extfieldsofstudy: Biology Medicine

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SRL-Coated PAMAM Dendrimer Nano-Carrier for Targeted Gene Delivery to the Glioma Cells and Competitive Inhibition by Lactoferrin.

Glioma, as a primary tumor of central nervous system, is the main cause of death in patients with brain cancer. Therefore, development of an efficient strategy for treatment of glioma is worthy. The aim of the current study was to develop a SRL peptide-coated dendrimer as a novel dual gene delivery system for targeting the LRP receptor, an up-regulated gene in both BBB and glioma cells. To perform this investigation, our newly developed nanocarrier (PAMAM-PEG-SRL) was used for gene delivery to C6 glioma cell lines. DNA (GFP) was loaded in these functionalized nanoparticles and their cellular uptake/distribution and gene transfection efficacy was evaluated by fluorescence and confocal microscopy. In vitro studies showed that SRL-modified nanoparticles have good transfection efficacy. Results revealed improved gene transfection efficiency of newly-synthesized delivery system. We also found that lactoferrin, as a LRP ligand, reduced the gene transfection efficacy of the delivery system due to its higher affinity compared to SRL peptides (Competitive inhibition). The present results suggest that the synthesized delivery system has the potential to be used as an alternative targeted drug delivery system for brain tumors.

Introduction
Glioma, as a primary tumor of the central nervous system, is responsible for most of brain cancer deaths (1). The ability of glioma cells to invade surrounding normal tissues makes complete surgical resection impossible, in turn leading to tumor recurrence (2). In view of this, there is an urgent need to develop an effective therapeutic delivery system to selectively deliver cytotoxic drugs to tumor cells while sparing normal cells. However, poor blood-brain barrier (BBB) penetration of drugs is a major obstacle for targeted delivery of drugs and genetic materials, such as DNA, to brain tumors. This has made the treatment of nervous system diseases rather difficult and complicated. The blood-brain barrier prevents the entry of most drugs and genetic materials (almost 98%) into the brain. Generally, only small (less than 400 Da) or highlipophilic particles are able to pass through the blood-brain barrier. In fact, poor blood-brain barrier penetration are considered to be the most common causes of treatment failure in patients with nervous system diseases (3, 4). Moreover, it seems to be important to prevent the intracellular accumulation of therapeutic agents in normal tissues and to increase the concentration of these agents at tumor sites (5).
Nowadays, nanotechnology provides solutions to such problems, so that a wide variety of nanoparticles and supramolecular nanodevices have been developed to improve the penetration of drugs and even genetic materials across different endothelial and epithelial barriers in a noninvasive manner (6, 7). PAMAM dendrimers, a class of the nanoparticles, have been considered as a promising system for gene delivery (8,9). More importantly, due to the presence of primary surface amines in this type of nanoparticles, PAMAM dendrimers have an intrinsic capacity to bind to a variety of negatively-charged agents, including gene segments, plasmids and several types of ligands (8). However, these carriers cannot easily permeate the BBB. One possible way that has been used to circumvent this problem is the specific binding of certain ligands to the surface of these nanoparticles to facilitate selective passage of nanoparticles across the BBB.
A dual-targeting strategy is a newlyintroduced method recently developed for the targeted delivery of therapeutic agents to the brain, in which nanocarriers decorated with only one targeted ligand on their surface are applied to target the glioma cells. In this approach, nanocarriers are able to deliver therapeutic and imaging agents to tumors by targeting specific receptors overexpressed on the surface of the BBB and glioma cells (10, 11). A variety of ligand types has been used for this purpose, including lactoferrin (11), Angiopep (9), Chlorotoxin (12), RGD (13), LDLR-mediated peptide-22 (14) and T7 peptide (15). Among them, lactoferrin and Angiopep target the LRP present on the surface of brain capillary endothelial cells (BCECs) (9, 11). Lipoprotein endocytosis is mediated by three structurally related cell surface receptors, which the presence of at least one copy of an NPXY motif in their structure leads to the receptor internalization into coated pits (16). Prototype of this family is low density lipoprotein (LDL)' receptor (17). These receptors bind to apoB-100 or apoE in plasma lipoproteins and remove them from plasma.
LRP, a member of the LDL receptor gene family, is much larger than the LDL receptor (16). The physiological role of LRP receptors include the permeability control of the BBB, regulation of vascular tone and postischemic lesion formation, in response to activated tissue-type plasminogen activator (18). LRP is expressed in all cells, and binds to more than 40 different types of ligands such as lipoproteins, protease/protease-inhibitor complexes, extracellular matrix proteins, viruses, growth factors and cytokines (19). Also, its demonstrated that lactoferrin is a specific ligand of LRP (16,20).
The unique feature of this protein is a high transcytosis rate (21).
It is well-documented that LRP is overexpressed on glioma cells (10,22,23). Therefore, LRP can be served as a potential dual targeting receptor to overcome the BBB and delivery of therapeutic agents to glioma cells.
More recently, we have developed a new nanocarrier based on the G4 PAMAM dendrimer, synthesized by binding the SRL peptide (CLSSRLDA) to G4 PAMAM dendrimers (24). The SRL peptide, designed by Pasqualini and Ruoslahti, exhibited high penetration efficiency into brain capillary endothelial cells (BCECs) (25). In our previous study, we showed that the SRL peptide was found to be a LRP ligand, and nanocarriers modified by this ligand were able to internalize into the BCECs by the clathrin/caveolin energy-dependent endocytosis mechanism (24).
Because of the fact that C6 glioma cells overexpressed LRP, we sought to attach the SRL peptide (with the CLSSRLDA sequence) on the surface of G4 PAMAM dendrimers, as a dual targeting delivery system, for targeting LRPs present on the C6 glioma cell line. Also, in this study the inhibitory effect of lactoferrin on the entrance of PAMAM-PEG-SRL/DNA complexes to C6 glioma cells was evaluated.
The C6 glioma cell line was purchased from National Cell Bank of Iran (NCBI), Pasteur Institute, Tehran, Iran. C6 glioma cells were cultured in DMEM (Sigma-Aldrich), supplemented with 10% FBS, 100 U/mL penicillin and 100 g/mL streptomycin at 37 o C in a humidified incubator containing 5% CO2. All the cells used in this study were extended between 10 to 15 passages.

Synthesis of PAMAM derivatives, and Characterization of Nanoparticles
PAMAM derivatives were synthesized according to the same methods described in the previous papers (27,28). In brief, PAMAM-PEG-SRL at 1:6:3 ratio synthesized as previously described. And their size and physicochemical characteristics was evaluated by NMR, Zeta sizer and UV spectroscopy (24).

Preparation
of Dendrimer/DNA Nanoparticles PAMAM and PAMAM-PEG-SRL were freshly synthesized, and diluted in distilled water. Then, a DNA solution (100 mg DNA/mL in 50 mM Sodium sulfate solution) was added to nanoparticle solution at specific weight ratio (1:10, PAMAM/DNA w/w), and gently vortexed at 37 o C for 30 sec. Afterwards, the complex was incubated at room temperature for 30 min to allow self-assembly formation. Agarose gel electrophoresis was carried out to verify the formation and stability of the nanoparticle-DNA complex (24).

Cellular uptake of dendrimers
BCECs and C6 glioma cells were seeded at a density of 2×10 4 cells/well in 24-well plates (Corning-Coaster, Sigma, USA), for 48 hours. Cell confluence and morphology were checked under a fluorescent microscope throughout the experiment. Afterwards, the C6 glioma cells were incubated at a 1 µM concentration of BODIPY-labeled PAMAM or 0.2, 0.5 and 1.0 µM concentrations of BODIPY-labeled PAMAM-PEG-SRL at 37 o C for 30 minutes with a final concentration of 1 µM. Furthermore, to study the energy dependency of nanoparticles internalization into target cells, C6 glioma cells were exposed to BODIPY-labeled PAMAM-PEG-SRL at a density of 1µM at 4 o C. The culture supernatant was removed, and cells were then washed three times with cold PBS (pH 7.4), and examined using a fluorescent microscope (Olympus, Osaka, Japan). To quantitatively analyze the cellular uptake of PAMAM-PEG-SRL at different concentrations, the cells BCECs and C6 glioma were cultured at a density of 8×10 4 cells/well in 6-well plates (Corning-Coaster, Sigma, USA) for 72 h. After checking the cell confluence and morphology, the cells were incubated at a 1 µM concentration of BODIPY-labeled PAMAM-PEG-SRL for 30 minutes. As noted in the previous section, the cells were washed three times with PBS (pH 7.4), and then, the treated cells were trypsinized and centrifuged for 8 minutes at 1600 rpm to obtain the cellular pellet. The pellet was resuspended in PBS (pH 7.4), and analyzed by a flow cytometer (FACS Calibur, BD Biosciences, Bedford MA, USA) equipped with an Argon Ion Laser (488 nm) as a stimulating source of fluorescent. For each sample, 10000 events were collected and analyzed using WinMDI software. Cells cultured under normal conditions and live cells were considered as controls and gates, respectively. In general, the only cells at the gate were analyzed. The mean density of fluorescent cells was calculated as a histogram plot (27).

