Effects of free long-chain fatty acids on thermophilic anaerobic digestion

Summary Low concentrations of the long-chain fatty acids oleate and stearate inhibited all steps of the anaerobic thermophilic biogas process during digestion of cattle manure. The lag phase increased when the concentrations of oleate and stearate were 0.2 g/l and 0.5 g/l, respectively, and no growth was found at concentrations of 0.5 g/l for oleate and 1.0 g/l for stearate. The toxic effect of these acids was permanent as growth did not occur when inhibited cultures were diluted to a non-inhibitory concentration. No adaptation to the fatty acids toxicity was observed by pre-exposing the cultures to non-inhibitory concentrations and the inhibitory response was the same as for cultures not pre-exposed to the fatty acids. Oleate was less inhibitory when added as a neutral oil in the form of the glycerol ester. This indicates that it is the free fatty acid that influences the bacterial activity. Correspondence to: B. K. Ahring     

Effect of the clay mineral bentonite on ammonia inhibition of anaerobic thermophilic reactors degrading animal waste

Addition of bentonite or the waste product bentonite-bound oil counteracted to some extent the inhibitory effect of ammonia during thermophilic anaerobic digestion of cattle manure. In continuously-fed reactor experiments, addition of bentonite or bentonite-bound oil delayed the onset of the inhibition and aided process recovery after initial inhibition. The effect was observed only when the ammonia concentration was increased gradually, indicating that the major effect of bentonite and BBO was not through a direct antagonistic effect towards ammonia but through an increased process resistance to toxic compounds. In batch experiments bentonite had a similar stimulatory effect leading to a decreased lag phase and increased methane production rate in ammonia inhibited reactors.     

Effects of mixing on methane production during thermophilic anaerobic digestion of manure: lab-scale and pilot-scale studies

The effect of mixing on anaerobic digestion of manure was evaluated in lab-scale and pilot-scale experiments at 55 degrees C. The effect of continuous (control), minimal (mixing for 10 min prior to extraction/feeding) and intermittent mixing (withholding mixing for 2h prior to extraction/feeding) on methane production was investigated in three lab-scale continuously stirred tank reactors. On comparison to continuous mixing, intermittent and minimal mixing strategies improved methane productions by 1.3% and 12.5%, respectively. Pilot-scale studies also supported the lab-scale results with an average 7% increase in biogas yields during intermittent mixing compared to continuous mixing. The effect of mixing intensities (minimal, gentle or vigorous) in batch assays at 55 degrees C showed that when the process was overloaded by high substrate to inoculum ratio (40/60), gentle (35 times per minute) or minimal mixing (10 min mixing before feeding) was advantageous compared to vigorous mixing (110 times per minute). On the other hand, under low substrate to inoculum ratio (10/90), gentle mixing was the best. The study thus indicated that mixing schemes and intensities have some effect on anaerobic digestion of manures.     

Effect of temperature and temperature fluctuation on thermophilic anaerobic digestion of cattle manure

The influence of temperature, 50 and 60 degrees C, at hydraulic retention times (HRTs) of 20 and 10 days, on the performance of anaerobic digestion of cow manure has been investigated in completely stirred tank reactors (CSTRs). Furthermore, the effect of both daily downward and daily upward temperature fluctuations has been studied. In the daily downward temperature fluctuation regime the temperatures of each reactor was reduced by 10 degrees C for 10 h while in the daily upward fluctuation regime the temperature of each reactor was increased 10 degrees C for 5 h. The results show that the methane production rate at 60 degrees C is lower than that at 50 degrees C at all experimental conditions of imposed HRT except when downward temperature fluctuations were applied at an HRT of 10 days. It also was found that the free ammonia concentration not only affects the acetate-utilising bacteria but also the hydrolysis and acidification process. The upward temperature fluctuation affects the maximum specific methanogenesis activity more severely as compared to imposed downward temperature fluctuations. The results clearly reveal the possibility of using available solar energy at daytime to heat up the reactor(s) without the need of heat storage during nights, especially at an operational temperature of 50 degrees C and at a 20 days HRT, and without the jeopardising of the overheating.     

