Ahmed Mahdy

Enzymatic cell disruption of microalgae biomass in biorefinery processes

When employing biotechnological processes for the procurement of biofuels and bio‐products from microalgae, one of the most critical steps affecting economy and yields is the “cell disruption” stage. Currently, enzymatic cell disruption has delivered effective and cost competitive results when compared to mechanical and chemical cell disruption methods. However, the introduction of enzymes implies additional associated cost within the overall process. In order to reduce this cost, autolysis of microalgae is proposed as alternative enzymatic cell disruption method. This review aims to provide the state of the art of enzymatic cell disruption treatments employed in biorefinery processes and highlights the use of endopeptidases. During the enzymatic processes of microalgae life cycle, some lytic enzymes involved in cell division and programmed cell death have been proven useful in performing cell lysis. In this context, the role of endopeptidases is emphasized. Mirroring these natural events, an alternative cell disruption approach is proposed and described with the potential to induce the autolysis process using intrinsic cell enzymes. Integrating induced autolysis within biofuel production processes offers a promising approach to reduce overall global costs and energetic input associated with those of current cell disruption methods. A number of options for further inquiry are also discussed. Biotechnol. Bioeng. 2015;112: 1955–1966.

Protease cell wall degradation of Chlorella vulgaris: Effect on methane production

In order to optimize the enzymatic dosage and microalgae biomass loads subjected to enzymatic hydrolysis prior anaerobic digestion of Chlorella vulgaris, organic matter solubilisation and methane production were investigated. Experimental data using protease dosage of 0.585 AU g DW(-1) showed that increasing biomass loads up to 65 g L(-1) did not affect markedly the hydrolysis efficiency (51%). Enzymatically pretreated biomasses subjected to anaerobic digestion enhanced methane production by 50-70%. The attempt of decreasing the enzymatic dosages revealed diminished hydrolysis efficiency concomitantly with a decreased methane production enhancement. In agreement with the good results observed for organic matter conversion into biogas, total nitrogen mineralization was attained for enzymatically pretreated biomass. Despite the high protein content of the biomass and the biocatalyst used in the present study no ammonia inhibition was detected.

Enzymatic pretreatment of Chlorella vulgaris for biogas production: influence of urban wastewater as a sole nutrient source on macromolecular profile and biocatalyst efficiency.

Two biocatalysts, namely carbohydrases and proteases, were assessed for organic matter solubilisation and methane yield enhancement of microalgae biomass. This study evidenced Chlorella vulgaris carbohydrate accumulation (40% on VSS basis) when grown in urban wastewater. Despite of the carbohydrate prevailing fraction, protease pretreatment showed higher organic matter hydrolysis efficiency (54%). Microscopic observation revealed that carbohydrases affected slightly the cell wall while protease was not selective to wall constituents. Raw and pretreated biomass was digested at 1.5 kg tCOD m(-3) day(-1) organic loading rate (OLR1) and 20 days hydraulic retention time (HRT). The highest methane yield (137 mL CH4 g COD in(-1)) was achieved in the reactor fed with protease pretreated C. vulgaris. Additionally, anaerobic digestion was conducted at OLR2 (3 kg tCOD m(-3) day(-1)) and HRT (15 days). When compared to raw biomass, methane yield increased 5- and 6.3-fold at OLR1 and OLR2, respectively. No inhibitors were detected during the anaerobic digestion.

Ammonia tolerant inocula provide a good base for anaerobic digestion of microalgae in third generation biogas process.

This study investigated the ability of an ammonia-acclimatized inoculum to digest efficiently protein-rich microalgae for continuous 3rd generation biogas production. Moreover, we investigated whether increased C/N ratio could alleviate ammonia toxicity. The biochemical methane potential (BMP) of five different algae (Chlorella vulgaris)/manure (cattle) mixtures showed that the mixture of 80/20 (on VS basis) resulted in the highest BMP value (431mLCH4 gVS-1), while the BMP of microalgae alone (100/0) was 415mLCH4 gVS-1. Subsequently, anaerobic digestion of those two substrates was tested in continuous stirred tank reactors (CSTR). Despite of the high ammonium levels (3.7-4.2g NH4+-NL-1), CSTR reactors using ammonia tolerant inoculum resulted in relatively high methane yields (i.e. 77.5% and 84% of the maximum expected, respectively). These results demonstrated that ammonia tolerant inocula could be a promising approach to successfully digest protein-rich microalgae and achieve a 3rd generation biogas production.

Acclimation to extremely high ammonia levels in continuous biomethanation process and the associated microbial community dynamics

Acclimatized anaerobic communities to high ammonia levels can offer a solution to the ammonia toxicity problem in biogas reactors. In the current study, a stepwise acclimation strategy up to 10g NH4+-N L-1, was performed in mesophilic (37±1°C) continuously stirred tank reactors. The reactors were co-digesting (20/80 based on volatile solid) cattle slurry and microalgae, a protein-rich, 3rd generation biomass. Throughout the acclimation period, methane production was stable with more than 95% of the uninhibited yield. Next generation 16S rRNA gene sequencing revealed a dramatic microbiome change throughout the ammonia acclimation process. Clostridium ultunense, a syntrophic acetate oxidizing bacteria, increased significantly alongside with hydrogenotrophic methanogen Methanoculleus spp., indicating strong hydrogenotrophic methanogenic activity at extreme ammonia levels (>7g NH4+-N L-1). Overall, this study demonstrated for the first time that acclimation of methanogenic communities to extreme ammonia levels in continuous AD process is possible, by developing a specialised acclimation AD microbiome.