Gene transfer efficiency into C6 glioma cells
The Plasmid DNA was covalently labeled using the fluorescent dye ethidiummonoazide bromide (EMA). Briefly, the Plasmid DNA solution (1 mg/mL in TE buffer, pH 7.0) was diluted in EMA solution until reaching a concentration of 0.1 mg/mL, and then incubated for 30 min in darkness. Then, the solution was precipitated by the addition of ethanol to a final concentration of 30% (v/v). The pellet was collected by centrifugation at 12000 rpm for 20 min, and resuspended in 50 mM sodium sulfate. EMA-DNA solution was used to prepare nanoparticles by the same method. C6 glioma cells were seeded at a density of 2.0 ×10 4 cells/well in 24-well plates, and then incubated for 48 hours. Subsequently, the nanoparticles (PAMAM or PAMAM-PEG-SRL) containing EMA-DNA were added to the cells (27). Thirty minutes later, the culture supenatant was removed, and the remaining cells were washed 3 times with PBS (pH 7.4), and observed under a fluorescent microscope.
Cellular uptake mechanism of dendrimer/ DNA nanoparticles C6 glioma cells were seeded at a density of 2×10 4 cells/well in 24-well plates, and cultured using the above method. After monitoring the morphology and confluency of the complex filipin (0.5 mg/mL), phenylarsine oxide (2.5 mM), colchicines (2.5 mM) or lactoferrin (1mg/ ml) were added to each well, and incubated for 10 minutes (27). Subsequently, the supernatants were removed, and an equal volume of the nanoparticle PAMAM-PEG-SRL containing EMA-DNA were added to the above mixtures. After 30 minutes, the supernatant was removed, and the cells were washed three times with cold PBS (pH 7.4), and observed under a fluorescent microscope.

Confocal microscopy
The intracellular distribution of the nanoparticles was studied using a confocal microscopy. C6 glioma cells were seeded at a density of 10 4 cells/well in a 35 mm glassbottom culture dish, and cultured at 37 o C under an atmosphere containing 5% CO2 for 48 hours, recultured in serum-free media, and then assessed 15 and 60 min after exposed to the PAMAM-PEG-SRL/DNA complex (5 µg DNA/ well, N/P=10). After this time, the cells were washed with cold PBS, and subjected to 50nM LysoTracker Green (Molecular probes Inc.) and DAPI for 30 and 10 minutes, respectively. Afterwards, the cells were washed three times with cold PBS, fixed with paraformaldehyde and observed under confocal laser scanning microscopy (29).

Qualitative assessment of Green Fluorescence protein expression (GFP) in transfected cells.
C6 glioma cells were cultured at a density of 1.0 × 10 6 cells/well in 6-well plates in 2 mL of culture media for 18 h to reach 50% confluence for gene therapy experiments. Prior to transfection, the medium was replaced with a serum-free medium, and the cells were then incubated for 4 h with the complexes PAMAM-PEG-SRL/DNA and PAMAM/DNA in the presence or absence of lactoferrin (8 micrograms per well, 8 μg/well) (Figure 8). The medium was replaced with a fresh medium containing 10% FBS, and then incubated again for 24 hours. GFP expression levels were detected under a fluorescent microscope (Olympus, Osaka, Japan) (30).
Luciferase Activity Assay. C6 glioma cells were cultured in a 24/ well plate at the appropriate confluence for 24 h, and allowed to grow 50% confluence at the transfection time. Prior to transfection, the medium was replaced with serum-free medium, and the cells were then incubated with PAMAM-PEG-SRL/pDNA complexes (4 μg /well) at a specific weight ratio under standard conditions for 4 h. Subsequently, the medium was replaced with fresh a medium containing 10% serum, and incubated again for 24 h.
To measure the luciferase activity, cells were washed with PBS two times, and 100 to 200 µL of cell lysis buffer was added to each well for 5 minutes at 37 °C. Cell lysis solution was centrifuged at 1200 rpm for 5 min, 10 µL of the supernatant was mixed with 25 µL of both luciferase substrate and ATP solution, and luciferase activity was measured with Sirius luminometer (Autolumat LB953, EG and G, Berthold, Germany). Small amounts of the protein were determined by the bicinchoninic acid assay (Bio-Rad Laboratories, Hercules, CA). Relative light units (RLUs) were normalized with the concentration of the proteins extracted from the cells. All transfection experiments were performed as triplicate. The transfection activity was reported by RLUs (31). Lipofectamine was used as a control to compare each sample.

Statistical analysis
All quantitative assessments were carried out in quadruplicate, and the experiments were repeated 3 times. The data were expressed as mean ± SD. Statistical analysis was performed by the one-way ANOVA, using the statistical software.

In-vitro uptake of DNA-containing particles by C6 glioma cells
The EMA-labeled nanoparticles were used for the study of cellular uptake, and the fluorescent images were analyzed by a fluorescent microscope. To investigate the concentration dependence of nanoparticles internalization into C6 glioma cells, the cells were measured after a 30-min cell exposure to different concentrations (0.2 to 1 µM) of EMAlabeled PAMAM-PEG-SRL/DNA. As it can be seen in Figure 1, an increase in the complex concentration up to 1 µM (250 µg/mL) resulted in an outstandingly higher cellular uptake of PAMAM-PEG-SRL/DNA ( Figure 1G, H), demonstrating the dose-dependency entrance behavior of the complexes. At the 1 µM (250 µg/ ml) concentration of PAMAM-PEG-SRL/DNA, there was an equal amount the positive-BODIPY cells (data not shown), while mean fluorescent intensity increased from 66 for BCEC to 132 for C6 glioma cells ( Figure 2B). An increase in mean fluorescent intensity is due to the higher expression level of LRP in C6 glioma cells than BCEC, therefore which can easily be targeted by the SRL peptide (as a LRP ligand) present on the surface of nanoparticles.
The cellular uptake of EMA-labeled PAMAM-PEG-SRL/DNA at 4 o C was much less than 37 o C that could be considered as another reason for energy-dependency of nanoparticles cellular uptake, endocytosis ( Figure 3C, D) (32). Results also indicated that the cellular uptake of the modified nanoparticles is dose-dependent.
The exact cellular uptake mechanism of DNA-containing particles Similar to our previous experiment (24), the results from this study showed that a ratio of 1:6:3 showed the highest level of uptake (data not shown) amongst the three PAMAM:PEG:SRL compositions used here (1:2:1, 1:4:2 and 1:6:3 molar ratios). Furthermore, it was shown that the cellular uptake of PAMAM-PEG-SRL/DNA (Figure 3) was more than that of PAMAM/ DNA. Our data also showed that the use of inhibitors such as phenylarsin oxide, filipin, or lactoferrin ( Figure 3) resulted in the inhibition of cellular uptake in the PAMAM-PEG-SRL/DNA complex. These inhibitors were used to study the mechanism blocking the entry of nanoparticles into the cells. Among them, lactoferrin and phenylarsin oxide have the highest level of inhibition ( Figure 3). Lactoferrin, as a ligand, is known to have a high affinity to LRP (20, 32). As depicted in Figure 3, Lactoferrin, as an LRP inhibitor, is able to inhibit gene delivery, and shows the increased inhibition of nanoparticle entry to cells ( Figure 3E, F).

Cellular localization of PAMAM-PEG-SRL/ DNA nanoparticles
The confocal microscopy was used to study the intracellular distribution of PAMAM-PEG-SRL/DNA nanoparticles. By definition, endocytosis is a process where molecules are internalized as vesicles into the cytoplasm, socalled endosomes (33). As a general rule, drugs should escape endosome to be able to act in the cell; otherwise, they will end in lysosomes, where they will be destroyed. Therefore, escape in early stages (endosomal escape) is necessary for drugs entered into the cells via endocytosis. The cellular uptake of EMA-labeled PAMAM-PEG-SRL/DNA complexes occurred in a timedependent manner. As shown in Figure 4. the small red dots represent DNA, whereas the small green dots indicate LysoTracker green. Because LysoTracker green marks the endosomes or lysosomes, the green and red spots are colocalized inside endosomes and lysosomes after 15 min ( Figure 4E-H), indicating that DNA has entered into the cell through endocytosis (as endolysosome cargo in endosomes and lysosomes). After 60 min, however, the red dots, the larger part of DNA illustrated in Figure 4I-L, were dispersed throughout the whole cell, as can be found while entering the nucleus). This shows that nanoparticles have escaped endosomes and lysosomes (32). This phenomenon has been described as proton sponge theory (34, 35). Based on this theory, secondary and tertiary amines of cationic carriers resist endosomal acidification by capturing and absorpting protons. This function leads to a delay in lysosomal fusion to the endosomes, and enables counterions (e.g. Cl−and H2O) to flood the endosomes. Entrance of water into the endosomes result in the swelling and rupture of endosomes, and the release of cargo into the cytoplasm (36). Although the mechanism underlying the pDNA entrance to the nucleus is not yet clearly understood, it seems that pDNA released from late endolysosomes enters into the nucleus by the diffusion process (29).

Qualitative analysis of In-Vitro transfection efficiency in C6 glioma cells
To qualitatively analyze in vitro gene expression efficiency of the synthesized nanoparticles, the C6 glioma cells were exposed to PAMAM-PEG/pDNA and PAMAM-PEG-SRL/pDNA with or without lactoferrin ( Figure  5). Our data showed that, without lactoferrin, PAMAM-PEG-SRL/pDNA induces higher GFP formation than PAMAM-PEG/pDNA ( Figure  5C, D). Meanwhile, the presence of lactoferrin decreased the GFP expression in C6 glioma cells treated with PAMAM-PEG-SRL/pDNA ( Figure  5B). This phenomenon occurred possibly due to the higher affinity of lactoferrin to LRPs than the SRL peptide. Finally, lactoferrin was found to have no effect on PAMAM-PEG/pDNA.

Quantification of gene transfection efficiency in C6 glioma cells
Gene transfer efficiency in cancer cells was investigated by using luciferase gene expression, and the results were reported as the lighting Lux unit per mg of protein (RLU/mg). As illustrated in Figure 6 A and B. the optical expression of the PAMAM-PEG-SRL nanoparticles at a polymer/DNA ratio of 10:1, and Lipofectamine reached 2.38×10 5 and 2.1×10 5 RLU/mg of the protein, respectively. However, in the presence of lactoferrin, the expression level decreased to 8×10 4 RLU/mg protein, which might be due to the competitive inhibition effect of lactoferrin on the uptake of SRL modified nanoparticles.

