Ips typographus, was quantified when weakly defended hosts  were attacked. In many of the replicates, colonization began with the insects rapidly scattering over the available surface and then randomly filling the gaps until a regular distribution was established, which resulted in a constant decrease in nearest-neighbour distances to a minimum below which attacks were not initiated. The scattered distribution of the first attacks suggested that the trees were only weakly defended. A minimal theoretical distance of 2.5 cm to the earlier settlers (corresponding to a density of 3.13 attacks dm−2) was calculated, but the attack density always remained lower, between 0.4 and 1.2 holes dm−2, according to our observations.Bark beetles use aggregation pheromones to promote group foraging, thus increasing the chances of an individual to find a host and, when relevant, to overwhelm the defences of healthy trees. When a male beetle finds a suitable host, it releases pheromones that attract potential mates as well as other ‘spying’ males, which result in aggregations on the new host. To date, most studies have been concerned with the use of aggregation pheromones by bark beetles to overcome the defences of living, well-protected trees. How insects behave when facing undefended or poorly defended hosts remains largely unknown. The spatio-temporal pattern of resource colonization by the European eight-toothed spruce bark beetle,  Hedden & Gara  (Three of the 16 segments exhibited particularly low numbers (<30 holes) despite having colonization dynamics similar to those of the other segments, and significant bark decay was observed on two of them. Thus, these three segments were discarded from our analysis, which ultimately considered a total of 13 segments.2.2.This part of the study focused on three spruces in Smuid  that were windfelled in the winter of 2014–2015 and remained on the ground. Roots were partially in contact with the soil, and the trees were pruned up to, but excluded, the crown before the observations began. No pheromone dispenser was added, and colonization occurred spontaneously over the trees. New entrance holes were marked with numbered pins every 1–3 days, depending on the weather conditions, during the main flight period from 5 June 2015 to 8 July 2015.2.3.mageJ software (v. 1.49t)  (N) were used. The significance level for all statistical tests was α = 0.05.When the normality of the distributions was confirmed (Shapiro–Wilk test), the results were presented as the mean ± s.d. (2.4.1.Asegment (in dm2), as a proxy of its maximum carrying capacity, and the number of attack holes, H, at each time step. The associated attack density, hereafter referred to as segment density, was defined as Λsegment = H/Asegment (holes dm−2).Colonization dynamics were analysed by considering the entire surface available on each segment of area, 2.4.2.complete spatial randomness  whose area was defined as Ahull (dm2). The attack density on the convex hull, hereafter referred to as hull density (λhull), was computed at different times during colonization as λhull = H/Ahull (holes dm−2).The R  package rocesses ). Spatia2.4.3.G-function (nearest distance between two attack holes) and the L-function , which were calculated from the observed patterns to an envelope of Monte Carlo simulations of homogeneous Poisson processes  (N = 13). Fifty per cent of the final number of holes occurred within the first 4 days  (N = 13), and 90%  were observed at day 14.The temporal colonization dynamics of the segments was nonlinear: the colonization rate rose for a short period and then constantly decreased until the number of entrance holes reached a plateau (N = 13) a. The prHEND stands for the number of entrance holes counted at the end of the observations and β controls the steepness of the curve with high β values leading to shorter durations to reach HEND . There was no relationship between β and HEND . The colonization dynamics at the tree scale (i.e. with H(t) being the sum of the attacks on each tree) showed similar profile and fitting curves .These colonization dynamics can be fitted to the following function:HFIRST), followed by a second stage from day 12 to day 45 (end of observations) leading to a second plateau (HSECOND with HFIRST + HSECOND = HEND). However, most of the attacks occurred during the first stage . There was no relationship between the values of HFIRST and HSECOND .Two successive stages can be observed on almost every segment: a first stage from day 0 (initiation of observations) to day 11, during which the number of entrance holes reached a quasi-plateau , which indicates that the observed colonization dynamics are directly influenced by insect availability; the increase in the number of landing males from the first to the second measurement probably explains the short increase in the rate of attack observed at the beginning of colonization (figure 1a). In addition, the landing rate strongly correlates with the average temperature measured during the observations , which is known to influence the emergence and flight behaviour of the beetles. These results indicate that the two plateaus observed in the global shape of the colonization dynamics only result from a beetle shortage, but the stagnation of the number of entrance holes, despite the observation of a third landing stage on the 31st day (figure 1b), suggests that other regulating mechanisms may be at work, or could reflect at least partly the activity of re-emerging beetles caught in the landing traps while trying to leave the trees.The daily colonization intensity was well correlated to the number of males landing during all the observations (figure 1Asegment) showed a linear relationship with the area of the convex hull , while the final number of entrance holes was not related with the area of the segments . This means that the probability of colonization per unit area is not equal between segments. By contrast, the final numbers of holes and the areas of the convex hulls were related , which agreed with the complementary analysis  and indicated that for density values similar to those observed in our observations, the number of holes influences the area of the convex hull in a predictable fashion. The following analysis indicated that the different trees are characterized by different intrinsic receptivities that can explain these differences.The area of the segments (HFIRST) and the final number of holes (HEND) on each segment, we simulated the