Azo dye reduction by mesophilic and thermophilic anaerobic consortia

The reduction of the azo dye model compounds Reactive Red 2 (RR2) and Reactive Orange 14 (RO14) by mesophilic (30 degrees C) and thermophilic (55 degrees C) anaerobic consortia was studied in batch assays. The contribution of fermentative and methanogenic microorganisms in both temperatures was evaluated in the presence of the fermentative substrate glucose and the methanogenic substrates acetate, H2/CO2, methanol, and formate. Additionally, the effect of the redox mediator riboflavin on electron shuttling was assessed. We concluded that the application of thermophilic anaerobic treatment is an interesting option for the reductive decolorization of azo dyes compared to mesophilic conditions. The use of high temperature may decrease or even take the place of the need for continuous redox mediator dosage in bioreactors, contrarily to the evident effect of those compounds on dye reduction under mesophilic conditions. Both fermenters and methanogens may play an important role during reductive decolorization of dyes, in which mediators are important not only for allowing the different microbes to participate more effectively in this complex reductive biochemistry but also for assisting in the competition for electrons between dyes and other organic and inorganic electron acceptors.     

Growth media in anaerobic fermentative processes: The underestimated potential of thermophilic fermentation and anaerobic digestion

Fermentation and anaerobic digestion of organic waste and wastewater is broadly studied and applied. Despite widely available results and data for these processes, comparison of the generated results in literature is difficult. Not only due to the used variety of process conditions, but also because of the many different growth media that are used. Composition of growth media can influence biogas production (rates) and lead to process instability during anaerobic digestion. To be able to compare results of the different studies reported, and to ensure nutrient limitation is not influencing observations ascribed to process dynamics and/or reaction kinetics, a standard protocol for creating a defined growth medium for anaerobic digestion and mixed culture fermentation is proposed. This paper explains the role(s) of the different macro- and micronutrients, as well as the choices for a growth medium formulation strategy. In addition, the differences in nutrient requirements between mesophilic and thermophilic systems are discussed as well as the importance of specific trace metals regarding specific conversion routes and the possible supplementary requirement of vitamins. The paper will also give some insight into the bio-availability and toxicity of trace metals. A remarkable finding is that mesophilic and thermophilic enzymes are quite comparable at their optimum temperatures. This has consequences for the trace metal requirements of thermophiles under certain conditions. Under non-limiting conditions, the trace metal requirement of thermophilic systems is about 3 times higher than for mesophilic systems.     

Mesophilic versus thermophilic anaerobic digestion of cattle manure: methane productivity and microbial ecology

In this study, productivity and physicochemical and microbiological (454 sequencing) parameters, as well as environmental criteria, were investigated in anaerobic reactors to contribute to the ongoing debate about the optimal temperature range for treating animal manure, and expand the general knowledge on the relation between microbiological and physicochemical process indicators. For this purpose, two reactor sizes were used (10 m³ and 16 l), in which two temperature conditions (35°C and 50°C) were tested. In addition, the effect of the hydraulic retention time was evaluated (16 versus 20 days).Thermophilic anaerobic digestion showed higher organic matter degradation (especially fiber), higher pH and higher methane (CH₄) yield, as well as better percentage of ultimate CH₄ yield retrieved and lower residual CH₄ emission, when compared with mesophilic conditions. In addition, lower microbial diversity was found in the thermophilic reactors, especially for Bacteria, where a clear intensification towards Clostridia class members was evident.Independent of temperature, some similarities were found in digestates when comparing with animal manure, including low volatile fatty acids concentrations and a high fraction of Euryarchaeota in the total microbial community, in which members of Methanosarcinales dominated for both temperature conditions; these indicators could be considered a sign of process stability.     

Mass spectrometric control of the thermophilic anaerobic digestion process based on levels of dissolved hydrogen

Summary The continuous and simultaneous monitoring of dissolved CH₄ and H₂ in samples from a laboratory scale thermophilic anaerobic digester contents by use of a silicone rubber-covered probe has enabled control of methanogenesis: regulation of the hydrogen signal in a closed feedback loop was by controlled addition of the carbon source. Dissolved hydrogen became apparent in this system at a lower loading rate than was obtained for a mesophilic anaerobic digestion system (Whitmoreet al., 1986). Controlling the supply of glucose (25 mM) at a dilution rate of 0.02 h^–1 and at progressively lower preset hydrogen levels allowed methanogenesis to be significantly prolonged before inhibition of the process occurred.     