Discussion
Glioma, one of the most common primary brain tumors, is categorized into four groups according to WHO classifications (37). Although its prevalence remains lower than other solid tumors, glioma has a very weak prognosis (38). Development of an effective therapeutic delivery system is crucial to selectively deliver cytotoxic drugs to the tumor cells while sparing normal cells. A dual-targeting delivery system, currently thought to be a promising approach for gene delivery to brain tumors, confers a series of advantages, such as high efficiency, low toxicity, stability and high transfection efficiency. These advantages may provide new opportunities in enhanced delivery of therapeutic agents across the blood-brain barrier to treat brain diseases (11,15). A variety of ligand types has been used for dual-targeting, such as lactoferrin (11), Angiopep (9), Chlorotoxin (12), RGD (13), LDLR-mediated peptide-22 (14), and T7 peptide (15). More recently, we have synthesized the SRL-modified PAMAM nanocarrier for gene delivery to the brain (24). In addition, we have  demonstrated that the SRL peptide serves as a LRP ligand. Because C6 glioma cells are able to overexpress LRP (10), we sought to use our newly-synthesized nanocarrier as a new dual targeting system for gene delivery to the glioma cells. In the present study, PAMAM G4 serves as a primary gene delivery vehicle, while SRL through residues added to the end of N binds to the dendrimer using NHS-PEG-MAL.
The cellular absorption of SRL-modified nanoparticles is dependent upon dose and energy contents (Figure 1 and 3).
Nowadays, the non-invasive gene delivery to the brain remains a challenge. However, receptor mediated transcytosis (RMT) is a potential way to successfully transfer drugs and genes across the BBB using endogenous or synthetics ligands (39,40). Over the past few years, numerous attempts have been made to produce the targeted LRP peptides (angiopep-2) (27, 41) being able to identify other types of this ligand (LDLR) (42, 43). The physiological role of LRP receptors is to control BBB permeability, vascular tone and the postischemic lesion formation as a response to activated tissue-type plasminogen activator (18). LRP is expressed on all cells, and associated with more than 40 different types of ligands such as lipoproteins, protease/proteaseinhibitor complexes, extracellular matrix proteins, viruses, growth factors and cytokines (19). A fast transcytosis rate is the unique feature of this protein (21). Lipoprotein endocytosis is mediated by three structurally related cell surface receptors, which the presence of at least one copy of an NPXY motif in their structure leads to the receptor internalization into coated pits (16). LRP is one member of the LDL receptor gene family. In our recent experiment, we showed that SRL peptide has a competitive inhibitory effect on the cellular uptake of PAMAM-PEG-SRL nanoparticles, consistent with the findings of Pasqualini and our recent work (24, 25).
As we shown in our recent paper, the entry of PAMAM-PEG-SRL/DNA complexes can be affected by different mechanisms (24). It is likely that uptake mechanisms can be affected by complex formation. Therefore, to study the entry route of PAMAM-PEG-SRL/DNA nanoparticles to C6 glioma cells, we attempted to use endocytosis inhibitors such as, filipin complex (endocytosis inhibitor mediated through caveolae) (44), colchicine (macropinocytosis inhibitor) (45) and phenylarsine oxide (endocytosis inhibitor mediated through clathrin). The findings showed that all the inhibitors were able to prevent the entry of nanoparticles into the cells the same BCECs as in our previous work, indicating involvement of both routes with the mediation of the receptor (RMT) in the cellular uptake of these nanoparticles. Among the inhibitors, the highest level of inhibition was found through the phenylarsine oxide pathway (Figure 3), observed in nanoparticles modified by other LRP ligands, such as lactoferrin and angiopep-2 (46-48). After comparing the sequences of the identified LDL ligands, such as angiopep-2 (TFFYGGSRGKRNNFKTEEY) (27), LDLRmediated peptide-22 ( MPRLRGC) (42), and the SRL peptide (CLSSRLDA) (owing the fact that both linear forms of the peptide had the same cellular uptake characteristics, as mentioned earlier), we suggest that these three peptides indicated a relatively similar section (underlined letters), and this amino acid sequence seems to play an important role in identifying LDL receptors. However, more investigations are required in this area to clarify the exact mechanism. Furthermore, our qualitative and quantitative gene transfection analysis showed that SRLs has a very low affinity to LRPs, as compared to lactoferrin. On the other hand, SRL-modified nanoparticles, compared to lipofectamine, exhibited high efficiency as a standard carrier, while not significant.
Finally, as LRP receptors are expressed in some other types of cancers such as colorectal carcinoma, the current nanoparticles may be used for increased delivery and targeting of anticancer drugs to these sites. It is obvious that for such kind of delivery, special consideration and enhancement techniques are required (49).

Conclusion
A new kind of modified G4 PAMAM conjugating on the surface of the SRL peptide was prepared and demonstrated to be efficient for glioma targeting gene delivery in vitro. In vitro qualitative and quantitative experiments by using the confocal microscopy, fluorescent microscopy, flow cytometry and AFM showed that PAMAM-PEG-SRL is able of targeting the C6 glioma cells with higher capability than BCECs. PAMAM-PEG-SRL was internalized by C6 glioma cells through clathrin and caveolaemediated endocytosis, similar to BCECs. LRPmediated endocytosis may be the main cellular mechanism for SRL-modified nanoparticles internalization. We believe that PAMAM-PEG-SRL has a promising potential for an efficientdual targeting gene delivery system to target the brain and glioma.


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The Roles of Microtubule-Based Transport at Presynaptic Nerve Terminals

Targeted intracellular movement of presynaptic proteins plays important roles during synapse formation and, later, in the homeostatic maintenance of mature synapses. Movement of these proteins, often as vesicular packages, is mediated by motor complexes travelling along intracellular cytoskeletal networks. Presynaptic protein transport by kinesin motors in particular plays important roles during synaptogenesis to bring newly synthesized proteins to establish nascent synaptic sites. Conversely, movement of proteins away from presynaptic sites by Dynein motors enables synapse-nuclear signaling and allows for synaptic renewal through degradation of unwanted or damaged proteins. Remarkably, recent data has indicated that synaptic and protein trafficking machineries can modulate each other’s functions. Here, we survey the mechanisms involved in moving presynaptic components to and away from synapses and how this process supports presynaptic function.

INTRODUCTION
Neurons possess the capacity to modify their synapses throughout life (Holtmaat and Svoboda, 2009). Being often located significant distances away from the neuronal cell body, biomolecules destined to function at synapses must usually be transported over extended distances before reaching their destinations. Likewise, biomolecules earmarked for degradation or involved in retrograde signaling must be returned to the cell body. Both processes are critically dependent on the intracellular trafficking machinery that is powered by cytoskeletal-based molecular motors. This review will examine the roles of the microtubule-based transport machinery as an integral set of nanomachines supporting the function of presynaptic terminals.

MOLECULAR COMPLEXITY OF SYNAPSES AND CHALLENGES FOR SYNAPTIC TRAFFICKING
Presynaptic terminals play critical roles in mediating neurotransmitter release during synaptic transmission (Figure 1). To fulfill these roles, mature terminals possess hundreds of different proteins that are intricately assembled into molecular nanomachines involved in various aspects of presynaptic function (Morciano et al., 2009;Südhof, 2012;Boyken et al., 2013; FIGURE 1 | Generalized representation of trafficking events to and from the presynapse. (1) Most presynaptic proteins are synthesized in the cell body and transit the Golgi apparatus where they are sorted to specific transporting vesicles (for example, synaptic vesicle precursor transport vesicles (STVs) or Piccolo-Bassoon transport vesicles (PTVs)) and delivered to presynapses by anterograde kinesin motors. Upon reaching their destinations, several mechanisms are known to mediate cargo unloading including: (a) post-translational modifications (e.g., phosphorylation) of kinesin motors, adaptors and cargoes or (b) via the action of GTPases and their effectors. Released cargoes can then be used to build new presynaptic sites (synaptogenesis), maintain existing synapses or contribute to synaptic plasticity. (2) Degradation and turnover of excess or damaged proteins by the autophagy pathway also contributes to presynaptic homeostatic maintenance. Autophagosomes formed here are returned to the cell body by retrograde Dynein motors. (3) Retrograde transport can also be used to convey extracellular signals received at presynaptic terminals to the nucleus where they modulate gene expression. These signals impact the growth and survival of neurons and mediate their response to injury. An example of such responses can be evoked by the binding of neurotrophins (for example NGF) to presynaptic Trk receptors. Activation of these receptors evokes local signaling response at presynapse. Alternatively, receptor-ligand complexes are endocytosed and form signaling endosomes containing other activated signaling molecules. These endosomes are then transported by Dynein to the cell body to induce changes in gene expression or directly regulate signaling cascades. Sizes of molecules and vesicles are not drawn to scale. Wilhelm et al., 2014). In particular, synaptic vesicles (SVs) and active zones (AZs) are key components involved in this process. Elegant proteomic analysis of SV composition by Takamori et al. (2006) demonstrated that, despite its small size, SVs exhibit remarkable complexity and diversity in their protein content. Proteins directly involved in vesicle exocytosis such as the SNARE proteins (syntaxins, synaptobrevins and SNAPs), the calcium sensor synaptotagmin, V-ATPase and vesicular neurotransmitter transporters (VGLUT and VGAT) were all detected. Similarly, AZs that demarcate sites of neurotransmitter release by recruiting SVs and voltage-gated Ca 2+ channels also comprise of a large ensemble of proteins including AZ proteins such as RIM, RIM-BP, Munc13, ELKS/CAST/ERC and α-liprin that participate in SV docking and priming (Südhof, 2012;Chua, 2014). While this complexity and diversity of proteins in the presynapse has long been appreciated, we are only starting to unravel the principles underlying the movement of these cargoes to and away from these sites.

