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Biofuels from microalgae—A review of technologies for production, processing, and extractions of biofuels and co-products

01 Feb 2010-Renewable & Sustainable Energy Reviews (RENEWABLE AND SUSTAINABLE ENERGY REVIEWS)-Vol. 14, Iss: 2, pp 557-577
TL;DR: In this article, the authors reviewed the technologies underpinning microalgae-to-bio-fuels systems, focusing on the biomass production, harvesting, conversion technologies, and the extraction of useful co-products.
Abstract: Sustainability is a key principle in natural resource management, and it involves operational efficiency, minimisation of environmental impact and socio-economic considerations; all of which are interdependent. It has become increasingly obvious that continued reliance on fossil fuel energy resources is unsustainable, owing to both depleting world reserves and the green house gas emissions associated with their use. Therefore, there are vigorous research initiatives aimed at developing alternative renewable and potentially carbon neutral solid, liquid and gaseous biofuels as alternative energy resources. However, alternate energy resources akin to first generation biofuels derived from terrestrial crops such as sugarcane, sugar beet, maize and rapeseed place an enormous strain on world food markets, contribute to water shortages and precipitate the destruction of the world's forests. Second generation biofuels derived from lignocellulosic agriculture and forest residues and from non-food crop feedstocks address some of the above problems; however there is concern over competing land use or required land use changes. Therefore, based on current knowledge and technology projections, third generation biofuels specifically derived from microalgae are considered to be a technically viable alternative energy resource that is devoid of the major drawbacks associated with first and second generation biofuels. Microalgae are photosynthetic microorganisms with simple growing requirements (light, sugars, CO 2 , N, P, and K) that can produce lipids, proteins and carbohydrates in large amounts over short periods of time. These products can be processed into both biofuels and valuable co-products. This study reviewed the technologies underpinning microalgae-to-biofuels systems, focusing on the biomass production, harvesting, conversion technologies, and the extraction of useful co-products. It also reviewed the synergistic coupling of microalgae propagation with carbon sequestration and wastewater treatment potential for mitigation of environmental impacts associated with energy conversion and utilisation. It was found that, whereas there are outstanding issues related to photosynthetic efficiencies and biomass output, microalgae-derived biofuels could progressively substitute a significant proportion of the fossil fuels required to meet the growing energy demand.

Summary (13 min read)

Jump to: [Bu çalışmanın amacı, fiziksel ve kimyasal ön arıtımın tarım atıkları (mısır artıkları ve][135℃ sıcak ön arıtım yöntemi uygulanan sığır gübresinde kümülatif biyogaz üretiminde][AKNOWLEDGEMENTS][LIST OF TABLES][LIST OF FIGURES][1.1. Background of Study][1.2. Statement of Problem][1.4. Research Hypothesis][1.5.1. Biofuels][1.6. Biogas Production through Anaerobic Digestion (AD)][1.6.1. Hydrolysis][1.6.2. Acidogenesis][1.6.3. Acetogenesis][1.6.4. Methanogenic Phase][1.7. Biogas Composition][1.8 Environmental Factors Influencing Anaerobic Digestion][1.9 Inhibition][1.10 Reactor types][1.11 Pretreatment of Lignocellulosic Biomass][I. Physical Pretreatment][II. Chemical Pretreatment][2. MATERIALS AND METHODS][2.1.1 Maize Straw (Corn Stover)][2.1.3 Cattle Manure][2.1.5 Inoculum][2.2 Pretreatment studies][2.2.2 Comminution][2.2.3 Liquid hot water (LHW) treatment][2.2.4 Microwave pretreatment][2.3. Chemical Pretreatment][2.3.1. Alkaline Pretreatment][Alkaline protocol][2.3.2 Acid Pretreatment][Acid Protocol][2.4 Biochemical Methane Potential (BMP) Test][2.5.1 Measurement of pH and Electrical Conductivity][2.5.2 Total Solids (TS)][2.5.3 Volatile solids (VS)][Procedure][2.6.1 Biogas Measurement (carbon dioxide concentration)][3.1 Electrical Conductivity and pH][3.2. Effects of Pretreatment on Solubility][3.3. Comparisms of the Solubility of Substrates][3.4. The Relationship between Reducing Sugar and Methane production][3.5.1. Acid Pretreatment of Maize Straw][3.5.2. Alkaline Pretreatment of Maize Straw][3.5.3. Microwave pretreatment][3.5.4. Liquid Hot Water treatment][3.6. Comparisms of Total Methane Production from Pretreatments of Maize Straw][3.7. Pretreatment of Cattle Manure][3.7.1. Acid pretreatment of Cattle Manure][3.7.2. Alkaline Pretreatment of Cattle Manure][3.7.3. Microwave Pretreatment of Cattle Manure][3.7.4. Liquid Hot Water Pretreatment of Cattle Manure][Manure][3.9: Comparisms between the total biogas production from current study and][Substrate CH4 yield (mL/gVS) Reference] and [4. CONCLUSION AND RECOMMENDATIONS]

Bu çalışmanın amacı, fiziksel ve kimyasal ön arıtımın tarım atıkları (mısır artıkları ve

  • 50 oranında karıştırılmıştır, also known as Biokütle örnekleri çamurla 50.
  • Karışımın pH değeri 7.0-7.5 değerlerine ayarlanmış ve örnekler 37oC inkübatörde tutulmuştur.
  • Mısır samanı için en verimli ön arıtım metodu alkali ön arıtım olmuştur.
  • Asidik ön arıtım yöntemi mısır atıklarından farklı olarak sığır gübresi çalışmalarında daha verimli olmuştur.

