, a stable archaeal community (similarity higher than 84 %) was observed and maintained throughout the continuous operation. This community exhibited high LCFA tolerance and high acetoclastic and hydrogenotrophic activity. Cloning and sequencing results showed that Methanobacterium- and Methanosaeta-like microorganisms prevailed in the system and were able to tolerate and endure during prolonged exposure to high LCFA loads, despite the previously reported LCFA sensitivity of methanogens.     

Inhibition of anaerobic digestion process: a review

Anaerobic digestion is an attractive waste treatment practice in which both pollution control and energy recovery can be achieved. Many agricultural and industrial wastes are ideal candidates for anaerobic digestion because they contain high levels of easily biodegradable materials. Problems such as low methane yield and process instability are often encountered in anaerobic digestion, preventing this technique from being widely applied. A wide variety of inhibitory substances are the primary cause of anaerobic digester upset or failure since they are present in substantial concentrations in wastes. Considerable research efforts have been made to identify the mechanism and the controlling factors of inhibition. This review provides a detailed summary of the research conducted on the inhibition of anaerobic processes. The inhibitors commonly present in anaerobic digesters include ammonia, sulfide, light metal ions, heavy metals, and organics. Due to the difference in anaerobic inocula, waste composition, and experimental methods and conditions, literature results on inhibition caused by specific toxicants vary widely. Co-digestion with other waste, adaptation of microorganisms to inhibitory substances, and incorporation of methods to remove or counteract toxicants before anaerobic digestion can significantly improve the waste treatment efficiency.     

Inhibition of the anaerobic digestion process by linear alkylbenzene sulfonates

Linear Alkylbenzene Sulfonates (LAS) are the most widely used synthetic anionicsurfactants. They are anthropogenic, toxic compounds and are found in the primarysludge generated in municipal wastewater treatment plants. Primary sludge is usuallystabilized anaerobically and therefore it is important to investigate the effect of thesexenobiotic compounds on an anaerobic environment. The inhibitory effect of LinearAlkylbenzene Sulfonates (LAS) on the acetogenic and methanogenic step of theanaerobic digestion process was studied. LAS inhibit both acetogenesis from propionate and methanogenesis from acetate and hydrogen and it is shown that the propionate-utilising bacteria are more sensitive to the presence of LAS than the acetoclastic methanogens. It has been proven that the inhibition intensity depends on the solids concentration and thus the term ``biomass specific LAS concentration' has been introduced in order to describe the phenomenon better. Conclusively, it is believed that the inhibitory effect of LAS is the main reason that anaerobic microbial enrichments on LAS have not been succeeded yet. Also, the inhibition caused by LAS on the acetogenic and methanogenic step of the anaerobic digestion process should be seriously taken into consideration when wastewater from a surfactant producing industry is to be treated biologically or enter a municipal wastewater treatment plant that employs anaerobic technology. The upper allowable biomass specific LAS concentration should be 14 mg LAS (gVSS)^-1.     

Persistence of <Emphasis Type="Italic">Methanosaeta</Emphasis> populations in anaerobic digestion during process instability

Anaerobic digestion is a sustainable technology for the treatment of organic waste and production of biogas. Acetoclastic methanogenesis accounts for the majority of methane production in anaerobic digestion. Therefore, sustaining robust acetoclastic methanogens is important for stable process performance. Due to faster growth kinetics at high acetate concentrations, it has been considered that Methanosarcina would be more prevalent than Methanosaeta in unstable anaerobic digestion processes which frequently experience high acetate levels. Methanogen population dynamics were monitored in multiple continuous anaerobic digesters for 500 days. Results from quantitative polymerase chain reaction analysis show that Methanosaeta dominated over Methanosarcina in anaerobic digestion at high acetate levels up to 44 mM, suggesting the potential of Methanosaeta as a robust and efficient acetoclastic candidate for resilient anaerobic methane conversion. Further efforts are needed to identify mechanisms contributing to the unexpected competitiveness of these methanogens at high acetate levels observed in this study.     