MICROTUBULE-BASED MOTORS AS NANOMACHINES TO TRANSPORT PRESYNAPTIC CARGOES
Diffusion can be effective for migration of small cargoes within very short distances (Encalada and Goldstein, 2014). However, the process becomes highly inefficient with increasing cargo size and distance to be travelled. The substantial gap separating most synapses and the neuronal cell body necessitates the employment of the intracellular trafficking machinery to shuttle presynaptic proteins between these sites. In general, Kinesin and Dynein motor protein families are involved in delivering and removing vesicular content to and from presynaptic sites, respectively (Hirokawa et al., 2010;Kevenaar and Hoogenraad, 2015). Both motor families utilize microtubule cytoskeletal networks to move cargo. Microtubules are inherently polar and the plus ends of microtubule tracks point away from the cell body in axons (Kapitein and Hoogenraad, 2011). Thus, in axonal transport, Kinesin motors are primarily responsible for moving cargoes from sites of synthesis to presynaptic sites, while Dynein motors mediate movement of biomolecules from synapses towards the cell body. In comparison, Myosin motors are generally regarded to be involved in short-range actin-based transport and will not be covered here (Cingolani and Goda, 2008;Hirokawa et al., 2010).

ANTEROGRADE TRANSPORT TO THE PRESYNAPSE INVOLVES KINESIN MOTORS
Many synaptic proteins traffic through the Golgi apparatus en route to synapses after their synthesis in the cell body (Figure 1; Sytnyk et al., 2004;Sann et al., 2009). Kinesin motors play important roles not only during the Golgi export of these proteins but also during subsequent trafficking to their destinations (Akhmanova and Hammer, 2010). Presynaptic proteins transported by Kinesin motors include SV and AZ proteins. The precise mechanisms governing the sorting and loading of cargoes onto their respective motors remain unclear but phosphorylation of cargoes, adapters and motors has been implicated as an important regulatory step in this process (Sato-Yoshitake et al., 1992;Lee and Hollenbeck, 1995;Morfini et al., 2002;Chua et al., 2012).
The trafficking of two broad categories of presynaptic cargoes has been studied. Synaptic vesicle precursor transport vesicles (STVs) carrying SV proteins were shown to be delivered to presynaptic sites by Kinesin-3 motors (KIF1A and KIF1Bβ; Yonekawa et al., 1998;Ahmari et al., 2000;Zhao et al., 2001;Sabo et al., 2006). Loss of KIF1A and KIF1Bβ in neurons caused significant reductions in SVs and their proteins in presynaptic terminals. On the other hand, vesicular transport of presynaptic membrane proteins, such as SNAP-25 and syntaxin-1, are mediated by Kinesin-1 motors (Diefenbach et al., 2002;Su et al., 2004;Chua et al., 2012). AZ proteins including Bassoon, Piccolo and RIM are also co-transported with SNAP-25 and synataxin-1 as Piccolo-Bassoon transport vesicles (PTVs; Shapira et al., 2003;Cai et al., 2007). Interestingly, emerging evidence suggests that presynaptic cargoes are probably not as discretely grouped and an appreciable cargo overlap during transport does exist (Goldstein et al., 2008;Maas et al., 2012;Wu et al., 2013).

DELIVERING CARGOES TO SPECIFIC SYNAPSES
Site-specific release of cargoes is one of the least understood steps in trafficking of presynaptic components. Given that an average mammalian neuron possesses thousands of synapses (Beaulieu and Colonnier, 1985), the transport machinery has to discriminate between the varying needs of different presynaptic sites and deliver the appropriate cargoes to these sites accordingly. The simplest explanation could be that cargoes are released upon reaching the ends of microtubule tracks at the presynapse (Rizzoli, 2014). These cargoes would be then redistributed between neighboring presynaptic sites by diffusion, retrograde transport on the same microtubule track or other mechanism (Darcy et al., 2006;Opazo et al., 2010;Staras et al., 2010). Nevertheless, release of cargo caused exclusively by discontinuity of microtubules cannot fully explain specificity of targeted delivery of presynaptic proteins. Local activation of signaling cascades involved in the arrest of anterograde transport can contribute to this process. One such pathway relates to the small GTPase Rab3 which is a marker of mature SVs and is co-transported with STVs to the presynapse (Fischer von Mollard et al., 1990;Schlüter et al., 2004). DENN/MADD is a Rab3 guanine nucleotide exchange factor (GEF) previously shown to regulate neurotransmitter release (Yamaguchi et al., 2002). DENN/MADD was shown to bind both KIF1A and KIF1Bβ and promote anterograde transport of vesicles associated with Rab3 in the GTP-bound state (Niwa et al., 2008). Either locking Rab3 in the GTP-bound state or deletion of the DENN domain containing the putative enzymatic GEF activity interferes with the transport of these vesicles to distal axons. These results suggest that local factors that induce GTP hydrolysis and cycling of Rab3 between its nucleotide bound states could act as a trigger to release cargoes at distal presynaptic sites.
Phosphorylation of motors and cargoes has been implicated in synaptic cargo transport (Sato-Yoshitake et al., 1992;Vagnoni et al., 2011;Chua et al., 2012). Similarly, phosphorylationdephosphorylation cycles of these proteins are also likely to affect synapse specific delivery of cargoes. One possible candidate for this is GSK3β. Phosphorylation of kinesin light chains by GSK3β does not affect the binding of kinesin to microtubules but instead releases membrane cargoes from the motor (Morfini et al., 2004). GSK3β is a general inhibitor of anterograde trafficking but is selectively active in growth cones (Morfini et al., 2004;Polleux and Snider, 2010;Weaver et al., 2013). Local activation GSK3β can therefore contribute to the release of kinesin cargoes and formation of synapses de novo along the leading edge of growing axon.
During synaptogenesis, both PTVs and STVs move bidirectionally along axonal processes and coordinated trafficking of a portion of STVs and PTVs to specific sites along axons has been documented (Ahmari et al., 2000;Shapira et al., 2003;Sabo et al., 2006;Bury and Sabo, 2011). Pausing of STVs and PTVs coincide with cargo deposition in response to a hierarchy of cues from synaptogenic molecules (such as trans-synaptic adhesion molecules) and initiates the formation of nascent presynaptic sites (Lucido et al., 2009;Siddiqui and Craig, 2011;Chia et al., 2013). Coordination between the small GTPase Arl8 and the Mitogen-activated protein (MAP) kinase signaling pathway has been implicated in facilitating sitespecific unloading of these cargoes to developing presynaptic sites in C. elegans (Wu et al., 2013). In this regard, Arl8 has been proposed to prevent undesirable premature assembly of presynaptic proteins during axonal transport while signaling through JKK-1/JNK-1 promotes capturing and integration of these proteins to the presynapse. It remains to be determined if Arl8 also serves a similar function during synaptogenesis in mammalian neurons.
In addition to the involvement of specific signaling pathways, recent studies suggest that some aspects of synaptic delivery could be inherently stochastic in nature. In mature synapses, a fraction of recycling SVs is distributed between mature synapses-a phenomenon that occurs in the scale of minutes (Darcy et al., 2006). Remarkably, this exchange occurs not only between neighboring boutons but also between remote synapses. Activity-dependent regulation of synapsin-1 phosphorylation at position Ser-551 by Cdk5 modulates clustering and dispersion of SVs between different presynaptic sites (Verstegen et al., 2014). This would indicate that redistribution of some synaptic components can occur following their delivery to synapses by Kinesin motors. In Drosophila, dense core vesicles destined for presynaptic terminals are initially observed to oscillate between proximal and distal axonal regions, with movement mediated by both Kinesin and Dynein motors . While undergoing retrograde movement to the neuronal soma, a portion of these vesicles becomes recruited to presynaptic terminals along the axons. Remarkably, these retrograde moving vesicles are preferentially captured by distal as compared to proximal presynaptic sites. Similar observations have also been made for SV precursor transport vesicles in C. elegans (Maeder et al., 2014). Whether or not an undefined mechanism is responsible for mediating the gradient decline in synaptic capture remains to be determined.

RETROGRADE TRAFFICKING IS MEDIATED BY DYNEIN
In addition to delivering cargoes, transport of biomolecules away from presynaptic sites towards the cell body also plays important regulatory as well as homeostatic roles for presynaptic function (Figure 1). Retrograde movement of specific messengers in response to synaptic activity can trigger transcription of genes in the nucleus whose products contribute to long-term modifications of synaptic behavior or as a response to injury (Panayotis et al., 2015). Furthermore, sustained and optimum presynaptic function requires removal and degradation of worn out or damaged presynaptic proteins or organelles for turnover and degradation (Bezprozvanny and Hiesinger, 2013). In both instances, Dynein mediates the retrograde movement of these cargoes (Hirokawa et al., 2010;Roberts et al., 2013).