135℃ sıcak ön arıtım yöntemi uygulanan sığır gübresinde kümülatif biyogaz üretiminde

  • Mikrodalga ön arıtım yöntemi de sığır gübresi çalışmalarında verimli olmuştur.
  • Biyogaz, ön arıtma, lignoselüloz, metan, mısır saman, sığır gübresi, also known as Anahtar Kelimeler.


  • I will like to sencerely thank each and everyone who helped me both morally, physically and financially in this thesis.
  • Your criticisms, corrections and advice have been key to the sucessful completion of this research.
  • This has truly been an amazing and unforgetable experience, I am grateful.
  • To my fellow classmates and friends who have become family, Luis Alfredo Carmona Arrieta, Ece Kendir and Ezgi Güzel thank you for the helpful ideas and encouragement throughout this research.
  • To my dearest mother; Aishatou El Hadji Shera and Father Abdul Karim, words will never be enough to describe who you are and what you have done for me.


  • 13 Table 1. 5 Ammonia nitrogen concentration and its effects on anaerobic treatment.
  • 22 Table 1. 7 Acid and Alkaline pretreatment methods for lignocellulosic feedstock.
  • 73 Table 3. 15 Cumulative methane production from maize straw in current study.


  • 58 Figure 3. 16 Cumulative biogas production from liquid hot water pretreatment of maize straw.
  • 59 Figure 3. 18 Comparative methane production from pretreatments of maize straw.
  • 65 Figure 3. 23 The cumulative methane production from alkaline pretreatment of cattle manure.

1.1. Background of Study

  • Biogas is composed of a combination of gases usually methane (CH4) and carbon dioxide (CO2) produced from the degradation of organic materials by anaerobic bacteria.
  • There are large amounts of organic wastes produced on a day to day basis in both rural and urban environments which makes their use for the production of bioenergy a feasible and cost effective method.
  • Agriculture remains the leading contributor to the economy of Cameroon.
  • Pretreatment is aimed at making the process faster but even more important to augment the biogas yield of lignocellulosic biomasses.
  • Cellulose contains microfibrils held together by hemicellulose with pectin or only pectin in some instances, concealed with lignin which gives rise to the formation of a complicated structure that is very difficult to breakdown by chemical or biological substances (Yi Zheng et al 2014).

1.2. Statement of Problem

  • Humanity’s heavy reliance on fossil materials like coal for its energy supply has led to serious environmental concerns.
  • Some renewables such as agricultural products like seeds, sugars, vegetable oils etc. are food crops and therefore are needed for human consumption which leads to competition while third generation biofuels like algae are very expensive to grow, maintain and harvest.
  • They also contaminate water sources and produce co-products whose 3 environmental impacts are not fully known presently.
  • This has led to the exploitation of what is known as second generation biofuels (Lignocellulosic substrates) as one of the most suitable options.

1.4. Research Hypothesis

  • The presence of lignocellulose in biomasses such as agricultural residues decreases their degradation by anaerobic microorganisms and hence biomethane production potential.
  • Therefore, the application of acid, alkaline, microwave, comminution and liquid hot water increases their solubility and hence they can be degraded more efficiently by anaerobic bacteria.
  • The faster breakdown of biomasses leads to enhanced rates of anaerobic digestion and a diminished hydraulic retention time.
  • Increased solubility will also result in more complete breakdown and thus a higher biogas production.

1.5.1. Biofuels

  • Biofuels generally refer to compact materials (such as wood chips, pellets etc.), fluids like oils, biodiesel and ethanol, or gaseous fuels such as biohydrogen, biogas, and biosyngas that are mainly obtained from biomass sources (Cecilia Sambusiti 2013).
  • The production of 1st generation biofuels is controversial because of the significant environmental and economic restrictions.
  • Second generation biofuels can also be obtained from farm residues or wastes like corn stover, animal manure amongst others.
  • They also produce relatively large amounts of carbohydrates, fats and oils which have a high potential for various biofuels production (Singh et al. 2011).
  • Microalgae are regarded as a useful alternative biomass which is independent from the major problems related to second and first generation biofuels (Dragone et al., 2010).

1.6. Biogas Production through Anaerobic Digestion (AD)

  • It results in the production of biogas with CH4 and CO2 as its principal constituents and traces of other gases in minute quantities generally considered as impurities.
  • It is a well-established technique used to manage wastes and produce fuels both for domestic and industrial purposes.
  • Recently, there has been increased interests and research in anaerobic digestion especially in the production of biomethane using lignocellulosic residues such as maize straw (Panagiotopoulos et al., 2009).
  • The four main stages are shown in figure 1.1. 7 Table 1. 1 Methane yield from anaerobic digestion of different agricultural wastes (Adapted from Merlin G. and Boileau H. 2013).
  • Type of agricultural waste CH4 yield (mL/gVS).