Inhibition of Methanogenesis from Acetate in Granular Sludge by Long-Chain Fatty Acids

The effect of four saturated long-chain fatty acids (caprylic, capric, lauric, and myristic) and one unsaturated long-chain fatty acid (oleic) on the microbial formation of methane from acetate was investigated in batch anaerobic toxicity assays. The tests were carried out with granular sludge from an upflow anaerobic sludge bed reactor. In this sludge, Methanothrix spp. are the predominant acetoclastic methanogens. Lauric acid appeared to be the most versatile inhibitor: inhibition started at 1.6 mM, and at 4.3 mM the maximum specific acetoclastic methanogenic activity had been reduced to 50%. Caprylic acid appeared to be only slightly inhibitory. Oleic acid was almost as inhibitory as lauric acid. Although adsorption of the inhibitor on the cell wall might play an important role in the mechanism of inhibition, the inhibition was found to be correlated with concentration rather than with the amount per unit of biomass. In practical situations, as in anaerobic waste treatment processes, synergism can be expected to enhance the inhibition of methanogenesis. In the present research a background concentration of lauric acid below its MIC strongly enhanced the toxicity of capric acid and (to an even greater extent) myristic acid.     

"Candidatus Cloacamonas acidaminovorans": genome sequence reconstruction provides a first glimpse of a new bacterial division

Many microorganisms live in anaerobic environments. Most of these microorganisms have not yet been cultivated. Here, we present, from a metagenomic analysis of an anaerobic digester of a municipal wastewater treatment plant, a reconstruction of the complete genome of a bacterium belonging to the WWE1 candidate division. In silico proteome analysis indicated that this bacterium might derive most of its carbon and energy from the fermentation of amino acids, and hence, it was provisionally classified as "Candidatus Cloacamonas acidaminovorans." "Candidatus Cloacamonas acidaminovorans" is probably a syntrophic bacterium that is present in many anaerobic digesters. This report highlights how environmental sequence data might provide genomic and functional information about a new bacterial clade whose members are involved in anaerobic digestion.     

Organic Matter Degradation Kinetics in an Anaerobic Thermophilic Fluidised Bed Bioreactor

This paper describes the thermophilic anaerobic biodegradation of wine distillery wastewater (vinasses) in a laboratory fluidised bed reactor (AFB) with a porous support medium. The experimental protocol was defined to examine the effect of increasing organic loading rate on the efficiency of AFB and to report on its steady-state performance. Moreover, in order to evaluate treatment efficiency and to investigate fermentation kinetics in an AFB reactor, experimental data were used to estimate the ‘active biomass’ concentration using an autocatalytic kinetic model proposed in this paper, since viable biomass in AFB reactors is very difficult to measure experimentally. The AFB reactor was subjected to a program of steady-state operation over a range of hydraulic retention time (HRTs) of 2.5–0.37 days and organic loading rate (OLRs) up to 5.88 kgCOD/m³/day in order to evaluate its treatment capacity. The AFB reactor was initially operated with organic loading rate of 5.88 kgCOD/m³/day and HRT of 2.5 days. The chemical oxygen demand (COD) removal efficiency was found to be 96.5% in the reactor while the methane content of biogas produced in the digester reached 1.08 m³/m³digester/day. Over 94 days operating period, an OLR of 32 kgCOD/m³/day at a food-to-micro-organisms (F:M) ratio of 0.55 kgCOD/kgVS_att/day was achieved with 81.5% COD removal efficiency in the experimental AFB reactor. At this moment, the methane content of biogas produced in the digester reached 9.0 m³/m³digester/day. The proposed kinetic model is able to estimate kinetic constants of the biodegradation process: non-biodegradable substrate (S_nb) and active adhered biomass concentration (X_a). The parameters of the model were obtained by the curve-fitting method to the proposed kinetic model using the COD as substrate of the anaerobic process and assuming a maximum specific μ_max: 0.72 per day. The comparison of the measured concentration of volatile attached solids (VS_att) with the estimated ‘active’ biomass concentration indicated that extremely high ‘active biomass’ concentrations can be maintained in the system because biofilm thickness is limited by the liquid flow rate applied. This is due to the fact that the anaerobic fluidised bed system retains the growth support medium in suspension by drag forces exerted by upflowing wastewater, and the distribution of biomass holdup (in the form of a biofilm) is thus relatively uniform.     

The effect of pharmaceuticals on the kinetics of methanogenesis and acetogenesis

In this study, the widely used anaerobic digestion model (ADM1) was used in order to simulate the inhibition of three pharmaceuticals, propranolol hydrochloride, ofloxacin and diclofenac sodium, on two groups of microorganisms, acetogens and acetoclastic methanogens, the most sensitive microorganisms groups involved in the anaerobic digestion process. The specific maximum consumption rate and saturation constant of acetate and propionate degraders were estimated through fitting the model to experimental data taken from continuous and batch experiments. A modified non-competitive inhibition function was used, and the inhibition constants were estimated using data from Batch experiments conducted at various concentrations of pharmaceuticals using enriched cultures with propionate and acetate degraders. It was found that propranolol hydrochloride was the most inhibitory pharmaceutical to both microorganisms groups.