SYNAPSE-NUCLEAR SIGNALING
Communication between synaptic termini and cell body is vital for modulation of synaptogenesis, long-term potentiation, axonal growth and responses to mechanical damage (Panayotis et al., 2015). Synapse-to-soma signaling can be divided into fast response component, that involves propagation of Ca 2+ ion waves through neurites as a signal, and slow response component, which utilizes retrograde transport of signaling molecules to the cell body. Ca 2+ signaling from the postsynapse to soma is well studied and has been demonstrated to be involved in modulation of gene expression (Bading et al., 1993;Greer and Greenberg, 2008;Hagenston and Bading, 2011). This method of signaling may also play a significant role following injury (Ziv and Spira, 1995;Wolf et al., 2001) and during axon growth (Yamane et al., 2012). Calcium waves can be propagated to soma, for example, through axon-specific ERlike structures (Merianda et al., 2009), or locally induce other signaling pathways (Ghosh-Roy et al., 2010) to evoke responses in the cell body.
In comparison to the fast response, which is most efficient for synapses located closer to the cell body, the slow signaling component involves retrograde transport of activated signaling molecules (for example kinases or receptors) to the nucleus using Dynein motors (Schmieg et al., 2014;Panayotis et al., 2015). Among these, the best studied are the neurotrophins, a group of extracellular signaling proteins including NGF, BDNF and NT-3/4 (Huang and Reichardt, 2001). At the presynapse, binding of neurotrophins to their receptors (either tropomyosin receptor kinase (Trk) family or p75 NTR receptors) induces their autophosphorylation and activates downstream signaling pathways through MAPK, PLCγ and PI3K (Delcroix et al., 2003;Howe and Mobley, 2005;Schmieg et al., 2014). Interestingly, activation of Trk receptors depends on the type of neurotrophin and synaptic terminus it is applied to Kuruvilla et al. (2004). Stimulation of axonal terminus by NT-3 in sympathetic neurons promotes axonal growth by local activation of TrkA independent of TrkA internalization. In contrast, NGFmediated activation of TrkA induces its endocytosis and cotransport with downstream signaling molecules pERK1/2 and pAkt to the soma (Kuruvilla et al., 2004). Multiple signaling molecules can be transported to the soma on common vesicles. This phenomenon led to the hypothesis of the signaling endosome (Schmieg et al., 2014), a dynamic organelle that co-transports numerous signaling molecules from the axon periphery to the cell body. Transporting multiple signaling molecules on a single vesicle fulfills two functions. Firstly, it potentially allows interactions to occur between components of the different signaling pathways carried by the vesicle that can result in either enhancement or inhibition of the eventual response. Second, vesicular transport of kinases or receptors preserves them in the active, often phosphorylated, state along the way. A large amount of data demonstrates that interruption of Dynein-mediated retrograde transport of neurotrophins leads to ablation of their signaling (Purves, 1976;Salehi et al., 2006;Sharma et al., 2010). For instance, disruption of BDNF-induced TrkB signaling by perturbing snapin-Dynein interaction, which prevents their retrograde transport to the nucleus, was recently shown to reduce dendritic outgrowth . Signaling endosomes are assumed to carry a range of signaling molecules and markers that define its fate during transport to the soma. In agreement with this, early endosomal markers Rab5 and EEA1 colocalize with TrkA at distal tips of neurites (Howe et al., 2001;Delcroix et al., 2003). Upon maturation, signaling endosomes exchange Rab5 for Rab7, which is a late endosome marker. This event is crucial for targeting TrkA to the cell body as overexpression of dominant negative Rab7 in PC12 cells disrupts transport of neurotrophin receptors complexes (Deinhardt et al., 2006).
In the cell body, signals derived from presynaptic sites are converted into specific responses, such as changes in gene expression. For instance, the downstream target of NGF, ERK5 can activate transcription factor MEF2D, which in turn induces expression of anti-apoptotic protein bcl-w, promoting survival of the developing sensory neurons (Pazyra-Murphy et al., 2009). Transcription factors can also be transported to nucleus directly from distal axonal regions. In support of this, several studies indicate that local translation of importins, proteins indispensable for nuclear transport of cytoplasmic proteins, can take place in injured axons (Hanz et al., 2003;Yudin et al., 2008;Perry et al., 2012). Knockout of importin β1 in axons led to a substantial change in the transcriptome of neurons inflicted with axonal injury (Perry et al., 2012). Studies of retrograde signaling from postsynapse revealed that importins bind to transcription factors and mediate their transport to the soma using Dynein motors (Meffert et al., 2003).

SYNAPSE HOMEOSTASIS AND TURNOVER OF PRESYNAPTIC PROTEINS
Presynaptic proteins are subjected to turnover during homeostatic and activity-dependent plasticity (Lazarevic et al., 2011;Cohen et al., 2013). Apart from proteasome-dependent degradation, autophagic degradation of presynaptic components is emerging as an important strategy to deal with unwanted biomolecules (Yamamoto and Yue, 2014;Menzies et al., 2015). Presynaptic components targeted for degradation by autophagy include SVs and α-synuclein (Friedman et al., 2012;Binotti et al., 2015). However, details concerning how these components are directed to sites of degradation remain unclear. Efficient clearance of autophagosomes is challenging in neurons since many of these vesicles are formed at the distal axons located far from lysosomes that are largely present in the soma (Yamamoto and Yue, 2014;Menzies et al., 2015). Retrograde transport of autophagosomes is an essential step towards the fusion of both types of vesicles thereby enabling completion of the degradation process. An important question is to understand how neurons employ particular sorting mechanisms to achieve this.
A recent study reported that retrograde movement of autophagosomes at distal sites in neuronal axons involves binding of JIP1, a Kinesin-1 activator, to LC3 present on autophagosomes (Verhey and Hammond, 2009;Fu et al., 2014). This interaction prevents JIP1 from activating Kinesin-1. Moreover, JIP1 recruits the Dynein motor complex by binding p150(Glued), a component of the dynactin complex which is an integral Dynein subunit shown to be important for initiating retrograde trafficking from synaptic sites (Allan, 2011;Lloyd et al., 2012;Fu and Holzbaur, 2013). Intriguingly, abrogation of JIP1 binding to LC3 by deleting the LC3-interaction region on JIP1 inhibits fusion of the autophagosomes with lysosomes-an essential step towards the eventual degradation of autophagosomal content. Because this deletion also impairs retrograde trafficking of autophagosomes, it remains to be clarified if the inhibition is related to the transport defect or if indeed that JIP1 does participate in a yet understood role in the autophagy pathway.
An alternative route for the recruitment of Dynein to autophagosomes appears to be facilitated by snapin. Previous data indicated that a complex of Dynein and snapin resides in late endosomes and is responsible for driving their retrograde transport (Cai et al., 2010). A proportion of autophagosomes was observed to fuse with late endosomes to form amphisomes, a known intermediate step in the autophagic pathway (Lamb et al., 2013;Yamamoto and Yue, 2014;Cheng et al., 2015). In doing so, these autophagosomes acquire the ability to move in a retrograde fashion via the Dynein-snapin complex present on late endosomes. Thus, the study provides additional insights into trafficking routes taken by autophagsomes en route for degradation.

PART OF A TEAM: COOPERATION BETWEEN KINESIN AND DYNEIN MOTORS IN MOVING PRESYNAPTIC PROTEINS
Although protein transport has been conventionally viewed to be unidirectional, Kinesin and Dynein motors are known to bind simultaneously to most synaptic cargoes (Encalada and Goldstein, 2014). Simultaneous binding of cargoes to motors of opposing directionality confers several advantages. First of all, retrograde movement of degenerated synaptic components or endocytosed material would be only possible if Dynein is available at the distal parts of the axon. This would necessitate Dynein travelling with cargoes in anterograde direction to enable distal localization (Yamada et al., 2010). Secondly, microtubule tracks along axons are not contiguous. Microtubule breaks or clogging of the axon can happen along the path of cargo transport and switching between motors of opposite polarity may allow the cargo to bypass the hindrance. Finally, many studies indicate that opposite-polarity motors activate one another and such an interaction is essential for efficient cargo transport in either direction (Barkus et al., 2008;Ally et al., 2009;Uchida et al., 2009). Nevertheless, it remains unclear what determines which motor becomes dominant in a given environment. Specific regulatory mechanisms are likely to determine the directionality of cargo movement. For instance, adaptor protein Huntingtin (Htt) together with huntingtin-associated protein-1 (HAP1) links BDNFcontaining vesicles to Dynein motor. Upon IGF signaling and consequent activation of the Akt pathway, Htt becomes phosphorylated at serine 421 which in turn recruits KHC to the cargo and favors its anterograde transport (Colin et al., 2008).

PRESYNAPTIC PLASTICITY AND INTRACELLULAR TRAFFICKING
Synapses undergo constant remodeling (plasticity changes) in accordance to their history of activity which is a basis for learning and memory (Holtmaat and Svoboda, 2009). Such remodeling can occur as subtle changes in synaptic composition or overt modifications to synaptic morphology (Lazarevic et al., 2011;Weyhersmüller et al., 2011). Recent studies indicate that in addition to constitutive transport to synapses, the trafficking machinery can itself also respond to changes in synaptic activity and contribute to synaptic plasticity and remodeling. In particular, activity-enhanced trafficking of ionotropic glutamate receptors to post-synaptic density contributes to synaptic plasticity (Yin et al., 2011;Hoerndli et al., 2013Hoerndli et al., , 2015. Similarly, activity-mediated bidirectional modulation of synaptic behavior and trafficking has also been observed at the presynapse. Repetitive stimulation of cultured neurons increases formation of new presynaptic terminals that is dependent on trafficking of presynaptic components by Kinesin-1 (Cai et al., 2007). Likewise, higher levels of the Kinesin-3 motor KIF1A corresponding with increased trafficking of presynaptic cargoes by Kinesin-3 motors has also been observed in mice placed in an enriched environment in a learning paradigm (Kondo et al., 2012). Direct morphological changes have also been described. Prolonged activation of photoreceptor synapses causes loss of T-bars at presynaptic sites-a phenomenon associated with the removal of AZ proteins liprin-α, Bruchpilot by the Kinesin-3 motor Imac (Sugie et al., 2015).