1.6.1. Hydrolysis

  • The breakdown of simple compounds like carbohydrates is relatively faster while the hydrolysis of oils, fats and proteins can take days to complete.
  • Lignocellulosic biomasses are only broken down partially and slowly hence initial pretreatment is required to increase the dissolution and biogas production.
  • During hydrolysis, long chain carbohydrates present in insoluble structures such as cellulose, hemicellulose, and starch are hydrolysed by hydrolases, producing short chain monomers.
  • The degree of hydrolysis is also reliant on the availability of enzymes i.e. if the bacteria is able to produce the required enzymes, the dissolution is relatively faster.

1.6.2. Acidogenesis

  • Monomers resulting from the hydrolysis of complex polymers are taken up by various obligatorily and facultative bacteria and are further broken down to short chained organic acids containing between one and five carbon atoms, molecules (which include but not limited to propionic acid, butyric acid, acetate etc.), NO2, alcohols, hydrogen sulfide, H2, and carbon dioxide.
  • The concentration of the hydrogen ions produced during acidogenesis influences the kind of fermentation output that will be formed.
  • Microorganisms belonging to the group of coli- aerogenes convert pyruvate into formate, ethanol, acetate, acetoin and butanediol.
  • During the breakdown of cysteine, hydrogen sulfide is produced resulting in a foul odour of rotten eggs.

1.6.3. Acetogenesis

  • The acetogenic phase makes use of the products of the acidogenic phase and this is because methane producing bacteria are unable to utilize the products of acidogenesis directly, thus they have to be processed further during the acetogenic phase, before they can be transformed into biomethane.
  • The creation of acetate by the decomposition of propionic acid, butyric acid etc. (long chain fatty acids) is thermodynamically enabled and can run on its own with when the partial pressure of the hydrogen is very low.
  • This means that the condition necessary for acetogens to acquire the energy they need to survive and grow is very low concentrations of hydrogen and as a result, they enter a symbiotic relationship with methane producing microorganisms which survive only in environments with high hydrogen partial pressure.
  • The methane producing bacteria only have the ability to process hydrogen, acetate and carbon dioxide.

1.6.4. Methanogenic Phase

  • This is the fourth and final step in anaerobic digestion during which biomethane is formed in the absence of oxygen.
  • It generally involves methane producing bacteria that generate methane, either from hydrogen and carbon dioxide or acetate.
  • Here, the carbon present in the substrate is transformed into carbon dioxide in the liquid mixture as a bicarbonate ion (HCO3 -) plus hydrogen and methane.
  • Methanogenic microbes are selective in the type of substrate they each degrade.
  • Generally, about 10 65-70% of the methane generated within anaerobic digesters originates from the acetate.

1.7. Biogas Composition

  • As mentioned earlier, the main constituents of biogas are methane and carbon dioxide.
  • 11 Table 1. 3 Physical and Chemical properties of methane gas Source: Samir K. (2008) Biogas might also possess some impurities like hydrogen sulfide, carbon dioxide, particulates and siloxanes.
  • It will also lead to corrosion of the gas pipes if it mixes with hydrogen sulfide found within the biogas because of the production of acids.
  • This refers to organic silicon polymers used in diverse industrial, medical, body care/aesthetics, commercial, and food products.
  • Their properties such as high volatility makes them to escape as gases during the anaerobic breakdown of municipal solid waste and municipal sludge.

1.8 Environmental Factors Influencing Anaerobic Digestion

  • Anaerobic bacteria are very vulnerable to alterations of environmental parameters such as temperature, alkalinity, pH, nutrients etc.
  • The Anaerobic digestion systems are more susceptible to changes than aerobic systems for the same magnitude of change from required environmental conditions.
  • According to a study carried out by Speece, trace elements like cobalt, iron etc. in minute quantities such as the mg/L dose and vitamin B12 in μg/L concentrations lead to an increase in methane creation (Samir K. 2008).

1.9 Inhibition

  • During anaerobic digestion, certain compounds formed from the metabolism of the anaerobic bacteria hinders the biocenosis (that is an association of various types of microorganisms forming a microbial community) and can even be toxic in high concentration.
  • Some examples of inhibitors include sulfide, ammonia and long-chain fatty acids .
  • If the ammonia nitrogen concentrations is above 3,000 mg/L, the NH4 + itself becomes toxic independent of the pH (McCarty 1964) as seen in the table 1.5; Table 1. 5 Ammonia nitrogen concentration and its effects on anaerobic treatment.
  • Wastes from industrial sites such as petrochemical industries, coal gasification plants, tanneries etc. have high amounts of sulphides.
  • The organic acids found in anaerobic digesters are short chain fatty acids, long chain fatty acids, and volatile fatty acids.

