Enzyme research and applications in biotechnological intensification of biogas production

Biogas technology provides an alternative source of energy to fossil fuels in many parts of the world. Using local resources such as agricultural crop remains, municipal solid wastes, market wastes and animal waste, energy (biogas), and manure are derived by anaerobic digestion. The hydrolysis process, where the complex insoluble organic materials are hydrolysed by extracellular enzymes, is a rate-limiting step for anaerobic digestion of high-solid organic solid wastes. Biomass pretreatment and hydrolysis are areas in need of drastic improvement for economic production of biogas from complex organic matter such as lignocellulosic material and sewage sludge. Despite development of pretreatment techniques, sugar release from complex biomass still remains an expensive and slow step, perhaps the most critical in the overall process. This paper gives an updated review of the biotechnological advances to improve biogas production by microbial enzymatic hydrolysis of different complex organic matter for converting them into fermentable structures. A number of authors have reported significant improvement in biogas production when crude and commercial enzymes are used in the pretreatment of complex organic matter. There have been studies on the improvement of biogas production from lignocellulolytic materials, one of the largest and renewable sources of energy on earth, after pretreatment with cellulases and cellulase-producing microorganisms. Lipids (characterised as oil, grease, fat, and free long chain fatty acids, LCFA) are a major organic compound in wastewater generated from the food processing industries and have been considered very difficult to convert into biogas. Improved methane yield has been reported in the literature when these lipid-rich wastewaters are pretreated with lipases and lipase-producing microorganisms. The enzymatic treatment of mixed sludge by added enzymes prior to anaerobic digestion has been shown to result in improved degradation of the sludge and an increase in methane production. Strategies for enzyme dosing to enhance anaerobic digestion of the different complex organic rich materials have been investigated. This review also highlights the various challenges and opportunities that exist to improve enzymatic hydrolysis of complex organic matter for biogas production. The arguments in favor of enzymes to pretreat complex biomass are compelling. The high cost of commercial enzyme production, however, still limits application of enzymatic hydrolysis in full-scale biogas production plants, although production of low-cost enzymes and genetic engineering are addressing this issue.     

生物强化技术在生物质沼气制备过程中的应用及研究进展

生物强化技术通过为特定的生物过程"设计"微生物,进而作为一种提升反应系统活力和性能的手段被应用于生物质沼气制备过程,以便加快发酵系统启动时间、增加原料利用率、缩短酸败系统的恢复时间、降低高有机负荷的抑制作用等。本文针对以木质纤维素为原料的沼气制备中的生物强化技术,从生物强化菌剂的构建及标准、生物强化作用的影响因素、生物强化作用机制的探究等几个方面来阐述目前国内外生物强化技术在生物质沼气制备过程中的应用与研究进展,以及存在的问题和解决方案。     

The effect of various pre‐treatment methods of chromium leather shavings in continuous biogas production

During leather manufacture, high amounts of chromium shavings, wet by‐products of the leather industry, are produced worldwide. They are stable towards temperatures of up to 110°C and enzymatic degradation, preventing anaerobic digestion in a biogas plant. Hitherto, chromium shavings are not utilized industrially to produce biogas. In order to ease enzymatic degradation, necessary to produce biogas, a previous denaturation of the native structure has to be carried out. In our projects, chromium shavings were pre‐treated thermally and mechanically by extrusion and hydrothermal methods. In previous works, we intensively studied the use of these shavings to produce biogas in batch scale and significant improvement was reached when using pre‐treated shavings. In this work, a scale‐up of the process was performed in a continuous reactor using pre‐treated and untreated chromium shavings to examine the feasibility of the considered method. Measuring different parameters along the anaerobic digestion, namely organic matter, collagen content, and volatile fatty acids content, it was possible to show that a higher methane production can be reached and a higher loading rate can be used when feeding the reactor with pre‐treated shavings instead of untreated chromium shavings, which means a more economical and efficient process in an industrial scenario.     