FUTURE PERSPECTIVES
In the fast changing environment of synaptic communication, neurons must cope with the varying demands of different synapses. Emerging evidence indicates that the intracellular trafficking machinery does not only respond passively to replenish or remove biomolecules from synapses but can also pro-actively respond to the needs of individual synapses. Key questions that remain to be clarified include: (1) What mechanisms govern the loading of cargoes to their motors and, upon reaching their destinations, unloading the cargoes; (2) How heterogeneous is the composition of different cargo subsets and is the composition of these subsets regulated over the course of the lifespan of the neuron; and (3) What are the mechanisms regulating specificity of targeting to different synapses? Noteworthy, impairment of intracellular trafficking has been identified in a range of neurodegenerative disorders such as Alzheimer's and ALS where synapse loss presents itself as an early feature during disease onset (Millecamps and Julien, 2013). A better understanding of mechanisms governing the specificity of targeting to different synapses and the precise contribution of synaptic protein trafficking to synapse health will be important questions to address. Recent advances in optogenetics and super-resolution microscopy techniques, coupled with characterization of the molecular identities of cargoes carried by different motors will be important to bring us one step closer towards unraveling these intriguing questions (Ballister et al., 2015;Hell et al., 2015;van Bergeijk et al., 2015).

AUTHOR CONTRIBUTIONS
The manuscript was conceived by JJEC and written by JJEC, OY and TKD. OY and TKD contributed equally to the work.


extfieldsofstudy: Chemistry Medicine

text:
Enhancing anaerobic digestion of poultry blood using activated carbon

Graphical abstract

A B S T R A C T
The potential of using anaerobic digestion for the treatment of poultry blood has been evaluated in batch assays at the laboratory scale and in a mesophilic semi-continuous reactor. The biodegradability test performed on residual poultry blood was carried out in spite of high inhibitory levels of acid intermediaries. The use of activated carbon as a way to prevent inhibitory conditions demonstrated the feasibility of attaining anaerobic digestion under extreme ammonium and acid conditions. Batch assays with higher carbon content presented higher methane

Introduction
Anaerobic digestion is a well-known process for the production of biogas, a mixture of methane and carbon dioxide, which is currently used and exploited at a local level in an efficient way. However, when considering large scale usage of biogas, the capital investment and upgrade costs associated with methane valorisation make it unfeasible in some cases [1]. Furthermore, the accumulation of toxic compounds and anaerobic intermediaries may cause a severe decrease in biogas yields, therefore compromising plant feasibility.
The use of adsorbents in anaerobic digestion has been widely studied to avoid inhibitory stages during the processes associated with high ammonia levels or to prevent odour emissions from the treatment of livestock wastes [2,3]. Many studies have focused on the addition of natural zeolites and clays for treating nitrogen-rich wastes [4] or their post-treatment to remove phenolic compounds [5]. Recently, the combination of anaerobic digestion and adsorption processes has led to using industrial clay residues [6], zeolites synthesised from coal fly ash [7], and low-cost adsorbents such as biochar [8] in an attempt to reduce the cost of the process.
Traditionally, slaughterhouse wastes have been considered a suitable co-substrate in digestion systems, with several authors reporting a marked increase in biogas production and stable performance of digesters as long as certain operational constraints are taken into account [9][10][11]. High amounts of solid organic by-products are generated from poultry slaughterhouses. These wastes usually comprise poultry manure, feathers, blood, and intestinal wastes [12]. Slaughterhouse wastes present a high potential for energy valorisation; this is particularly true for gastrointestinal residues characterised by high-fat content [13]. However, the main problems that arise when digesting this type of waste are associated with foaming and flotation of sludge, along with ammonium inhibition due to the high protein content [14,15].
The number of studies dealing with the digestion of residual blood has increased in the recent years [10,16,17]. However, residual blood is a complex substrate with high nitrogen content; therefore, its use as co-substrate has been widely studied, but attempting its individual digestion can lead to various difficulties due to the accumulation of ammonium in the reactor. Nitrogen is an essential nutrient in biological processes, but excess nitrogen can cause ammonia inhibition, as frequently reported, with inhibitory levels noted to be around 4-6 g N/ L expressed as total ammonia nitrogen. It should also be taken into account, however, that particular characteristics of the process and substrate, such as pH condition, temperature, and type of seed sludge, among others, have a major effect on the degree of inhibition [18,19].
The digestion of nitrogen-rich wastes has been attempted with the aid of a carbon-rich substrate in order to increase the carbon to nitrogen (C:N) ratio. The digestion of abattoir wastes with mixtures of food wastes and cheese whey was evaluated by Allen et al. [20], who reported an increase in digestion performance based on the higher capacity of the reactor to treat the organic matter, which was associated with an increase in carbon content. The treatment of slaughterhouse wastes containing residual blood and grease was also investigated by Ortner et al. [21]. These authors reported volatile fatty acid (VFA) build-up (>8.0 g/L) and high free ammonia levels. The decrease in the organic loading rate that was achieved in an attempt to lower the ammonium content in the reactor to values below 6 g/L resulted in a successful alternative for the recovery of the digestion process and gas yields. Similar results were also reported by Alvarez and Lide´n [22], who studied the co-digestion of slaughterhouse wastes containing residual blood from cattle and swine with food wastes. These authors reported on a decrease in biogas yield due to the accumulation of ammonia in the reactor.
To the author's knowledge, this paper is the first work focused on the anaerobic digestion of poultry blood as the sole substrate. The aim of the present study was to evaluate the digestion of residual blood under semi-continuous conditions. The effect on gas production and performance of the digester was evaluated when using granular and powder activated carbon as way to prevent ammonium and VFA inhibitory conditions. The digestion process was assessed with the aid of thermal analysis and scanning electron microscopy (SEM) for evaluating changes in the carbon surface and organic material.

Inoculum and substrate sources
The inoculum was obtained from a laboratory digester treating slaughterhouse waste adapted to an environment rich in ammonia. The acclimation procedure was performed based on the one described by Fierro and co-workers [23]. The reactor was fed with slaughterhouse waste with a hydraulic retention time (HRT) of 50 d, and then the feeding volume was increased to reach a 36 d HRT. The inoculum thus obtained was stored under ambient conditions to allow for the further release of biogas. The residual poultry blood was obtained from a local poultry slaughterhouse in Leo´n (Spain) and pasteurised (60 min, 70°C) prior to its use in digestion experiments. Characteristics of the inoculum and substrate are presented in Table 1.
Granular and powdered activated carbon (from Sigma-Aldrich) was used in batch and continuous digestion experiments. The granular activated carbon had a particle size of 12-20 mesh and a mean surface area of 600 m 2 /g. The powdered activated carbon had a particle size of 200-325 mesh, with an approximate surface area of 750 m 2 /g.

Batch digestion experiments
The batch digestion of blood was carried out in batch assays. Sixteen replicates were run over the course of 20 days. Two replicates were removed from the bath for liquid-phase analysis on days 1, 3, 7, 9, 11, 15, and 20. Digestion experiments for evaluating the effect of adding activated carbon were also performed under batch conditions. These experiments were carried out using different proportions of poultry blood and activated carbon. The mixtures were made using ratios of blood (total solids (TS)) to mass of activated carbon added, of 4.5, 3.0, and 1.5 [4]. This ratio expresses the amount of organic blood material added to the reactor (measured in terms of TS) and the mass of activated carbon added in terms of a proportion; in other words, for every 4.5 g of TS of residual blood, 1.0 g of activated carbon is added in the first case, 1.5 g in the second case, and 3.0 g, in the third case. Experiments were performed in 100 mL Erlenmeyer flasks incubated at 37 ± 1°C in a water bath under stirring conditions (200 rpm). The inoculum to substrate (I:S) ratio was kept constant for all batch experiments with a value of 2.0 to avoid adding an alkali solution for pH correction and prevent VFA overloading. Reactors were denoted as B_4.5, B_3.0, and B_1.5 based on the ratio of activated carbon added in the mixture. For each assay, 20 replicates were initially set and two replicates were withdrawn from the water bath on days 1, 3, 5, 8, 11, 15, 18, 22, 25, and 30. The volume of biogas produced was measured using liquid displacement bottles. Values obtained were corrected to standard temperature and pressure.
An additional batch experiment was performed using powdered activated carbon as an adsorbent at a ratio of 1.5 in order to evaluate any improvement in biogas production. This experiment was denoted Bp_1.5. In addition, three control assays were run in parallel to measure the background methane production from the inoculum. The residual biogas was subtracted from the total production in each case.
Cumulative biogas curves were fitted to a modified Gompertz equation (1). This model has been successfully tested for adjusting biogas data obtained from batch digestion assays using residual blood and co-substrates [10]: where P (t) is the cumulative biogas production (l), P max is the maximum biogas value obtained (mL), R max is the maximum biogas production rate (mL/d), y k is the lag-phase time (d), and e is 2.71. The software Origin 6.0 was used for fitting data to the equation and obtaining the model parameters P max , R max , and k.

Adsorption assay
Adsorption experiments were carried out using 100 mL Erlenmeyer flasks with magnetic stirrers at 37°C. These flasks contained 100 mL of a solution with a 5 g/L concentration of a single component. Adsorption tests were performed on acetic, propionic, butyric, and ammonia chloride solutions (reagents purchased from Merck), adding to each Erlenmeyer flask 0.5 g of granular activated carbon. The amount of activated carbon added was the same as that added to test B_1.5. The concentration of the different species was regularly measured during a 24 h period.

Semi-continuous anaerobic digestion
Semi-continuous digestion was carried out in reactors with a working volume of 900 mL. Reactors worked under static conditions using granular activated carbon in one case and powdered in the other. Reactors were denoted as RG when using granular carbon and RP when powdered activated carbon was added. Manual agitation was performed once a day before and after the feeding procedure. Reactors were kept at 37 ± 1°C and worked at an HRT of 36 d with an organic loading rate (OLR) of 1.15 g VS/L d. Reactors were manually fed every day using a ratio of poultry blood (TS content) and mass of activated carbon of 3.0. Reactors were evaluated for a 75 d period. Daily gas production was measured using a reversible device with liquid displacement and a wet-tip counter. Gas composition was analysed by gas chromatography. TS, VS, pH, alkalinity, ammonia, chemical oxygen demand (COD), and VFAs were routinely analysed.