1.10 Reactor types

  • There are verities of biogas plants used to produce biomethane from agricultural residues such as maize straw and animal manure.
  • They are classified based on their feeding method as batch plants, semi-batch plants and continuous plants.
  • The substrates are degraded my anaerobic microorganisms without adding or removing anything until the biomass is completely exhausted and gas production ceases.
  • In these types of reactors, materials empty automatically through the overflow whenever new materials are added from the inlet pipe.
  • Semi batch reactors have a better biogas 20 yield than batch reactors and also, the gradual addition or removal of biomass and digestate assists in controlling temperature.

1.11 Pretreatment of Lignocellulosic Biomass

  • Lignocellulose simply means the complex conformation of plants cell walls, often regarded as the most bounteous biomass on the surface of the earth.
  • Cellulose is the major constituent of lignocellulosic biomass.
  • The branches of hemicelluloses form a grid with cellulose microfibrils while also interacting with lignin, resulting in a cellulose-hemicellulose-lignin structure which is very rigid in nature (Zhang et al. 2014).
  • The amorphous and branched structure of hemicellulose makes it easily broken down by chemical and biological agents.
  • The main objective of pretreatments is to alter the properties of the cell walls to improve the contact between the sugars and the methanogens and enzymes.

I. Physical Pretreatment

  • Physical pretreatments are used to lessen the crystallinity of cellulose thus increasing the total surface area for enzyme action and also increase the pore size of lignocelluloses.
  • It also plays the role of downgrading the extent of polymerization of the cellulose molecules present in the lignocellulose.
  • Physical pretreatment methods are those that do not use microorganisms or chemicals to alter the structures of lignocellulosic biomasses such as 22 agricultural waste.
  • The Microwave refers to a type of electromagnetic radiations which are nonionizing having frequencies between the infrared and radio waves region of the electromagnetic spectrum.
  • Also, prolonged time of microwave treatment has been found to increase the disintegration of polysaccharides (Jendrzejczyk et al., 2019).

II. Chemical Pretreatment

  • It involves the use of chemical substances such as ionic liquids, bases and acids to change the properties and structures of lignocellulosic substrates to increase their rates of biodegradation.
  • It changes the structural and chemical features of the cell walls to make them more accessible to methanogens.
  • The application of acid pretreatment on lignocelluloses is one of the most efficient methods used in solubilizing hemicelluloses and increasing the accessibility of cellulose to bacteria.
  • Alkaline pretreatment on the other hand is effective in the dissolution and elimination of lignin from the biomass sample.
  • The effects, advantages and disadvantages of acid and alkaline pretreatments are given in table 1.7.


  • There will be a general characterization of the substrates followed by the measurements of physicochemical parameters and the pretreatment studies.
  • It includes the description of the pretreatment types, conditions and the procedure for the preparation of solutions used in pretreatment and how it is applied to each biomass.
  • The final part of the chapter is the description of the Biochemical methane potential test and the formulae for the calculation of biogas and methane produced in the experiments.
  • As stated earlier, three physical pretreatment methods will be used viz.
  • Comminution, microwave, and hydrothermal (liquid hot water) pretreatment, and two chemical pretreatment techniques i.e. acid and alkaline pretreatment on two of the main agricultural wastes produced in Cameroon; maize straw and cattle manure.

2.1.1 Maize Straw (Corn Stover)

  • Maize straw or stover consists of the leaves, cobs, and stalks that are left over after harvest and the removal of grains from the cobs.
  • Maize straw is mostly utilised in the production of bioethanol through fermentation, and as forage for dairy animals in some regions of the world.
  • It equally has the potential for biogas production through anaerobic digestion.
  • It was then taken to the laboratory and preserved in a refrigerator at 4oC for a few days before samples were prepared for pretreatment and biomethane production.
  • First 27 of all, the grains were removed, and then the cob, leaves and stalk cut into small pieces using a knife.

2.1.3 Cattle Manure

  • Cattle manure refers to the indigestible components of grass, leaves etc. which has passed through the cow’s digestive system.
  • According to the American society of agronomy, one cow, based on the size and age can produce between 20 and 50 kilograms of manure each day (American Society of Agronomy, 2015).
  • In the absence of appropriate disposal methods, it has the potential to cause negative impacts to public health and the environment because it can contaminate water bodies in its vicinity with pathogens present in the dung.
  • The cattle manure used in this study was obtained from a biogas plant located at Sincan in the outskirts of Ankara.

2.1.5 Inoculum

  • Sludge seed used for the biochemical methane potential (BMP) test was also collected from the same biogas plant where cattle manure was obtained.
  • The total and volatile solids of the sludge seed were measured following the standard methods (APHA 2005).
  • Before using the sludge seed for the BMP test, it was activated by adding a small amount of glucose and placing it in an incubator at a temperature of 37oC for forty eight hours.
  • The glucose is used by the bacteria as food to keep them alive while stored at the mesophilic temperature range which they require for optimal growth.

