باستمرار، أثار الهاضم اللاهوائي لتحويل النفايات العضوية إلى الغاز الحيوي: إعداد النظام الأساسي وعملية

Summary

Laboratory-scale anaerobic digesters allow scientists to research new ways of optimizing existing applications of anaerobic biotechnology and to evaluate the methane producing potential of various organic wastes. This article introduces a generalized model for the construction, inoculation, operation, and monitoring of a laboratory-scale continuously stirred anaerobic digester.

Abstract

Anaerobic digestion (AD) is a bioprocess that is commonly used to convert complex organic wastes into a useful biogas with methane as the energy carrier ^1-3. Increasingly, AD is being used in industrial, agricultural, and municipal waste(water) treatment applications ^4,5. The use of AD technology allows plant operators to reduce waste disposal costs and offset energy utility expenses. In addition to treating organic wastes, energy crops are being converted into the energy carrier methane ^6,7. As the application of AD technology broadens for the treatment of new substrates and co-substrate mixtures ⁸, so does the demand for a reliable testing methodology at the pilot- and laboratory-scale.

Anaerobic digestion systems have a variety of configurations, including the continuously stirred tank reactor (CSTR), plug flow (PF), and anaerobic sequencing batch reactor (ASBR) configurations ⁹. The CSTR is frequently used in research due to its simplicity in design and operation, but also for its advantages in experimentation. Compared to other configurations, the CSTR provides greater uniformity of system parameters, such as temperature, mixing, chemical concentration, and substrate concentration. Ultimately, when designing a full-scale reactor, the optimum reactor configuration will depend on the character of a given substrate among many other nontechnical considerations. However, all configurations share fundamental design features and operating parameters that render the CSTR appropriate for most preliminary assessments. If researchers and engineers use an influent stream with relatively high concentrations of solids, then lab-scale bioreactor configurations cannot be fed continuously due to plugging problems of lab-scale pumps with solids or settling of solids in tubing. For that scenario with continuous mixing requirements, lab-scale bioreactors are fed periodically and we refer to such configurations as continuously stirred anaerobic digesters (CSADs).

This article presents a general methodology for constructing, inoculating, operating, and monitoring a CSAD system for the purpose of testing the suitability of a given organic substrate for long-term anaerobic digestion. The construction section of this article will cover building the lab-scale reactor system. The inoculation section will explain how to create an anaerobic environment suitable for seeding with an active methanogenic inoculum. The operating section will cover operation, maintenance, and troubleshooting. The monitoring section will introduce testing protocols using standard analyses. The use of these measures is necessary for reliable experimental assessments of substrate suitability for AD. This protocol should provide greater protection against a common mistake made in AD studies, which is to conclude that reactor failure was caused by the substrate in use, when really it was improper user operation ¹⁰.

Introduction

Anaerobic digestion (AD) is a mature technology involving the biologically mediated conversion of complex organic waste substrates into useful biogas with methane as the energy carrier. There are many benefits of anaerobic treatment, including minimal energy and nutrient inputs and reduced biosolids production compared to aerobic treatment ¹⁰. In addition, the versatility of the mixed microbial community inherent to these systems renders a wide variety of organic substrates suitable as feedstocks ^11,12. Indeed, it is due to these benefits that a growing number of applications for AD are being adopted outside of conventional municipal wastewater treatment, particularly in the industrial, municipal (e.g., food waste), and agricultural sectors ^4,7,13. AD experienced its first major proliferation beginning in the 1980s in response to the national energy crisis of the previous decade. As the world faces a growing global energy crisis, coupled with environmental degradation, greater focus is now being placed on biofuel technologies and the waste-to-energy concept in particular. For example, in the U.S., anaerobic digestion can generate 5.5% of the total electrical power needs ⁸.