installation of 1614 entrance holes  within 13 compartments with areas similar to those of the segments, using an SSI with r = 0 . The comparison of the observed densities at the end of the colonization process with those obtained by 10 000 simulations indicated that colonization was not random among segments , which suggests that the variability in the plateau value HEND between observations was due to either a heterogeneous distribution of the flying insects around the segments and/or a difference in susceptibility between the segments.To investigate the high variability in both the first plateau (Φ(t) is the instantaneous flow of flying insects at time t; iA is the area of the segment i; iα is the susceptibility of this segment; and iH is the cumulative number of entrance holes on this segment at time t.Therefore, we describe the colonization of the segments as follows:iΛ of segment i is iΛ = iH/iA. The homogeneous flow of flying insects, Φ(t), around the segments is indicated by a linear relationship between the segment densities, iΛ and jΛ, with a slope defined as the ratio of the susceptibility of each segment, iα/jα.Coupling the equation (3.3), the relative colonization of each segment compared with the others is described as follows:Λ values . During the first colonization stage, a linear relationship describes the link between the Λ of each couple at any time well , indicating that the density of flying insects is similar around each segment and that there is no amplification or competition between them. When the second colonization stage occurs, the relationship remains linear, but the value of the slope is significantly different from that in the first stage in 70% of the pairs . There was no significant relationship between both susceptibility values (αFIRST and αSECOND) in any given segment. Moreover, the variation in the susceptibility of the segments (Δα = αSECOND − αFIRST) was not related to the density at the end of the first colonization stage (ΛFIRST), which indicates that the number of entrance holes that occurred during the first stage did not influence, in a predictable fashion, the number of additional holes occurring during the second stage. There was a strong relationship between the initial receptivities (αFIRST) of the basal and upper segments of any given tree , indicating that each tree was characterized by its own intrinsic susceptibility.Each pair of segments shows a characteristic relationship between their 3.2.Λsmooth = 0.37 holes dm−2; Λrough = 2.33 holes dm−2; figure 2a). Moreover, the hole density on rough bark was linearly related to that on smooth bark at the same time step , indicating that the respective rates of discovery and infestation of both textures remained constant throughout the observations. This higher density of entrance holes on rough bark was observed on each segment (figure 2b). We did not find any relationship between the area of each texture or the ratio of rough bark with either plateau value of hole number (HFIRST and HSECOND) (linear regression: p > 0.05).The global density of entrance holes  at the end of the observations was 6.3 times higher on rough bark  and dclf tests indicated that nine of the 13 segments showed a regular attack distribution at the end of the observations (dclf test: p ≤ 0.05), while the other four were characterized by a random pattern . It is worth noting that the distribution of the entrance holes on three of these four segments, all of which were characterized by low density values, was close to a regular pattern . This suggests that, had colonization continued, the pattern on these four segments would have been characterized as regular.The pointwise envelopes b–d and dd) because the observed curve is under the CSR envelopes for short distances. This result was confirmed by the dclf test with p = 0.001. In this test, the integral deviation measure obtained from the data was rmax = 20 cm and G1(r), … ,NG(r), as defined in equation (2.1), and evaluated based on the N = 999 independent Poisson process simulations.Moreover, a statistical analysis considering all replicates together indicated that the data were more regular than under a CSR hypothesis d because3.3.2.detection = 0.69 attacks dm−2 , N = 9; figure 4b; Kruskal–Wallis p = 0.036; post hoc test: the only significant difference was between final densities of random and regular patterns; electronic supplementary material, table S3), with newcomers filling the empty spaces (this will be confirmed later). The density allowing the detection of a regular pattern was almost twice as high as the density recorded at the time of pheromone lure removal .While entrance hole patterns appeared to be randomly distributed at low densities (CSR), those observed at high densities were regular , suggesting the existence of a minimal density at which a regular pattern can be discriminated from CSR (λ3.3.3.dist) decreased monotonically with density following a power law of the form a). This statistically significant relationship was observed in each replicate . Figure 5a shows the mean curve fitted to the values of all segments combined .The mean distance to the nearest neighbour (NNist=γ×λα a. This s3.3.4.G(r) first crosses the lower CSR envelope indicates the existence of an inhibition mechanism regulating beetle establishment and provides a first approximation of the inhibition distance (MAD). Figure 5b shows that I. typographus iteratively fills the empty space using a spacing mechanism. Moreover, the minimum nearest-neighbour distance The distance at which the nearest-neighbour distance function 3.4.3.4.1.c,d shows this phenomenon: the mean nearest-neighbour distance between attacks at times up to t and attacks occurring at the next time step, t + 1, tends to increase with time (c). However, because new attacks are more numerous at the beginning of the infestation (a), the inverse trend is observed when the rate of new attacks between time step t and t + 1 is considered: this relationship indicates that new entrance holes appear closer to each other. This empirical description was confirmed by a complementary analysis based on statistical methods dedicated to the study of point process patterns  that indicated that there are interactions between the attacks (i.e. inhibition) throughout the entire colonization process. Moreover, it appeared that the sole filling of the empty areas by new entrance holes could explain the observed decrease in the nearest-neighbour distance ; the average