2.2 Pretreatment studies

  • This study tests the effects of physical and chemical pretreatments on biogas generation from maize straw and cattle manure.
  • Three physical pretreatment methods were applied which include comminution, microwave pretreatment and hydrothermal pretreatment.
  • Use of a knife and blender to chop maize straw into tiny pieces of sizes between 0.2-2mm by grinding.
  • Liquid hot water 105oC for 30mins 120oC for 30mins 135oC for 30mins Microwave 300W for 2 to 4 minutes 31 b) Chemical Pretreatments Table 2. 7 Summary of Pretreatment conditions for acid and alkaline pretreatments.

2.2.2 Comminution

  • This pretreatment method was only applied on maize straw as it is not necessary for cattle manure.
  • First of all, the maize grains were removed from the cob.
  • The leaves, the cob and the stem were then cut into small pieces with the use of a knife.
  • The pieces were then placed in a blender and a small amount of water added to them to facilitate the blending process.
  • It was then placed in a plastic dish, covered and placed in a refrigerator at 4oC for use in other pretreatment methods and for the biochemical methane potential (BMP) test.

2.2.3 Liquid hot water (LHW) treatment

  • 5 % (w/w) total solids i.e. 4.81g cattle manure was weighed on an electronic balance and 95% (v/v) medium of deionized water was measured in a measuring cylinder and mixed with the cattle manure in a 100mL beaker.
  • The mixture was closed using an aluminium foil to prevent the solution from spilling out during bubbling.
  • 32 5% total solids (6.25g) of the blended maize straw was carefully weighed on an electronic balance.
  • For both biomasses, samples were done in duplicates and placed in an autoclave at three different temperatures i.e. 105oC, 120oC and 135oC for 30 minutes each.
  • The filtered liquid was put in 50mL falcon tubes and placed in a refrigerator.

2.2.4 Microwave pretreatment

  • Microwave pretreatment was applied to both biomasses: maize straw and cattle manure.
  • Exactly 4.81g of cattle manure and 6.25g of maize straw were weighed on an electronic balance and put in 100mL beakers.
  • They were then closed using aluminium foils and placed in the microwave at a power of 300W.
  • One set was performed for 2 minutes and another set for 4 minutes.
  • After 2, and 4 minutes respectively, the beakers were removed from the microwave, allowed to cool down, centrifuged and sifted using cellulose acetate membrane filters of pore size 0.45μm.

2.3. Chemical Pretreatment

  • Two chemical pretreatment methods were used in this study.
  • The procedures for this pretreatments are explained in the sections that follow;.

2.3.1. Alkaline Pretreatment

  • Alkaline pretreatment was performed using dilute solutions of sodium hydroxide (0.2M, 0.3M and 0.4M) representing concentrations of 0.8%w/v, 1.2%w/v and 1.65w/v respectively.
  • Heating is recommended because it increases the reaction rate hence increasing the efficiency of the pretreatment process.
  • Sodium hydroxide has been chosen for this study because it is most effective in increasing the digestibility and 33 fractionation of agricultural residues, it increases sugar production, elimination of lignin and the high rate of biomass utilization.
  • A stirring rod was used the stir the pellets until they were dissolved completely.
  • The mixture was then moved into 1000mL volumetric flasks.

Alkaline protocol

  • 81g of cattle manure and 6.25g of maize straw were weighed on an electronic balance.
  • Secondly, 47.5mL of 0.2M, 0.3M and 0.4M solutions of sodium hydroxide were measured using a measuring cylinder.
  • One set of samples were treated in an autoclave at 120oC while there was no heat for the other set.
  • The samples were then sifted using glucose acetate membrane filters with pore sizes of 0.45μm, put into 50mL falcon tubes and stored in a refrigerator.
  • For both samples, the measurements were performed in replica and the average of the outcomes were taken in order to minimize random errors.

2.3.2 Acid Pretreatment

  • Acid pretreatments were performed on both biomasses using dilute solutions of sulphuric acid.
  • Sulphuric acid was chosen because of its high reactivity and its effectiveness in the breakdown of celluloses to their constituent sugars such as glucose.
  • Distilled water was then added drop wise from a water bottle until it reached the 1000mL marks of the volumetric flasks.
  • The solutions were mixed properly and stored at room temperature for use in the biomass pretreatments.

Acid Protocol

  • Exactly 4.81g of cattle manure and 6.25g of maize straw were measured on an electronic balance using 100mL beakers.
  • 5mL of the 0.2M, 0.3M and 0.4M sulphuric acid solutions were measured using a measuring cylinder.
  • For both concentrations, the samples were performed in duplicates.
  • Maize straw samples were treated in mild heat for 50 hours and also in an autoclave at 120oC for half an hour while cattle manure was treated just once; with the autoclave at 120oC for 30 minutes under pressure.
  • The beakers were then closed and kept in an oven at 70oC for 50 hours and the other set placed in an autoclave at 120oC for 0.5hour.

2.4 Biochemical Methane Potential (BMP) Test

  • 5% biomass and 95% medium were used in the BMP test for both substrates.
  • The pH of the mixture was neutralised to between 7.0 and 7.5 using 1M hydrochloric acid and 1M sodium hydroxide solutions.
  • The bottles were kept in an incubator at 37oC and the biogas produced was measured once every day until the biomass became exhausted and no gas was produced anymore.
  • The ideal gas equation was used to calculate the quantity of biogas generated in mL of methane per gram volatile solid.
  • The results of the BMP tests during this study are given in chapter three.