Analytical techniques
Kjeldahl nitrogen (KN), TS, volatile solids (VS), COD, alkalinity, ammonium, and pH were measured in accordance with standard methods [24]. Free ammonia (FA) was calculated based on the equilibrium equation (2) based on Bonmatı´and Flotats [25]. Total ammonia (TAN) values were measured using the ion selective electrode.
Organic matter was measured using the Walkley-Black method [26], and the total organic carbon (TOC) content was calculated from the organic matter value, using a correlation factor of 1.72. Biogas composition was analysed using a gas chromatograph (Varian CP 3800 GC) equipped with a thermal conductivity detector. A column 4 m long, packed with HayeSepQ80/100, followed by a molecular sieve column 1 m long, was used to separate CH 4 , CO 2 , N 2 , H 2 , and O 2 . The carrier gas was helium, and the columns were operated at 331 kPa at a temperature of 50°C. VFAs were analysed using a gas chromatograph (Varian CP 3800 GC) equipped with a Nukol capillary column (30 m Â 0.25 mm Â 0.25 lm) from Supelco (Bellefonte, PA, USA) and a flame ionisation detector. The carrier gas was helium. The temperature of the injector was 250°C, and the temperature of the oven was initially set at 150°C for 3 min and thereafter increased to 180°C . Samples were previously centrifuged (20 min, 3500g) and the supernatant was cleaned using the procedure described by Cuetos et al. [27].
Inoculum, activated carbon, and digestate samples from the semi-continuous reactors were collected for thermal analysis. Thermogravimetric analysis was performed using a Setaram TGA92 analyser. Five milligrams of sample was used in each experiment. Analyses were carried out under an air flow of 100 mL/min at a heating rate of 15°C/min from room temperature ($22°C) to 850°C. The mass loss (TG) and derivative curves (DTG) were represented as a function of temperature.
The surface of activated carbon and solids obtained after dismantling the reactors was analysed by SEM. Digestates samples were obtained after sedimentation (3 d) of the reactor liquor. Solids were dried at 105°C and ground using a ball mill Retsch MM200. Samples were sputter-coated with gold in high vacuum (0.05-0.07 mbar) conditions with a coater Blazers SCD 004. The samples were examined using a JOEL JSM 6840 LV scanning electron microscope.

Batch digestion experiments
Residual blood used in batch digestion experiments as a substrate presented a low C:N ratio (as shown in Table 1). The production of biogas obtained from the digestion assay of poultry blood was 46.5 L/kg VS. This system was characterised by high pH values throughout the digestion assay (around 8.8). Furthermore, the concentration of ammonium was about 4500 mg/L, resulting in the presence of high levels of free ammonia (with an average value of 1813 mg/L). These conditions translated into a severe inhibitory stage, thus explaining the low biogas yield. VFA build-up was attained on the fourth day of the experiment, with the acetic content reaching a value of 2184 mg/L, while acid species corresponding to C3-C5 forms reached an average value of 350 mg/L. The high and rapidly attained ammonia and acid concentrations probably prevented further degradation of the organic material.
On the other hand, the addition of granular activated carbon resulted in successful digestion of the residual blood. Experiments presented similar values of final cumulative biogas production (see Fig. 1(a)), with the difference in the pro-cess being associated with initial stages of the batch experiment. A clear improvement is easily observed when comparing gas data with those of the control experiment (Blood).
Experiments with lower contents of activated carbon demonstrated a lower rate at the beginning of the cumulative curve (B_4.5 and B_3.0). The lower gas production rate of these experiments is related to the VFA build-up, which adds to the negative effect caused by the high ammonia content in these reactors. During the first 10 days, VFAs presented high values, which in turn explain the low degradation rate of the substrate ( Fig. 1(b) and (c)). However, the anaerobic microflora could circumvent this stage, assimilating the whole amount of VFA initially produced. The addition of a larger amount of activated carbon resulted in a higher biogas production rate during the first five days of the batch experiment, which was in line with the lower amount of VFAs measured. The biochemical methane production value obtained from experiment B_1.5 was 317.4 ± 31.8 mL CH 4 /g VS. A similar value was obtained when using powdered activated carbon. From the curves presented in Fig. 1(a), it is clear that the form of carbon used (either granular or powder) does not greatly affect the gas production rate.
The presence of a larger amount of granular activated carbon not only affected the initial amount of VFA accumulated in the system but also affected the concentration of ammonium in the reactor. Although the initial value was similar in the three experiments, the average value obtained during the experimental period was much lower for the B_1.5 system (Fig. 1(e)). This behaviour aided in reducing the inhibitory stage associated with protein conversion and therefore results in a better digestion performance. The B_1.5 system had a higher methane production rate during the first 10 days (34 mL biogas/d, calculated as the slope of the curve during the first 10 days) because of lower levels of accumulated inhibitory substances during this batch test. These inhibitory conditions were prevented by the increase in the amount of activated carbon added.
Results of the application of the modified Gompertz model to gas production curves obtained from the different experimental sets are presented in Table 2. All data sets presented a good fit to the model, as observed from the high R 2 values. There is a reduction in k values due to the increase in the amount of activated carbon added to the reactor. This factor also affected the gas production rate; those experiments with higher amounts of activated carbon also presented higher R max values.
The pH of the three systems was about 8.0, with this value being lower than that obtained for the batch experiment digesting blood and having a strong effect on the ionic species present in the solution (e.g., ammonia form). The pH in the digestion system is affected by the equilibrium species of carbonates, ammonium, and VFA, with ammonia levels substantially influencing the buffer capacity of the solution [28]. In the present study, the adsorption capacity of activated carbon also plays a crucial role in the final pH value attained, thereby relieving the inhibitory conditions responsible for preventing the degradation of the substrate.   experiments. Although the concentration curves represented in Fig. 2 could not be fitted to any particular adsorption model, the results indicate a great capacity for retaining ammonium, which is one of the major inhibitors when digesting residual blood. Ammonium levels could be reduced to around 3000 mg/L, and this value obtained after 24 h of the experiments was similar to that obtained at the end of the batch digestion process when using the highest addition of activated carbon (B_1.5).

Results from adsorption assays
In the case of VFA, the effect of acetic and propionic acid was less pronounced, while butyric acid was highly retained by the adsorbent during the initial hours, finally reducing its concentration in solution after 24 h to about 3600 mg/L. However, in all cases, the concentration of any of VFA presents high variability during the time of the experiment. Just as in the previous case, VFA adsorption curves could not be fitted to any adsorption model, but the curves obtained were indicative of a mild retention of acids onto the activated carbon surface, which may have partially alleviated that at the inhibitory stages microorganisms are subject to when dealing with the digestion of residual blood.
The adsorption of water/organic mixtures is a complex phenomenon because of the nonuniformity of the adsorbent surfaces and specific interactions of polar molecules with oxygen-containing surface groups [29]. The adsorption equilibrium for organics on activated carbon is mainly dependent on the chemistry of the carbon surface. Heterogeneous oxygen groups play an important role in the adsorption process, as well as hydrogen bonding and the water adsorption effect [30,31]. Carboxylic functional groups of the organic acids are present in solution in their negative form (COO À ), experiencing repulsive electrostatic interactions with the negative carbon surface. In contrast to these repulsive forces are the formation of H-bonds by carboxylic groups in organic acids. Gun'Ko et al. [29] proposed the formation of a chain or a cluster of organic acids associated with the H-bonding mechanism, which could lead to pore blockage, similar to water adsorption. The erratic behaviour observed in Fig. 2b is the result of the net effect of these two opposing mechanisms.
The addition of adsorbents like biochar has been demonstrated to enhance the digestion process by reducing the lag phase and improving the resistance of anaerobic microflora to highly acidic conditions [32]. In the present experiment, a similar effect was observed with the addition of activated car-bon, yielding better performance even though a high VFA content was observed. Adding activated carbon to the digestion batch tests resulted in the complete digestion of the substrate, even with the lower dose of activated carbon. However, the economics of this approach are unfavourable because the addition of this adsorbent would increase operating costs of the plant by 50%, when considering calculations for an industrial digester (3500 m 3 ) based on the economic assumptions proposed by Fierro et al. [33] at a price of 2500 €/t of activated carbon.