Land cover changes with the development of anaerobic digestion for biogas production in France

Anaerobic digestion is developing in various countries worldwide to produce renewable energy. In addition, the resulting digestates provide readily available nutrients when applied to cropping systems as fertilizers. The introduction of a biogas plant on a farm can induce land cover changes, in relation to the production of feedstock for the biogas plant and/or to the modification of the farming system. The aim of this study was therefore to characterize and quantify the land cover changes in farms associated with biogas plants in France. We combined two national spatialized databases: the Land Parcel Identification System (yearly French land cover at the parcel scale with farm identifier per parcel) and the SINOE database (biogas plant location and year of start-up). We showed that, on average, the changes were limited, with an increase in maize areas (+3.4% of the total farm areas) compensated by a decrease in wheat and rapeseed areas (−1.8% and −1.9%, respectively), but with a certain variability. The French regulation and market limiting the use of dedicated energy crops seems to have limited land cover changes in France compared to other countries. However, we elaborated a typology of land cover changes and characterized five clusters of farms across the country. The main one (67% of the farms) corresponded to unchanged land cover after the introduction of a biogas plant. The four other clusters showed contrasting changes, for example, an increase or a decrease in grassland areas, a strong increase in maize areas, or a replacement of winter wheat by winter barley. The diversity and the driving factors behind these changes deserve to be better studied and understood through further farmer surveys.     

Feeding approaches for biogas production from animal wastes and industrial effluents

Different feeding approaches were applied to a 5 l anaerobic digester in order to improve the biogas production. During operation, the reactor was fed with a mixture (9.7% w/v total solids (TS) and 7.6% w/v volatile solids (VS) in average) of pig manure with fish oil waste and waste from bentonite of edible oil filtration process, at different intervals of 24, 12 and 4 h at 15 days of hydraulic retention time. Production and quality of the biogas were practically constant at 183.7 ml (average) of biogas per gram of volatile solids available in the reactor per day, and the best biogas composition was 73.6% v/v CH₄ and 26.4% v/v CO₂.     

添加厨余垃圾对剩余污泥厌氧消化产沼气过程的影响

为提高剩余污泥厌氧消化的沼气产量和甲烷含量,研究了厨余垃圾的不同添加量对剩余污泥厌氧消化性能的影响。结果表明,在35℃下,随着剩余污泥中厨余垃圾添加量的增加,厌氧消化系统中碳氮质量比（C／N）、胞外多聚物（EPS）等生理生化指标均有不同程度的改善。其中当剩余污泥与厨余垃圾质量比为2：1时,混合有机废弃物中沼气产量和甲烷含量均达到最大值,每克挥发性固体（VS）产生了156．56mL沼气,甲烷体积分数为67．52％,分别比剩余污泥单独厌氧消化时的产气量提高了5倍和1．5倍。     

Miscanthus for biogas production: Influence of harvest date and ensiling on digestibility and methane hectare yield

The 8,000 biogas plants currently in operation in Germany are mainly fed with biomass from annual crops. However, feedstock from perennial crops such as miscanthus is expected to be more environmentally benign. If miscanthus is to be used in greater amounts as a substrate for anaerobic digestion, storage will become a relevant topic, as a continuous supply of biomass throughout the year is necessary. The objective of this study was to identify the miscanthus harvest time that best balances the simultaneous achievement of high silage quality, high digestibility and high methane hectare yields. For this purpose, biomass from four miscanthus genotypes with varying senescence characteristics was harvested on three different dates in autumn 2017. Part of the biomass was ensiled, and the methane yield of both ensiled and non‐ensiled biomass was analysed in a biogas batch test to assess the effect of ensiling on the methane hectare yield and digestion velocity. The ensiled biomass was found to have an up to 7% higher substrate‐specific methane yield and also showed a higher digestion velocity than the non‐ensiled biomass. The silage quality was best when miscanthus was harvested in mid‐October, due to highest lactic acid content (average: 3.0% of DM) and lowest pH (average: 4.39) compared to the harvests in mid‐September and beginning of October. Mass losses during ensiling (as high as 7.6% of fresh matter for the M. sinensis genotype Sin55) were compensated for by a higher substrate‐specific methane yield (up to 353 Nml CH₄ (g oDM)^?1) in ensiled miscanthus. This resulted in non‐significantly different methane hectare yields for non‐ensiled (average: 4.635 Nm³ CH₄/ha) and ensiled miscanthus biomass (4.803 Nm³ CH₄/ha). A comparison of the four genotypes suggests that Miscanthus x giganteus is the most suitable genotype for ensiling as it had the best silage quality.     