2.5.1 Measurement of pH and Electrical Conductivity

  • After the pretreatments and filtration, the pH and electrical conductivity were obtained from a pH meter.
  • After stabilization, the pH value on the screen is read and recorded and the probe was removed and rinsed with deionized water.
  • This action was repeated for all samples and their duplicates.
  • To measure the electrical conductivity, the pH probe was rinsed and placed in a neutral solution while the conductivity probe was immersed in the solution.
  • At the end of these measurements, the solutions were again filled in 50mL falcon tubes and stored in a refrigerator at 4oC for subsequent use.

2.5.2 Total Solids (TS)

  • First of all the maize straw was chopped into fine pieces using a blender.
  • After that, two porcelain dishes were washed and dried in an oven at 105oC for one hour.
  • After 60 minutes, the two porcelain dishes were removed and kept in a desiccator to cool down and also to prevent them from trapping some moisture.
  • They were then labelled and placed one after the other on an electronic balance to get the weight of the empty dishes.
  • A known amount of biomass (cattle manure and maize straw) were carefully added to the dishes and the weight of the dish plus the biomass recorded.

2.5.3 Volatile solids (VS)

  • To determine the volatile solids of the biomasses, the dried biomass from the total solids measurement above was burned in an oven at 550oC for two hours.
  • The dishes were then removed and kept in a desiccator for one hour to cold down.
  • After cooling, the dishes and the ash left after burning at 550oC for 120 minutes were weighed on an electronic balance and the changes in weight between the dried dish and biomass and the dried dish and ash were recorded.
  • Calculations were made to obtain the volatile solids in percentage as the average between the two dishes for each biomass.
  • The calculated volatile solids in this study were as follows; 82.0% VS (in terms of total solids) for cattle manure and 51.6% VS for maize straw.


  • The DNS reagent containing 1% of the Dinitrosalicylic acid (DNS), 1% NaOH and 0.05% sodium sulphite was added to a beaker.
  • The Rochelle salt was placed in a 100mL beaker and dissolved using pure water and a stirring rod until it was wholly dissolved.
  • The beakers and stirring rods were rinsed into the flasks and deionized water added to the solution to make it to the 100mL mark in each of the volumetric flasks.
  • The biogas produced was calculated per gram VS while the carbon dioxide sensor was used to 41 quantify the percentage of carbon dioxide and hence methane in the biogas produced.

2.6.1 Biogas Measurement (carbon dioxide concentration)

  • The carbon dioxide content of the samples was measured once every week using the carbon dioxide sensor.
  • The sensor can measure both the carbon dioxide and oxygen content of a mixture of gases.
  • The sensor is started and carbon dioxide is selected on the top right corner of the sensor.
  • This is done in duplicates for each pretreatment method and condition and the average recorded as the percentage of the gas.
  • The main reason for measuring the carbon dioxide is to be able to calculate the quantities of the constituents (methane and carbon dioxide) of the biogas produced.

3.1 Electrical Conductivity and pH

  • The results of the pH and electrical conductivity in this study measured using the pH meter are given in table 3.1.
  • This suggests that sodium hydroxide and sulphuric acid pretreatment breaks down the biomass into finer particles producing more dissolved material in the solution and hence rendering it more biodegradable.
  • The pH of the samples was neutralised to between 7.0 and 7.5 after pretreatments.
  • It can also be seen that the samples treated with sulphuric acid and sodium hydroxide had the highest electrical conductivity while those treated with liquid hot water showed the lowest.

3.2. Effects of Pretreatment on Solubility

  • The solubility of the organic matter in the biomasses after pretreatment studies were determined by measuring the soluble sugar concentration they produced.
  • The test tubes were kept in bubbling hot water for 5 minutes for the colours to come out and then removed.
  • The colour intensities were measured using cells placed in a spectrophotometer at 575mµ and width of 0.06 mm.
  • The results obtained for each of the pretreatments and the untreated samples are summarised in tables 3.3 and 3.4. a) Maize Straw Table 3. 3 Amount of reducing sugar from pretreatment of maize straw.
  • Liquid hot water pretreatment produced the lowest amount of reducing sugars compared with the microwave, sulphuric acid and alkaline pretreatments.

3.3. Comparisms of the Solubility of Substrates

  • Comparatively, maize straw produced higher amounts of soluble sugar than cattle manure.
  • This shows that for reducing sugar production, the applied chemical and physical 47 pretreatments were more effective for maize straw than cattle manure.
  • It also means that for this study, maize straw has a higher biogas production potential than cattle manure.
  • Liquid hot water pretreatments increased the solubility of cattle manure more than maize straw while acid, alkaline and microwave pretreatments increased the solubility of maize straw more than cattle manure.