Effect of pre-aeration and inoculum on the start-up of batch thermophilic anaerobic digestion of municipal solid waste

In this study, a short pre-aeration step was investigated as pre-treatment for thermophilic anaerobic digestion of the organic fraction of municipal solid waste (OFMSW). It was found that pre-aeration of 48 h generated enough biological heat to increase the temperature of bulk OFMSW to 60 °C. This was sufficient self-heating of the bulk OFMSW for the start-up of thermophilic anaerobic digestion without the need for an external heat source. Pre-aeration also reduced excess easily degradable organic compounds in OFMSW, which were the common cause of acidification during the start-up of the batch system. Careful consideration however must be taken to avoid over aeration as this consumes substrate, which would otherwise be available to methanogens to produce biogas. To accelerate methane production and volatile solids destruction, the anaerobic digestion in this study was operated as a wet process with the anaerobic liquid recycled through the OFMSW. Appropriate anaerobic liquid inoculum was found to be particularly beneficial. It provided high buffer capacity as well as suitable microbial inoculum. As a result, acidification during start-up was kept to a minimum. With volatile fatty acids (VFAs-acetate in particular) and H₂ accumulation typical of hydrolysis and fermentation of the easily degradable substrates during start-up, inoculum with high numbers of hydrogenotrophic methanogens was critical to not only maximise CH₄ production but also reduce H₂ partial pressure in the system to allow VFAs degradation. In a lab-scale bioreactor, the combined pre-aeration and wet thermophilic anaerobic digestion was able to stabilise the OFMSW within a period of only 12 days. The stabilised inert residual material can be used as a soil amendment product.     

Rapid start-up of thermophilic anaerobic digestion with the turf fraction of MSW as inoculum

This study aims to determine suitable start-up conditions and inoculum sources for thermophilic anaerobic digestion. Within days of incubation MSW at 55 °C, methane was produced at a high rate. In an attempt to narrow down which components of typical MSW contained the thermophilic methanogens, vacuum cleaner dust, banana peel, kitchen waste, and garden waste were tested as inoculum for thermophilic methanogenesis with acetate as the substrate. Results singled out grass turf as the key source of thermophilic acetate degrading methanogenic consortia. Within 4 days of anaerobic incubation (55 °C), anaerobically incubated grass turf samples produced methane accompanied by acetate degradation enabling successful start-up of thermophilic anaerobic digestion. Other essential start-up conditions are specified. Stirring of the culture was not conducive for successful start-up as it resulted specifically in propionate accumulation.     

Single-stage, batch, leach-bed, thermophilic anaerobic digestion of spent sugar beet pulp

Spent sugar beet pulp as received was digested in a single-stage, batch, unmixed, leach-bed, laboratory scale thermophilic anaerobic digester. Biogasification of each 0.450 kg (wet weight) batch of spent pulp was initiated by inoculating with anaerobically digested liquor from previous run. The average methane yield was 0.336 m3 CH4 at STP (kgVS)(-1), the maximum methane production rate was 0.087 m3 CH4 at STP (kgVS)(-1)d(-1), average lag time to initiate methanogenesis was only 0.44 days and time required to achieve 95% methane yield was 8 days. The pH in the digesters ranged between 8.0 and 9.5. High rates of methane generation were sustained even at high pH values. The equivalent organic loading rate in the batch digesters was 4 kgCODm(-3)d(-1). The digestion process used here offers significant improvements over one-stage and two-stage systems reported in the literature with comparable performance as it is a single-stage system where the feedstock does not require size reduction, and mixing is not required in the digester.     

Hydrogen-dependent control of the continuous thermophilic anaerobic digestion process using membrane inlet mass spectrometry

The control of a thermophilic continuous anaerobic digestion system when subjected to potential inhibitory shock loadings was achieved through the regulation of dissolved H₂, monitored using membrane inlet mass spectrometry, by the controlled addition of carbon source. At a feed pump switching threshold equivalent to 1 μmol/1 H₂ a steady state rate of methanogenesis of approximately 40 μmol/1/min was obtained. Higher H₂ thresholds resulted in an inhibition of methanogenesis, but precise control of H₂ concentration was demonstrated with an oscillatory response of period 2·5–5·0 min.     