Simultaneous hydrogen utilization and in situ biogas upgrading in an anaerobic reactor

The possibility of converting hydrogen to methane and simultaneous upgrading of biogas was investigated in both batch tests and fully mixed biogas reactor, simultaneously fed with manure and hydrogen. Batch experiments showed that hydrogen could be converted to methane by hydrogenotrophic methanogenesis with conversion of more than 90% of the consumed hydrogen to methane. The hydrogen consumption rates were affected by both (hydrogen partial pressure) and mixing intensity. Inhibition of propionate and butyrate degradation by hydrogen (1 atm) was only observed under high mixing intensity (shaking speed 300 rpm). Continuous addition of hydrogen (flow rate of 28.6 mL/(L/h)) to an anaerobic reactor fed with manure, showed that more than 80% of the hydrogen was utilized. The propionate and butyrate level in the reactor was not significantly affected by the hydrogen addition. The methane production rate of the reactor with H₂ addition was 22% higher, compared to the control reactor only fed with manure. The CO₂ content in the produced biogas was only 15%, while it was 38% in the control reactor. However, the addition of hydrogen resulted in increase of pH (from 8.0 to 8.3) due to the consumption of bicarbonate, which subsequently caused slight inhibition of methanogenesis. Biotechnol. Bioeng. 2012; 109:1088–1094. © 2011 Wiley Periodicals, Inc.     

The fate of phosphate under anoxic conditions in biological nutrient removal activated sludge systems

The nitrogen removal potential of phosphate accumulating organisms under anoxic conditions has been evaluated using a laboratory scale sequencing batch reactor fed with synthetic wastewater and operated in a sequence of anaerobic, anoxic and aerobic periods. The phosphate uptake rate under anoxic conditions was lower than that under aerobic conditions. However, in the presence of an external substrate such as glucose and acetate, the fate of phosphate was dependent on the substrate type; phosphate release occurred in the presence of nitrate as long as acetate was present and glucose did not cause any phosphate release. The nitrate uptake rate was also much lower with glucose than acetate. The results implied that poly-hydroxyalkanoates could be oxidized by nitrate and phosphate uptake during the anoxic phase should be introduced into process modeling. © Rapid Science Ltd. 1998     

Residual phosphate concentration under nitrogen-limiting conditions regulates curdlan production in Agrobacterium species

We investigated the influence of inorganic phosphate concentration on the production of curdlan by Agrobacterium species. A two-step culture method was employed where cells were first cultured, followed by curdlan production under nitrogen-limiting conditions. In the curdlan production step, cells did not grow but metabolized sugar into curdlan. Shake-flask experiments showed that the optimal phosphate concentration for curdlan production was in the range of 0.1–0.3 g l⁻¹. As the cell concentration increased from 0.42 to 1.68 g l⁻¹ in shake-flask cultures, curdlan production increased from 0.44 to 2.80 g l⁻¹. However, the optimal phosphate concentration range was not dependent upon cell concentration. The specific production rate was about 70 mg curdlan g-cell⁻¹ h⁻¹ irrespective of cell concentration. When the phosphate concentration was maintained at 0.5 g l⁻¹ under nitrogen-limiting conditions, as high as 65 g l⁻¹ of curdlan was obtained in 120 h. Journal of Industrial Microbiology & Biotechnology (2000) 25, 180–183. Received 25 October 1999/ Accepted in revised form 21 July 2000     

In situ ammonia production as a sanitation agent during anaerobic digestion at mesophilic temperature