3.4. The Relationship between Reducing Sugar and Methane production

  • The relationships between the reducing sugar production and methane production from treated and raw samples of maize straw and cattle manure are given on figures 3.3 and 3.4.
  • Figure 3. 3 Relationship between measured reducing sugar and methane production from maize straw.
  • It shows that the pretreatment methods which produced the highest quantity of soluble sugar and biogas production are alkaline pretreatment and microwave pretreatments.
  • Acid and liquid hot water pretreatments produced the relatively lowest amounts of reducing sugar and while the methane production for acid pretreatment was relatively lower, the highest temperatures of liquid hot water pretreatments produced higher volumes of methane.
  • 4 Relationship between measured reducing sugar and biomethane production from cattle manure.

3.5.1. Acid Pretreatment of Maize Straw

  • In acid pretreatment, dilute concentrations of sulphuric acid assisted by heat were used.
  • As seen on the table 3.5, the biogas production increased rapidly from day 3 until it reached the point of maximum production between 15 and 18 days for the samples treated with 0.2M and 0.3M H2SO4 while the samples treated with 0.4M H2SO4 reached their highest production around the 9th day.
  • The samples treated with 0.4M concentration had the least production and the earliest maximum point.
  • The daily biogas production from acid pretreatment of maize straw is given on figure 3.5 50 Figure 3. 5 Daily methane production from acid pretreatment of Maize straw.
  • 6, there is an increase in biogas production in a logarithmic manner from day 2 untill it reached a maximum production after 15 days, then there was steady production for a few days before production started to fall.

3.5.2. Alkaline Pretreatment of Maize Straw

  • Alkaline/basic pretreatment was applied by using dilute sodium hydroxide solutions assisted by heat and pressure.
  • Three different concentrations of dilute sodium hydroxide solutions while heating in an autoclave at 120oC for thirty minutes were applied on the biomass.
  • Figure 3. 8 Daily methane production from alkaline pretreatment of maize straw.
  • After which it increased untill it reached maximum production on the 20th day and then decreased steadily.
  • Biogas production for the pretreated samples lasted about one week to ten days longer than the untreated and control samples.

3.5.3. Microwave pretreatment

  • The microwave pretreatment applied on maize straw used the same power/intensity of 300W and varying times.
  • The results obtained are highlighted in table 3.7.
  • It can be seen that the straw treated for 2 and 4 minutes generally had similar production rates which varied only slightly in their peaks as the time went on.
  • The cumulative biogas production is shown on figure 3.12.
  • 56 Figure 3. 12 The cumulative methane production from microwave pretreatment of maize straw.

3.5.4. Liquid Hot Water treatment

  • The other pretreatment method applied on the organic maize straw was the liquid hot water pretreatment.
  • The results obtained are summarised on the table 3.8 Table 3. 8 59 Figure 3. 15 Cumulative biogas production from liquid hot water pretreatment of maize straw.
  • It shows how the biogas production increased steadily within the first fourteen days before stabilising for about a week for the samples treated at a temperature of 120oC while the production for those treated at 135oC kept on increasing until around the 23rd day.

3.6. Comparisms of Total Methane Production from Pretreatments of Maize Straw

  • Table 3. 9 Comparative biogas production from pretreatments of maize straw.
  • 61 Figure 3. 17 Comparative methane production from pretreatments of maize straw.

3.7. Pretreatment of Cattle Manure

  • Four types of pretreatments were applied on cattle manure to investigate their effects on biogas production.
  • They include; acid pretreatment, alkaline pretreatment, microwave and liquid hot water pretreatments.
  • The microwave pretreatment was performed at the same intensity (300W) but at different exposure times.
  • In both cases, samples were duplicated and the average of the results taken.
  • The results obtained for the various pretreatment types are summarised in the following sections.

3.7.1. Acid pretreatment of Cattle Manure

  • Three different concentrations were tested for cattle manure.
  • Figure 3.19 shows the cumulative biogas production.
  • Figure 3. 20 Cumulative methane production from acid pretreatment of cattle manure.
  • The second most effective result was the samples treated with 0.2M sulphuric acid solution which lead to an average increase of 12% in biomethane production while the samples treated with 0.4M sulphuric acid resulted in a 4% increase in methane yield.
  • It indicates that the higher the acid concentration, the lower biogas was produced.

3.7.2. Alkaline Pretreatment of Cattle Manure

  • In alkaline pretreatment, three different concentrations of dilute sodium hydroxide solutions assisted with heat were used.
  • Heating was done in an autoclave at 120oC for thirty minutes.
  • The results obtained in the BMP tests are given in table 3.11 Table 3. 11 Results of the BMP test of alkaline treated cattle manure.

3.7.3. Microwave Pretreatment of Cattle Manure

  • 67 undigested plants remains found in cattle manure.
  • Production was then stable for about a week before it started falling steadily until it ceased.
  • Figure 3.24 shows the daily biogas production from two microwave pretreatment conditions applied on cattle manure.
  • Biogas production lasted over 32 days for both pretreatment conditions.
  • From the graph, it can be seen that the production of biogas increased daily from the second day until it reached its highest concentration between the 18th and 20th days.