Mesophilic and thermophilic anaerobic digestion of source-sorted organic wastes: effect of ammonia on glucose degradation and methane production

The wet organic fraction of household wastes was digested anaerobically at 37 °C and 55 °C. At both temperatures the volatile solids loading was increased from 1 g l⁻¹ day⁻¹ to 9.65 g l⁻¹ day⁻¹, by reducing the nominal hydraulic retention time from 93 days to 19 days. The volatile solids removal in the reactors at both temperatures for the same loading rates was in a similar range and was still 65% at 19 days hydraulic retention time. Although more biogas was produced in the thermophilic reactor, the energy conservation in methane was slightly lower, because of a lower methane content, compared to the biogas of the mesophilic reactor. The slightly lower amount of energy conserved in the methane of the thermophilic digester was presumably balanced by the hydrogen that escaped into the gas phase and thus was no longer available for methanogenesis. In the thermophilic process, 1.4 g/l ammonia was released, whereas in the mesophilic process only 1 g/l ammonia was generated, presumably from protein degradation. Inhibition studies of methane production and glucose fermentation revealed a K _i (50%) of 3 g/l and 3.7 g/l ammonia (equivalent to 0.22 g/l and 0.28 g/l free NH₃) at 37 °C and a K _i (50%) of 3.5 g/l and 3.4 g/l ammonia (equivalent to 0.69 g/l and 0.68 g/l free NH₃) at 55 °C. This indicated that the thermophilic flora tolerated at least twice as much of free NH₃ than the mesophilic flora and, furthermore, that the thermophilic flora was able to degrade more protein. The apparent ammonia concentrations in the mesophilic and in the thermophilic biowaste reactor were low enough not to inhibit glucose fermentation and methane production of either process significantly, but may have been high enough to inhibit protein degradation. The data indicated either that the mesophilic and thermophilic protein degraders revealed a different sensitivity towards free ammonia or that the mesophilic population contained less versatile protein degraders, leaving more protein undegraded. Received: 26 March 1997 / Received revision: 13 May 1997 / Accepted: 19 May 1997     

Comparison of two-stage thermophilic (68 degrees C/55 degrees C) anaerobic digestion with one-stage thermophilic (55 degrees C) digestion of cattle manure

A two-stage 68 degrees C/55 degrees C anaerobic degradation process for treatment of cattle manure was studied. In batch experiments, an increase of the specific methane yield, ranging from 24% to 56%, was obtained when cattle manure and its fractions (fibers and liquid) were pretreated at 68 degrees C for periods of 36, 108, and 168 h, and subsequently digested at 55 degrees C. In a lab-scale experiment, the performance of a two-stage reactor system, consisting of a digester operating at 68 degrees C with a hydraulic retention time (HRT) of 3 days, connected to a 55 degrees C reactor with 12-day HRT, was compared with a conventional single-stage reactor running at 55 degrees C with 15-days HRT. When an organic loading of 3 g volatile solids (VS) per liter per day was applied, the two-stage setup had a 6% to 8% higher specific methane yield and a 9% more effective VS-removal than the conventional single-stage reactor. The 68 degrees C reactor generated 7% to 9% of the total amount of methane of the two-stage system and maintained a volatile fatty acids (VFA) concentration of 4.0 to 4.4 g acetate per liter. Population size and activity of aceticlastic methanogens, syntrophic bacteria, and hydrolytic/fermentative bacteria were significantly lower in the 68 degrees C reactor than in the 55 degrees C reactors. The density levels of methanogens utilizing H2/CO2 or formate were, however, in the same range for all reactors, although the degradation of these substrates was significantly lower in the 68 degrees C reactor than in the 55 degrees C reactors. Temporal temperature gradient electrophoresis profiles (TTGE) of the 68 degrees C reactor demonstrated a stable bacterial community along with a less divergent community of archaeal species.     

Effects of nitrite inhibition on anaerobic ammonium oxidation

In order to assess the stability of nitrogen removal systems utilizing anaerobic ammonium oxidation (anammox), it is necessary to study the toxic effects of nitrite on these biochemical reactions. In this study, the effects of nitrite on anammox bacteria entrapped in gel carriers were investigated using batch and continuous feeding tests. The results showed that the nitrite concentration in a reactor must be less than 274-mg N/L in order to prevent a decrease in the anammox activity, which occurred when the gel carriers were soaked in nitrite solutions with concentrations greater than 274-mg N/L in a batch test. In a continuous feeding test, nitrite inhibition was not observed at low concentrations of nitrite. However, the anammox activity decreased to 10% when the nitrite concentration increased to 750-mg N/L over a 7-day period in the reactor. In addition, it was shown that the effects of nitrogen on the anammox reaction were reversible because the anammox activity completely recovered within 3 days when the influent nitrite concentration was decreased to less than 274-mg N/L.     