Aim: To measure the sanitizing effect of mesophilic (37°C) anaerobic digestion in high ammonia concentrations produced in situ. Methods and Results: Indicator organisms and salmonella were transferred to small‐scale anaerobic batch cultures and D‐values were calculated. Batch cultures were started with material from two biogas processes operating at high (46 mmol l^?1) and low (1·6 mmol l^?1) ammonia concentration. D‐values were shortened from c. 3 days to <1 day for the bacteria. MS2 had the same D‐value (1·3 days) independent of ammonia concentration whereas ΦX174 and 28B were faster inactivated in the control (1·1 and 7·9 days) than in the high ammonia (8·9 and 39 days) batch cultures. Conclusion: Running biogas processes at high levels of ammonia shortens the time to meet EU regulation concerning reduction of salmonella and enterococci (5 log). Unless a minimum retention time of 2 days, post‐treatment digestion is needed to achieve sufficient sanitation in continuous biogas processes. Significance and Impact of the Study: Running mesophilic biogas processes at high ammonia level produces residue with a high fertilizer value. With some stipulations concerning management parameters, such processes provide a method of bacterial sanitation without preceding pasteurization of the incoming organic waste.     

Energy cover crops for biogas production increase soil organic carbon stocks: A modeling approach

Energy cover crops for biogas production through anaerobic digestion (AD) are inserted between two primary crops. They replace either bare soil or nonharvested cover crops, and their management is usually intensified to produce more biomass. They allow the production of renewable energy as well as digestate, used as an organic fertilizer, without directly competing with food production. Because of the increased biomass production and export and of the return of a digested biomass to the soil, the impact of energy cover crops on soil organic carbon (SOC) is questioned. The objective of this paper was to study the difference in SOC stocks induced by the introduction of energy cover crops for AD coupled with the application of the resulting amount of digestate. We used the AD model Sys-Metha combined with the soil C model AMG to simulate SOC stocks for 13 case studies in France, with scenarios comparing different intercrop management practices, with or without cover crops, harvested or not. Our results indicated that the higher biomass production of energy cover crops (from 6.7 to 11.1 t DM ha⁻¹) in comparison with nonharvested cover crops (2 t DM ha⁻¹) or bare soil led to higher humified C input (belowground input and digestate), despite the high C fraction exported in AD. This resulted in an increase in SOC stocks in comparison with nonharvested cover crops or bare soil (from 0.01 to 0.12 t C ha⁻¹ year⁻¹ over 30 years). The uncertainties in the model parameters did not modify these results. However, in the case of equal biomass production between energy cover crops and nonharvested cover crops, SOC stocks would be lower with energy cover crops.     

生物燃气技术及工程的发展现状

生物燃气俗称沼气,是微生物群体在厌氧条件下协同发酵可降解有机废弃物的产物,传统能源供应的萎缩和增加可再生能源在能源消费中份额的需求使沼气的重要性越来越突出。经过不断研究与工程实践,已经开发出了不同的发酵工艺处理生活垃圾和工农业生产废弃物等有机质生产生物燃气,其中全混式中温发酵占主导地位,欧洲的技术处于领先水平,特别是德国的沼气发电、热电联产。结合作者多年沼气研究积累的经验,综述了沼气技术的最新进展,包括厌氧发酵菌群、消化反应器结构和发酵工艺,沼气生产和应用等,指出了今后发展的重点和方向。     

Perspectives of surface plasmon resonance sensors for optimized biogas methanation

Biogas production is becoming significantly viable as an energy source for replacing fossil‐based fuels. The further development of the biogas production process could lead to significant improvements in its potential. Wastewater treatment currently accounts for 3% of the electrical energy load in developed countries, while it could be developed to provide a source of nitrogen and phosphorus, in addition to energy. The improvement of anaerobic digestion (AD) detection technologies is the cornerstone to reach higher methane productivities and develop fully automatized processes to decrease operational costs. New sensors are requested to automatically obtain a better interpretation of the complex and dynamical internal reactor environment. This will require detailed systematic detection in order to realize a near‐optimal production process. In this review, optical fiber‐based sensors will be discussed to assess their potential for use in AD. There is currently a disparity between the complexity of AD, and online detection. By improving the durability, sensitivity, and cost of dissolved H₂ (as well as H₂S, acetic acid, ammonia, and methane) sensor technology, further understanding of the AD process may allow the prevention of process failure. The emergence of surface plasmon resonance (SPR) sensing with optical fibers coupled with the H₂‐sensitive metal palladium, allows detection of dissolved hydrogen in liquid. By implementing these SPR sensors into AD, improvements to the biogas production process, even at small scales, may be achieved by guiding the process in the optimum direction, avoiding the collapse of the biological process. This review intends to assess the feasibility of online, cost‐effective, rapid, and efficient detection of dissolved H₂, as well as briefly assessing H₂S, acetic acid, ammonia, and methane in AD by SPR.     