3.7.4. Liquid Hot Water Pretreatment of Cattle Manure

  • The fourth and last pretreatment method applied on the cattle manure was the liquid hot water pretreatment.
  • Samples were treated at two different temperatures under the same contact time.
  • The temperature was increased in 15oC intervals to find out the effects of heat variation on the production of biogas.
  • The studies were carried out at temperatures 120oC and 135oC.
  • The outcomes obtained from the BMP tests are given on table 3.13 70.


  • All in all, both chemical and the physical pretreatment methods applied in this study demonstrated high effectiveness in enhancing biomethane yield from cattle manure.
  • This is probably because high temperatures are required to breakdown the undigested remains of the grass straw, plants leaves and other substances present in the manure.
  • The pretreatment with the lowest biogas production was the acid pretreatment but it also had the lowest hydraulic retention time.

3.9: Comparisms between the total biogas production from current study and

  • Literature Compared to previous studies presented in tables 2.2 and 2.3 in chapter 2, it can be seen that the results in this study are relatively higher for maize straw but generally similar for cattle manure.
  • In the same study, maize silage was reported to produce biomethane 74 concentrations ranging between 379-390mL/gVS.
  • The comparative biomethane concentrations are portrayed in tables 3.15 and 3.16.
  • Table 3. 15 Cumulative methane production from maize straw in current study.
  • Table 3. 16 Cumulative methane production from cattle manure in literature.

Substrate CH4 yield (mL/gVS) Reference

  • Table 3. 17 Cumulative biomethane yield from cattle manure in literature.
  • Biogas production from animal manure, also known as Table 2.3.


  • The results obtained from this study lead to the following conclusions;.
  • As expected, all pretreatments increased biogas production for both cattle manure and maize straw although the degree of increase varied.
  • Acid pretreatment lasted shorter period compared to all the other pretreatments both for cattle manure pretreatment and maize straw pretreatment.
  • Two different temperatures were applied on the biomass (120oC and 135oC) with the samples treated at 135oC producing the highest concentration of biogas.
  • Lower temperatures especially for cattle manure are very ineffective.

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Frequently Asked Questions (18)
Q1. What is the main advantage of acid pretreatment?

Acid pretreatment contributes to the output of high amounts of reducing sugars with the application of mild temperatures leading to the hydrolysis of cellulose and hence increased biogas production. 

There are three major building blocks of lignocellulosic biomass namely; hemicellulose, lignin and cellulose, but also minute quantities of other components. 

The various chemical pretreatment types applied before anaerobic digestion in biogas production include; acid pretreatment, alkaline pretreatment, wet oxidation, catalysed steam explosion, oxidative pretreatment with peroxides and the use of ionic liquids. 

Biogas production from untreated straw lasted for fewer days probably because there were lower amounts of reducing sugars available for the bacteria to feed on. 

The production of 2nd generation biofuels is generally not very fruitful because of the necessity of pretreatment due to the presence of lignocellulose which adds to the operating cost, cost of transportation of feedstock and the acquisition of capital equipment. 

Biofuels generally refer to compact materials (such as wood chips, pellets etc.), fluids like oils, biodiesel and ethanol, or gaseous fuels such as biohydrogen, biogas, and biosyngas that are mainly obtained from biomass sources (Cecilia Sambusiti 2013). 

heat assisted acid pretreatment proved to be more effective especially for maize straw because test samples treated with acid without the application of heat failed to produce biogas probably because of a decrease in pH. 

The US, EU and other developing countries like China, Brazil, Thailand, Colombia and Indonesia, have successfully implemented industries that process and produce 1st generation biofuels like bioethanol and biodiesel. 

If the enzymes do not have access to the substrate (the case in lignocellulosic biomass) hydrolysis becomes the rate-limiting step (Karimi 2008). 

Examples of heavy metals that can lead to disturbances in biogas plants are copper, nickel cadmium, zinc (Dieter D. and Angelika S. 2008). 

The anaerobic digestion process is complex and includes a wide range of microbes acting in up to nine stages of transformation of organic matter. 

Major merits of using microalgae as a substrates for biofuel6production is its enormous oil content (approximately 40% on dry matter basis). 

Estimates show that in the production of 1 kilogram of corn grain, approximately one kilogram of maize straw is simultaneously produced (Koundinya, et al. 2017). 

due to the little or no lignin and hemicelluloses in algae biomass, there is a step up in the efficiency of the biomethane production process (Saqib et al., 2013). 

This means that the condition necessary for acetogens to acquire the energy they need to survive and grow is very low concentrations of hydrogen and as a result, they enter a symbiotic relationship with methane producing microorganisms which survive only in environments with high hydrogen partial pressure 

Three different concentrations of dilute sodium hydroxide solutions while heating in an autoclave at 120oC for thirty minutes were applied on the biomass. 

The method used to determine the reducing sugar loads for both the untreated and treated biomass samples used in the study was the G. Lorenz Miller method (G. L. Miller 1959) using the Dinitrosalicylic acid reagent (DNS). 

As seen above, the pretreatment condition which led to the maximum production of biomethane 0.3M acid solution with a total methane production value of 162.2mLCH4/gVS representing a 20% more methane production than the raw cattle manure.