Enzyme activity for monitoring the stability in a thermophilic anaerobic digestion of wastewater containing methanol

To maintain good conditions in a thermophilic methane bioreactor treating methanol as the main substrate, microbial enzyme activity in the reactor was investigated. In a preliminary study, seven enzymes were tested for their suitability as indicators using an acid-former, 22a originating from a digester with low efficiency, Methanosarcina sp. (CHTI-55) and Desulfotomaculum nigrificans (Delft 74). Among the tested strains, the activities of seven enzymes were the highest in 22a. Acidic phosphatase (ACP), glutamic-pyruvic transaminase (GPT) and α-amylase (AMY) were chosen as hopeful indicators for lab-scale tests and their activities were measured at the optimum temperature of 55°C. In the lab-scale test, reactor failure was induced by nitrogen deficiency or addition of dimethyl disulfide (DMDS) as an inhibitor. ACP, GPT and AMY outperformed the conventional parameters as indicators of any instability in the process.     

Manganese inhibition of microbial iron reduction in anaerobic sediments

Potential mechanisms for the lack of Fe(II) accumulation in Mn(IV)‐con‐taining anaerobic sediments were investigated. The addition of Mn(IV) to sediments in which Fe(III) reduction was the terminal electron‐accepting process removed all the pore‐water Fe(II), completely inhibited net Fe(III) reduction, and stimulated Mn(IV) reduction. In a solution buffered at pH 7, Mn(IV) oxidized Fe(II) to amorphic Fe(III) oxide. Mn(IV) naturally present in oxic freshwater sediments also rapidly oxidized Fe(II). A pure culture of a dissimilatory FE(III)‐ and Mn(FV)‐reducing organism isolated from the sediments reduced Fe(III) to Fe(II) in the presence of Mn(IV) when ferrozine was present to trap Fe(II) before Mn(IV) oxidized it. Depth profiles of dissolved iron and manganese reported in previous studies suggest that Fe(II) diffusing up from the zone of Fe(III) reduction is consumed within the Mn(IV)‐reducing zone. These results demonstrate that preferential reduction of Mn(IV) by Fe(III)‐reducing bacteria cannot completely explain the lack of Fe(II) accumulation in anaerobic, Mn(IV)‐containing sedments, and indicate that Mn(IV) oxidation of Fe(II) is the mechanism that ultimately prevents Fe(II) accumulation.     

Use of respirometer in evaluation of process and toxicity of thermophilic anaerobic digestion for treating kitchen waste

A thermophilic anaerobic digestion (TAnD, 55 degrees C) system was adopted to hydrolyze the kitchen waste for 3 days, which was then fermented for a hydraulic retention time (HRT) of 10 days. The TAnD system performed much better than a similar system without thermal pre-treatment. A bubble respirometer was employed to study the effects of thermal pre-treatment, which showed that pre-treatment at 60 degrees C yielded the highest Total COD (TCOD) removal efficiency (79.2%) after 300h reaction. Respirometer results also indicated that oil and grease (O and G) began to inhibit the TAnD system at a concentration of approximately 1000mg/L and the gas production was inhibited by 50% at a concentration of approximately 7500mg/L of sodium.     

Optimization of two-phase thermophilic anaerobic digestion of biowaste for hydrogen and methane production through reject water recirculation

The optimization of a two-phase thermophilic anaerobic process treating biowaste for hydrogen and methane production was carried out at pilot scale using two stirred reactors (CSTRs) and without any physical/chemical pre-treatment of inoculum. During the experiment the hydrogen production at low hydraulic retention time (3d) was tested, both with and without reject water recirculation and at two organic loading rate (16 and 21 kgTVS/m³d). The better yields were obtained with recirculation where the pH reached an optimal value (5.5) thanks to the buffering capacity of the recycle stream. The specific gas production of the first reactor was 51 l/kgVS_fed and H₂ content in biogas 37%. The mixture of gas obtained from the two reactors met the standards for the biohythane mix only when lower loading rate were applied to the first reactor, with a composition of 6.7% H₂, 40.1% CO₂ and 52.3% CH₄ the overall SGP being 0.78 m³/kgVS_fed.