Integrated biogas upgrading and hydrogen utilization in an anaerobic reactor containing enriched hydrogenotrophic methanogenic culture

Biogas produced by anaerobic digestion, is mainly used in a gas motor for heat and electricity production. However, after removal of CO₂, biogas can be upgraded to natural gas quality, giving more utilization possibilities, such as utilization as autogas, or distant utilization by using the existing natural gas grid. The current study presents a new biological method for biogas upgrading in a separate biogas reactor, containing enriched hydrogenotrophic methanogens and fed with biogas and hydrogen. Both mesophilic‐ and thermophilic anaerobic cultures were enriched to convert CO₂ to CH₄ by addition of H₂. Enrichment at thermophilic temperature (55°C) resulted in CO₂ and H₂ bioconversion rate of 320 mL CH₄/(gVSS h), which was more than 60% higher than that under mesophilic temperature (37°C). Different dominant species were found at mesophilic‐ and thermophilic‐enriched cultures, as revealed by PCR–DGGE. Nonetheless, they all belonged to the order Methanobacteriales, which can mediate hydrogenotrophic methanogenesis. Biogas upgrading was then tested in a thermophilic anaerobic reactor under various operation conditions. By continuous addition of hydrogen in the biogas reactor, high degree of biogas upgrading was achieved. The produced biogas had a CH₄ content, around 95% at steady‐state, at gas (mixture of biogas and hydrogen) injection rate of 6 L/(L day). The increase of gas injection rate to 12 L/(L day) resulted in the decrease of CH₄ content to around 90%. Further study showed that by decreasing the gas–liquid mass transfer by increasing the stirring speed of the mixture the CH₄ content was increased to around 95%. Finally, the CH₄ content around 90% was achieved in this study with the gas injection rate as high as 24 L/(L day). Biotechnol. Bioeng. 2012; 109: 2729–2736. © 2012 Wiley Periodicals, Inc.     

Effect of pH change on the performance and microbial community of enhanced biological phosphate removal process

An acetate-rich wastewater, containing 170 mg/L of total organic carbon (TOC), 13 mg/L of N, and 15 mg/L of P, was treated using the enhanced biological phosphate removal (EBPR) process operated in a sequencing batch reactor. A slight change of pH of the mixed liquor from 7.0 to 6.5 led to a complete loss of phosphate-removing capability and a drastic change of microbial populations. The process steadily removed 94% of TOC and 99.9% of P from the wastewater at pH 7.0, but only 93% TOC and 17% of P 14 days after the pH was lowered to pH 6.5. The sludge contained 8.8% P at pH 7.0, but only 1.9% at pH 6.5. Based on 16S rDNA analysis, 64.8% of the clones obtained from the sludge at pH 7.0 were absent in the pH 6.5 sludge. The missing microbes, some of which were likely responsible for the phosphate removal at pH 7.0, included beta-Proteobacteria, Actinobacteria, Bacteriodetes/Chlorobi group, plus photosynthetic bacteria and Defluvicoccus of the alpha-Proteobacteria. Among them, the last two groups, which represented 9.3% and 10.1% of the EBPR sludge at pH 7.0, have rarely been reported in an EBPR system.     

Hydrogen and methane production through two-stage mesophilic anaerobic digestion of olive pulp

The present study focused on the anaerobic biohydrogen production from olive pulp (two phase olive mill wastes, TPOMW) and the subsequent anaerobic treatment of the effluent for methane production under mesophilic conditions in a two-stage process. Biohydrogen production from water-diluted (1:4) olive pulp was investigated at hydraulic retention times (HRT) of 30 h, 14.5 h and 7.5 h while methane production from the effluent of hydrogenogenic reactor was studied at 20 d, 15 d, 10 d and 5 d HRT. In comparison with previous studies, it has been shown that the thermophilic hydrogen production process was more efficient than the mesophilic one in both hydrogen production rate and yield. The methanogenic reactor was successfully operated at 20, 15 and 10 days HRT while it failed when an HRT of 5 days was applied. Methane productivity reached the maximum value of 1.13 ± 0.08 L/L/d at 10 days HRT whereas the methane yield increased with the HRT. The Anaerobic Digestion Model no. 1 (ADM1) was applied to the obtained experimental data from the methanogenic reactor to simulate the digester response at all HRT tested. The ability of the model to predict the experimental results was evident even in the case of the process failure, thus implying that the ADM1 could be a valuable tool for process design even in the case of a complex feedstock. In general, the two-stage anaerobic digestion proved to be a stable, reliable and effective process for energy recovery and stabilization treatment of olive pulp.     

