The simultaneous application of two pretreatment methods can potentially enhance COD solubilization, VS reduction, and methane yield. The following combination of treatment methods have been studied with WAS:

— Thermochemical (alkali-, acid-thermal pretreatment)

— Ultrasonic plus thermal

— Irradiation-assisted methods

Thermal treatment of WAS after adjustment of the pH to 12 (121°C for 30 min) led to an increase in the COD solubilization. With NaOH, KOH, Mg(OH)2 , and Ca(OH)2, solubilization reached 51.8%, 47.8%, 18.3%, 17.1% after treatment at 121°C for 30 min [201] or 71.6%, 83.7%, 55.6%, 51.5% at 140°C for 30 min [225], respectively. Other authors reported a COD solubilization of 55% at pH 12 (NaOH) and 140°C for 30 min compared to 48% without alkali reagent [236].

Thermochemical pretreatment enhanced methane yield by 34 and 19% compared to untreated and chemically pretreated samples, respectively [201]. Combined ther­mochemical pretreatment of A. maxima had a stronger impact on increasing the amount of sCOD (Fig. 12a) [233]. A maximum solubilization value of 78% was achieved at pH 13 and temperature 150°C. In most cases, methanogenesis was inhibited compared to untreated fresh algae (Fig. 12b). At pH 11, the methane yield increased by 5%, 10%, and 20% at temperatures 50, 100, and 150°C, respectively. Overall, alkali-thermal pretreatment led to higher levels of solubilization and to larger methane yields com­pared to acid-thermal pretreatment. Strong inhibition of methanogens can be caused by ammonia, toxic chemicals, and/or fatty acids formed during pretreatment.

Wet oxidation is a pretreatment process when organic materials are treated by gaseous oxygen at high temperatures. It is able to convert poorly biodegradable lignocellulose to carbon dioxide, water, and carboxylic acid [253] . Newspaper bio­mass pretreated by wet oxidation (190°C) and fermented in a batch anaerobic reac­tor showed 59% lignin removal, 74-88% cellulose removal, and 59% of the total COD converted to methane [223]. A doubling of methane yield from raw yard waste after wet oxidation pretreatment was reported [254] .

Microwave irradiation can be used as a volumetrically distributed heat source, and it can be applied with acid or alkali pretreatment. Microwave-assisted acid pretreatment of herbal-extraction process residue enhanced biogas production by 65, 29, and 14% compared to nontreated, acid, or microwave pretreated samples [255, 256]. Microwave-assisted alkali pretreatment of switchgrass increased the cellulase hydrolysis yield by 53% [257]. Ultrasonic treatment (42 kHz for 120 min) after ther­mal treatment (121°C for 30 min) had no effect on COD solubilization. Solubilization enhancement was not statistically significant—19.4% vs. 18.4% (ultrasonic only) — and was comparable with the value for single thermal pretreatment of 17.6% [201].

5.1.1 Algal Metabolic Manipulations Through Environmental Factors

The algal biochemical composition has a dramatic impact on algal biomass digest­ibility and methane yield (Figs. 5 and 7). Improving the extent of algal biomass biodegradability in anaerobic digesters is a critical research need.

Metabolic manipulation is an effective tool for control and influence of algal growth rate and biochemical composition. Several environmental factors, such as availability of carbon dioxide, nutrients (nitrogen, phosphorus), trace metals, silicon for diatoms, level of irradiation, salinity, and temperature affect the enzyme activity and algal biochemical profile significantly.

Generally, stress conditions (nitrogen, phosphorus, or trace metals depletion, photo-oxidative stress, and high salinity) lead to increasing cellular lipid content. Exposure to stress conditions resulted in increasing lipid content on average from

25.5 to 45.7% in green microalgae, from 22.7 to 44.6% in diatoms, and from 27.1 to 44.6% in other oleaginous algae identified as chrysophytes, haptophytes, eustig — matophytes, dinophytes, xanthophytes, or rhodophytes [258]. A major disadvantage of using metabolic methods to modify the lipid content is a decrease in the algal growth/division rate and productivity [259, 260] . This can be countered by an increase in the overall calorific value and theoretical methane potential of the algal biomass due to accumulation of more reduced lipid compounds [261-263]. Li observed the highest lipid productivity at 5 mM nitrate while the highest lipid con­tent was observed at 3 mM nitrate [264]. Optimization of all environmental param­eters is necessary to achieve the highest biomass and lipid productivity [265] .