This has increased the demand for well-controlled experimental research at the pilot- and laboratory-scale to assess the suitability of new organic waste materials and waste mixtures for anaerobic digestion ¹⁴. We intend to provide a generic model for the construction, inoculation, operation, and monitoring of a laboratory-scale anaerobic digester that will be suitable for robust assessments. Anaerobic digesters exist in many different configurations. A few common configurations include the: continuously-stirred tank reactor (CSTR) with continuous influent feeding; continuously stirred anaerobic digester (CSAD) with periodic influent feeding; plug flow (PF), upflow anaerobic sludge blanket (UASB); anaerobic migrating blanket reactor (AMBR); anaerobic baffled reactor (ABR), and anaerobic sequencing batch reactor (ASBR) configurations ^9,15. The CSTR and CSAD configuration have been widely adopted for laboratory-scale experiments due to its ease of setup and favorable operating conditions. Because of continuous mixing, the hydraulic retention time (HRT) is equal to the sludge retention time (SRT). The SRT is the important design parameter for ADs. The configuration is also conducive to controlled experiments because of a greater spatial uniformity of parameters, such as chemical species concentrations, temperature, and diffusion rates. It should be noted, however, that the optimal full-scale configuration for an anaerobic digester depends on the particular physical and chemical qualities of the organic substrate among other nontechnical aspects, such as target effluent quality. For example, dilute waste streams with relatively high soluble organic content and little particulates, such as brewery wastewater, typically experience greater energy conversion in an high-rate upflow bioreactor configuration (e.g., UASB) rather than a CSAD configuration. Regardless, there are fundamental operating parameters that are essential to successful digestion and relevant to all configurations, which justify a generic explication of using this configuration.

Indeed, every AD system containing a diverse, open community of anaerobic microbes will serially metabolize the substrate to methane (the final end-product with the lowest available free energy per electron). The metabolic pathways involved in this process constitute an intricate food web loosely categorized into four trophic stages: hydrolysis; acidogenesis; acetogenesis; and methanogenesis. In hydrolysis, complex organic polymers (e.g., carbohydrates, lipids, and proteins) are broken down to their respective monomers (e.g., sugars, long-chain fatty acids, and amino acids) by hydrolyzing, fermentative bacteria. In acidogenesis, these monomers are fermented by acidogenic bacteria to volatile fatty acids (VFAs) and alcohols, which in acetogenesis, are further oxidized to acetate and hydrogen by homoacetogenic and obligatory hydrogen-producing bacteria, respectfully ⁵. In the final step of methanogenesis, acetate and hydrogen are metabolized to methane by acetoclastic and hydrogenotrophic methanogens. It is important to recognize that the overall AD process, by relying on an interconnected series of metabolisms by different groups of microbes, will depend on the successful function of each member before the system as a whole will perform optimally. The design and construction of an AD bioreactor system should always take into consideration the requirement to completely seal the bioreactor. Small leaks in the top of the bioreactor (separating the headspace) or in the gas-handling system may be difficult to detect, and therefore the system should be pressure tested before use. After ensuring a leak-free setup, failures with anaerobic digester studies often stem from errors during inoculation, culturing, and day-to-day operation. As a result, digesters have a reputation as being intrinsically unstable and prone to unexpected failure. Why is it then that full-scale digesters have been operated under stable conditions for decades ¹³? Failure is likely to stem from improper handling by the operator, especially during the startup period during which the microbial community must slowly acclimate to the organic waste composition and strength. Therefore, our goal is not only to provide a methodology for constructing an AD system, but to also elucidate the processes of inoculation, operation, and monitoring of these systems.

The first section of the article will explain how to construct the CSTR or CSAD system, while the second section will provide a procedure for digester inoculation with active methanogenic biomass. It is more practical and less time-consuming to inoculate digesters with active methanogenic biomass from the mixed-liquor or effluent of an operating digester that is treating a similar substrate than to attempt to develop a sufficient biomass from an incipient culture. The third section of the article will cover operating considerations, such as feeding substrate, decanting effluent, and troubleshooting various reactor problems. Feeding substrate and decanting effluent for this system will be conducted on a semi-continuous basis (i.e., periodic feeding and decanting while most of the biomass and mixed liquor stays in the bioreactor). The frequency in which the digester is fed/decanted is the prerogative of the operator. In general, feeding/decanting more frequently and at regular intervals will promote greater digester stability and consistency in performance between feeding cycles. The fourth section will introduce a basic monitoring protocol to be used during the experimental period. Several standard analyses, which are outlined in Standard Methods for the Examination of Water and Wastewater ¹⁶ (Table 1, 2), will be required for characterization of the substrate and proper system monitoring. In addition to the measured variables, an important aspect of monitoring is to check that the digester system components are functioning properly. Regular maintenance to the digester system will preempt major system problems that could otherwise jeopardize the long-term performance and stability of the digester. For example, a failure of the heating element, leading to a drop in temperature, could cause the accumulation of volatile fatty acids by reducing the metabolic rate of methanogens. This problem would be compounded if the system lacked sufficient alkalinity to maintain the pH above inhibitory levels for methanogens. It is also important to detect and close possible leaks after unexpected drops in biogas production rates. Therefore, duplication within the experimental design by, for example, running two bioreactors side-by-side under the exact operating conditions, is important to detect unexpected performance losses caused by system malfunctions, such as small leaks.