Semi-continuous digestion tests
The reactors were operated under semi-continuous conditions, and the results are shown in Fig. 3. Systems demonstrated low biogas production at the beginning of the study, which increased progressively during the first eight days of operation. Once a period equivalent to an HRT had elapsed, the biogas profile became stable, with an average specific methane production (SMP) value of 216 ± 12 mL CH 4 /g VS. The methane content in biogas ranged from 52 to 56% for both reactors. Although the SMP was far below the value obtained from batch tests, this result is in any case remarkable taking into account the adverse conditions in which the digestion was taking place.
Reactors presented an initial accumulation of acetic acid, which caused a serious build-up of VFAs during most of the operating period (Fig. 3). Values between 4000 and 5000 mg/ L were reached after 20 days of operation. However, a decreasing trend in the content of acetic acid was observed for both reactors when the experiment was near the end, with this trend starting at an earlier stage for the RG system (on day 45). In spite of this phenomenon, the reduction observed in acetic acid concentrations during the last days of the experiment was not associated with increased biogas production for any of the reactors.
The affinity of activated carbon for acetic acid was shown to be rather irregular, with a low adsorption capacity being obtained in particular hours of the experiment and higher adsorption levels being obtained at the end of the 24 h adsorption test. Therefore, it may be inferred that the adsorption of VFA may not play a main role in the improvement of digestion performance; other phenomena, such as favouring microbial metabolism, may be the reason behind the improved results.  The addition of activated carbon to anaerobic digesters has been evaluated by Xu et al. [34]. These authors reported the use of this adsorbent with different particle sizes, reporting an enhancement of methane production associated with the benefits caused by syntrophic metabolism of alcohol and VFAs. Therefore, the adsorption and faster degradation of VFAs observed in the present experiments lead us to expect positive effects on methane production.
The decreasing trend of acetic acid observed for both reactors may then be related to the ability of microorganisms to find proper protective sites. In the case of the RP system, this takes place around day 60, which may be associated with the lower amount of protective sites and this carbon offers to the anaerobic microflora because it is unable to efficiently remove this acid from the liquid phase. This increased amount of acetic acid found in the RP systems explains the observed instabilities in daily gas production at the end of the first HRT period.
In general, the free acetic acid levels attained in any of the reactors were much higher than the levels reported as inhibitory by Fukuzaki and co-workers [35] when evaluating the methanogenic fermentation of acetate. The RG system presented a maximum free acetic acid value of 95 lM on day 24, while the maximum value in the RP system was 69 lM on day 21.
Total VFA values were higher than 6 g/L for most of the experimental period for reactor RG and were close to this value for reactor RP. An inhibitory threshold of 6 g/L was reported by Siegert and Banks [36]; therefore, the digestion of blood was attained in extreme conditions for the anaerobic microflora. The ratio of VFA to total alkalinity (VFA/TA) is also shown in Fig. 3b and c. This ratio is considered to give a good indication of the stability of the digestion process when its value is below 0.4 units [27]. In the present study, the reactor supplemented with granular activated carbon presented values above this limit throughout the operation period, giving a clear indication of the severity of the inhibitory conditions experienced. On the other hand, the addition of powdered activated carbon (Fig. 3c) helped in reducing this ratio and reaching stability levels. This better performance may be influenced by the size of carbon particles used which allowed for an enhancement in the assimilation of acids with more than two carbon atoms.
In the case of C3-C5 acid forms, these acids demonstrated an increasing trend that was more pronounced in the RG system. The propionic acid concentration continuously increased during the time of the experiment for this reactor, reaching final values around 3000 mg/L (Fig 3b). Inhibitory effects of propionic acids have been reported, with a value of 900 mg/L being indicated as the threshold [37]. The presence of iso-forms has been associated with instabilities based on the different degradation rates of VFA and the inhibitory effects caused by high acetic and propionic levels on isoform degradation [38]. However, different reports have also been published indicating that high levels of propionic acid do not necessarily affect methane production in an adverse way [39]. In the present experiments, high levels of propionic acid and iso-forms were reached, and still a stable biogas production was obtained. The addition of powdered activated carbon into the reactor affected the behaviour of iso-forms. For this latter system, the difference between the initial and final values for the C3-C5 acid forms (in any form) was less, probably an indicator of better adsorption performance (Fig 3c).
The presence of the solid phase in the reactor may offer protection to microorganisms against these harsh environmental conditions, allowing for the stable behaviour of biogas evolution reported in Fig. 3. However, the different behaviours observed for these C3-C5 forms when the addition of the activated carbon is carried out in the powder form are not reflected in an improved performance. Fig. 3c shows the evolution of VFA in the RP system. Propionic acid has a mean value of 930 ± 270 mg/L, which is much lower than that obtained for reactor RG, while isovaleric shows a clear increasing trend with the final value being around 700 mg/L. The higher specific surface area of the powdered carbon probably offers a greater adsorption capacity for these acids; the SMP value obtained from this system, however, was not higher than that of the RG system, indicating that the lower VFA content obtained in this reactor was not enough to further improve the digestion process.
The values of pH measured during the working period for the two reactors were in the range of 7.0-7.5 with alkalinity values greater than 15 g/L. Values of VFA measured were those typical for start-up stages, acid phases, and systems subjected to overloading [40]. The ratio of VFA-to-alkalinity was close to 1.0 at the end of the study, which leads to considering the digestion as a failed anaerobic process. However, thanks to the buffering capacity provided by the high protein levels in the residual blood, stable pH values were obtained from the entire process.
The high ammonia levels reached during the digestion process helped maintain high pH values despite the high VFA build-up (See Fig 3d). However, these values can be considered inhibitory based on the results reported by Moestedt and coworkers [41] who set the threshold values as 1.0 g/L of NH 3 for evidencing negative effects of methane production. The different physical properties of the activated carbon used had no significant effect on ammonium evolution. Ammonium content presented a similar profile in both studied reactors, reaching levels around 8000 mg/L at the end of the second retention time.
The aggregation of cells is a key factor for efficient methanisation as a direct result of an efficient electron transfer between obligate H 2 -producing acetogens and methanogens. Direct interspecies electron transfer (DIET) is a syntrophic metabolism in which free electrons flow from one cell to another without being shuttled by reduced molecules such as molecular hydrogen or formate [42]. DIET has been suggested as the reason for obtaining better degradation rates of simple substrates and higher biogas yields in anaerobic systems when carbonbased conductive materials are added [43], as was demonstrated by Rotaru et al. [44] and Zhao et al. [45] when studying the use of activated carbon. In the present experiments, enhancement via DIET may be similarly relevant, as the improvement of digestion may not be completely explained by the adsorption phenomenon.

Results from thermal analysis and SEM
Because of the presence of carbon particles inside the reactor, the measurement of TS and VS was not useful. The digestate samples taken at the end of the process were analysed by means of thermal analysis and SEM. Fig. 4 shows the thermal profile of the inoculum sample and changes experienced by the original carbon sample and solids collected from the reactors at the end of the digestion. The mass losses experienced around 300 and 450°C are associated with the presence of microbial biomass and the residual organic material obtained from the digestion process; in particular, these mass losses are associated with the organic carbon content of the sample [46]. The profiles in Fig. 4b and c show the TG curves for the original carbons and the mixture of digestate and activated carbon obtained after the biological transformation. There is a relevant increase in the ash content after the digestion process due to the inorganic material accumulated in the reactor. This increase is typically observed in waste digestion processes as the mineralisation of the organic matter takes place [47]. Fig. 4 also shows the differences in DTG profiles for the same samples. For both reactors, the residual organic material mixed with the activated carbon causes the early loss of mass at around 200°C. It is also responsible for the interaction between digestate stable compounds and the activated carbon particles, which is observed as early oxidation of these latter particles in the DTG curves. Digestates are usually characterised as experiencing an early mass loss associated with labile compounds and a high-temperature oxidation, which is normally associated with the thermal degradation of either recalcitrant compounds or organic molecules with complex structure. These compounds may have already been present in the original material or they may have been generated during the microbial decomposition [48].
The SEM images also show the changes experienced by the carbon surface due to the presence of microorganism inside the reactor. The image shows the carbon surface before the digestion process and the surface of solid material obtained at the end of the semi-continuous operation. An increase in roughness is noticeable, being more evident in the case of the powder activated carbon (Fig. 4b and c).
The study by Xu et al. [34] on the use of activated carbon in anaerobic digesters reported the development of a layered structure of the anaerobic sludge granule, where the outer Fig. 4 Results from thermal analysis of inoculum sample (a), and thermal analysis -SEM images for granular activated carbon/RG digestate (b) and powdered activated carbon/RP digestate (c).
Enhancing anaerobic digestion of poultry blood layer was dominated by Bacteria and the inner one by Archaea. These authors also attributed the improvement in digester performance to the increased microbial population of methanogenic bacteria and syntrophic metabolism bacteria. Methanosarcina and Methanoculleus were the predominant species, along with Bacteroidales, Desulfuromonas, and Thermotogaceae, which were also found to be more abundant in the reactor operating with powder activated carbon.
In a different study, Zhao et al. [45] reported a change in microbial populations when evaluating anaerobic reactors for propionate/butyrate degradation with the aid of activated carbon. Methanosaeta and Methanosarcina species constituted a dominant part (81.49%) of the communities in their initial seed sludge, which significantly decreased when propionate and/or butyrate was used as the sole carbon source. However, they described no effects on the syntrophic metabolism of the substrate. On the other hand, Dang et al. [43] reported the main role of Methanosarcina (which are capable of DIET) when conductive materials are incorporated in anaerobic digesters. These authors highlight the benefits of accepting electrons from conductive materials by Methanosarcina, because the conversion of acetate to methane yields little energy, and this type of organism typically grows slowly on acetate. Electrons obtained via DIET might enhance their metabolism and even increase their ability to produce methane by acetate decarboxylation.
In the present study, the use of activated carbon allowed for digestion of the substrate, which was in no other way possible. The growth of microorganisms on the carbon surface probably promoted DIET; this phenomenon, in addition to the protective effect associated with mass transfer limitation of inhibitory compounds and the adsorption capacity of the activated carbon, aided in the degradation of the organic material by anaerobic microflora.

Conclusions
The addition of activated carbon to the digestion of residual blood greatly improved the digestion process due to its adsorption capacity for ammonium, resulting in lower levels of ammonium during batch digestion experiments. The presence of the solid phase (addition of granular and powdered activated carbon) probably acted as a protective layer for microorganisms, resulting in successful digestion under semicontinuous conditions. Although inhibitory levels of VFA and NH 4 + were reached, biogas production was maintained with low variations, and this behaviour may be explained by the presence of protective sites offered by the activated carbon particles.
Although specific methane productions were similar for the two semi-continuous reactors tested, the use of granular activated carbon resulted in higher accumulation of propionic and iso-forms. However, the use of powdered activated carbon resulted in better assimilation of C3-C5 species, probably indicating enhancement of syntrophic metabolism.

Conflict of Interest
The authors have declared no conflict of interest.

Compliance with Ethics Requirements
This article does not contain any studies with human or animal subjects.