Eliminating methanogenic activity in hydrogen reactor to improve biogas production in a two-stage anaerobic digestion process co-digesting municipal food waste and sewage sludge

Laboratory scale two-stage anaerobic digestion process model was operated for 280 days to investigate the feasibility to produce both hydrogen and methane from a mixture feedstock (1:1 (v/v)) of municipal food waste and sewage sludge. The maximum hydrogen and methane yields obtained in the two stages were 0.93 and 9.5 mL/mL feedstock. To eliminate methanogenic activity and obtain substantial hydrogen production in the hydrogen reactor, both feedstock and mixed liquor required treatment. The heat treatment (100 °C, 10 min) for feedstock and a periodical treatment (every 2-5 weeks, either heating, removal of biomass particles or flushing with air) for mixed liquor were effective in different extent. The methane production in the second stage was significantly improved by the hydrogen production in the first stage. The maximum methane production obtained in the period of high hydrogen production was more than 2-fold of that observed in the low hydrogen production period.     

过氧化氢预处理对褐煤物化性质及生物产气的影响

【目的】研究过氧化氢预处理对褐煤物化性质及生物产气的影响。【方法】以胜利5号褐煤为研究对象,利用正交试验对过氧化氢预处理褐煤条件进行优化,在最优条件下处理褐煤得到处理后的残煤和处理液,通过X射线衍射分析(X-ray diffraction, XRD)、扫描电镜分析(scanning electron microscopy, SEM)、比表面积分析及孔隙分析(brunauer-emmett-teller, BET)、气相色谱-质谱分析(GC-MS)、高效液相色谱分析(HPLC)等方法对原煤、残煤和处理液的物化性质进行比较分析。【结果】经过氧化氢预处理,褐煤的最优条件为过氧化氢浓度5.0%、预处理时间20 d、液固比30:1,处理液中总有机碳含量为105 mg/L。在最优条件下,过氧化氢处理后残煤表面裂痕、凹陷增多,表面结构变得松散;煤的芳香面网间距增加,芳环结构更加疏松,晶核结构变小;孔隙度和比表面积均增大。处理后残煤中的固定碳、C元素和镜质组的相对含量降低,而灰分、挥发分、O和H元素及惰质组含量增加,残煤中O=C-O、C=C、C=O官能团含量增加,而N-H、C-H官能团含量则减少。生物产气结果表明反应液和残煤产气量均低于原煤,分别减少了39.13%和94.46%。过氧化氢预处理主要作用于煤中镜质组,使其有机碳溶解,煤中大分子结构的官能团发生变化,改变煤的芳环结构,在氧化作用下煤结构中的小分子溶解进入处理液。处理液中有机物以短链脂肪酸为主。经生物产气后,反应液中小分子酸以及有机物种类减少,被微生物利用产气。而各产气试验组中优势菌门及优势菌属的菌群丰度呈现出显著差异,古菌中原煤产气组盐杆菌门(Halobacteriota)为优势菌门,甲烷八叠球菌属(Methanosarcina)为优势菌属;反应液产气组热变形菌(Thermoprotei)为优势菌门,深古菌属(Bathyarchaeia)为优势菌属;细菌中原煤产气组放线菌门(Actinomycetota)为优势菌门,Gaiellales为优势菌属;反应液产气试验组假单胞菌门(Pseudomonadota)为优势菌门,代尔夫特菌属(Delftia)为优势菌属。【结论】煤溶解有机碳可以被微生物利用产气,但是煤中有机组分的过氧化脱除导致生物产气量减少。