Protocol

1. Digester Construction Select a digester vessel that contains all the features shown in Fig. 1 (a cone is not necessary), and your desired working volume (typically between 1-10 L). If your digester vessel is not equipped with a heated-water jacket, place the digester in some other temperature-controlled environment, such as a heated water bath or incubation chamber. Secure the vessel in a vertical position in an area with sufficient horizontal bench space for placement of remaini…

Discussion

The anaerobic digestion system presented in this article provides a general introduction and some basic guidelines for the treatment of most substrates in an experimental context. The wide variety of substrate types, digester configurations, operating parameters, and also the unique ecology of the mixed-microbial community underlying these systems precludes outlining hard quantitative metrics, which can be applied universally. Despite all this variability, all anaerobic digestion systems follow a well-characterized ser…

Materials

Reactor Equipment	Company	Catalogue number	Comments
Heated Recirculator	VWR Scientific	13271-063 VWR	For use with a heating jacket reactor system
Variable Speed Electric Lab Stirrer	Cleveland Mixer Co.	(Model 5VB)	This mixer model facilitates mounting with a ring stand
Wet-Type Precision Gas Meter	Ritter Gasmeters	(Model TG-01)	This model needs a minimum flow of (0.1 L/h) and can handle a maximum flow of 30 L/h
Gas Bubbler	Chemglass	(Model AF-0513-20)
Gas Sampling Tube	Chemglass	(Model CG-1808)
Axial Impeller	Lightnin’	R04560-25 Cole-Parmer	Impeller blades with 7.9375 mm bore diameter
Impeller Shaft	Grainger	2EXC9 Grainger	1.83 m stainless steel rod with 7.9375 mm O.D. (needs to be cut to appropriate size)
Cast Iron Support Stands	American Educational Products	(Model 7-G16)	For mixer mounting
Three-Prong Extension Clamp	Talon	21572-803 VWR	For mixer mounting
Regular Clamp Holder	Talon	21572-501 VWR	For mixer mounting
Peristaltic Pump	Masterflex	WU-07523-80 Cole-Parmer	For effluent decanting
L/S Standard Pump Head	Masterflex	EW-07018-21 Cole-Parmer	For effluent decanting -accessory to peristaltic pump
L/S Precision Pump Tubing	Masterflex	EW-06508-18 Cole-Parmer	For effluent decanting – accessory to peristaltic pump
Analysis Equipment/Reagents	Company	Catalogue number	Comments
			pH Analysis
pH Meter	Thermo Fisher Scientific – Orion	1212000
			Total and Volatile Solids Analysis (Standard Methods: 2540-B,E)
Glass Vacuum Dessicator	Kimax	WU-06536-30 Cole-Parmer
Porcelain Evaporating Dishes	VWR	89038-082 VWR
Lab Oven	Thermo Fisher Scientific	(Model 13-246-516GAQ)
Medium Chamber Muffle Furnace	Barnstead/ Thermolyne	F6010 Thermo Scientific
			Total Volatile Fatty Acid Analysis (Standard Methods: 5560-C)
Large Capacity Variable Speed Centrifuge	Sigma	WU-17451-00 Cole-Parmer
Laboratory Hot Plate	Thermo Scientific	(Model HP53013A)
Large Condenser	Kemtech America	(Model C150190)
Acetic Acid Reagent [CAS: 64-19-7]	Alfa Aesar	AA33252-AK
			Chemical Oxygen Demand (Standard Methods: 5520-C)
COD Block Heater	HACH	(Model DRB-200)
Borosilicate Culture Tubes	Pyrex	(Model 9825-13)
Potassium Dichromate Reagent [CAS: 7778-50-9]	Avantor Performance Materials	3090-01
Mercury II Sulfate Reagent [CAS: 7783-35-9]	Avantor Performance Materials	2640-04
Ferroin Indicator Solution [CAS: 14634-91-4]	Ricca Chemical	R3140000-120C
Ammonium iron(II) sulfate hexahydrate [CAS: 7783-85-9]	Alfa Aesar	13448-36
			Gas Composition by Gas Chromatography Analysis
Gas Chromatograph	SRI Instruments	Model 8610C	Must be equipped with a thermal conductibility detector (TCD), using below mentioned column and carrier gas operated at an isothermal temperature of 105°C
Helium Gas	Airgas	He HP300	To be used as the carrier gas
Packed-Column	Restek	80484-800	To be used for N2, CH4, and CO2 separation