and observed distance to the nearest neighbour decreases with increasing density (density increases with time). Additional analysis suggested that the inhibition distance is barely affected by the age of the attacks .The spatio-temporal pattern of attack holes was similar between replicates. The first entrance holes were randomly located, covering almost the entire segment , and theFigure 5ith time c. Howeveestation a, the in3.4.2.X-axis (distance between the most distant projected holes) at the first measurement was close to that measured at the end of the observations; the spreads of holes along the X-axis at the first measurement (spread1) and the final pattern (spreadEND) showed a strong linear relationship . These patches of entrance holes covered almost the entire available length (seg.length) of each segment . It should be noted again that the location of the pheromone dispenser had no influence on hole locations .The spread of the entrance holes along the H1/HEND) ranged from 4% to 48% . The number of holes at this first measurement (H1) did not influence the spread of the holes over the X-axis. The number of holes quickly reached almost the maximum value . In addition, the coverage of the colonized patch quickly approached its maximal value: on day 3, the relative coverage was 73.1 ± 11.9% (N = 13), while 35.7 ± 11.6% (N = 13) of the final number of holes was recorded.The fraction of all entrance holes mapped that were present at the first measurement  in nine of 13 replicates. In the four replicates showing a statistical relationship, this trend was very mild considering the low R2 values (maximum = 0.21) and the small displacement of the centre of gravity of the entrance holes, X, through time . This indicates that the segment was filled with entrance holes without any preferential location during the entire observation period.Moreover, there was no relationship between the time and location of the entrance holes along the detection) seem unaffected by segment size .In terms of spatial pattern, the spread of entrance holes over the segment length (at first measurement or at the end of the observations), the mean nearest-neighbour distances . This theoretical analysis indicated that no plateau occurred within the range of the observed entrance hole densities and at inhibition distances compatible with our results . Hence, the inhibition distance controls the location of successive holes over a small range (a few centimetres), but does not significantly influence the infestation rate at the global scale  at common realistic densities.3.5.a,b and 5a; electronic supplementary material, table S1).The three whole trees showed the same colonization dynamics and similar spread characteristics as the segments, confirming that the pheromones used in the observations described here, or the splitting of each tree into segments, did not influence the spatio-temporal patterns , but this minimum was rarely reached in our observations.Our analyses show that colonization first occurs without any preferred location or direction; the insects apparently initially establish anywhere over almost the entire available surface. A regular pattern is then progressively achieved by randomly occupying the still-vacant areas, leading to a constant decrease in the distance to the nearest neighbour down to a minimum below which new entrance holes are not excavated. The theoretical values that we predicted agree well with those reported by Byers  , suggesting that other regulatory mechanisms operate before this theoretical density is reached. As many landings occurred almost simultaneously, the information perceived by new potential settlers must have been conveyed very rapidly by the first beetles that established.Our simulations  showed that a maximum density of 3.13 attacks dmcks dm−2 ,43–46) adm−2 [−2 ,44,46,47 [−2 [−2 a, suggesb) . Landing without attack has already been described by Anderbrant et al. [et al. [I. typographus has shown a relatively long survival capacity of more than 14 days [−1. These capacities probably allow the insects to be fairly selective in terms of their breeding sites. In addition, I. typographus is probably flexible in its acceptance of a host, and its previous experience could influence further choices, as reported for Ips pini by Wallin & Raffa [The mechanisms possibly regulating attack density have been thoroughly discussed by Byers , and thefigure 1t et al. , who rep [et al. . The fat 14 days  to more  14 days . A high  14 days , with up & Raffa . Under c & Raffa .et al. [I. typographus mostly explores crevices during host colonization, and similar observations have been reported for D. ponderosae by Safranyik & Vithayasai [Ips calligraphus by Haack et al. [Bark texture plays a strong role in the choice of attack locations. The rough bark at the base of the branches exhibits higher entrance hole densities than the smooth bark covering the rest of the segments. There is no indication that the colonization process could end due to the saturation of the preferred rough bark, and the ratio of densities on each bark type remains stable, suggesting that the discovery of rough bark is due to random, non-oriented walks on the hosts. This frequent settlement around branches is likely to be caused by irregularities in these tissues that favour the initiation of an entrance hole. Paynter et al.  report tthayasai  and Ferrthayasai , and fork et al. . Such thk et al. –58, coulk et al. ,60. The Although a number of unsolved issues should be explored further , our results strongly suggest that, when weakened hosts are involved, the colonization behaviour of facultative tree-killing bark beetles does not fit the widely accepted paradigm of ‘mass-attack against tree defence’, but rather corresponds to the expected outcome of group foraging for scarce resources with mass-colonization only being an unnecessary consequence of mass-foraging, with costs and benefits that vary widely from those associated with the mass-killing of HTs.


Introduction: Bone graft is harvested from bone and used to stimulate bone healing due to its properties. The aims of the study were to compare the quantity of graft harvested from proximal tibia and iliac crest, and the complications at these donor sites.Materials and Methods: This was a prospective study carried out on all consecutive patients who had orthopaedic procedures that required bone grafting at the study centre, from April, 2015 to March, 2016.Results: During the study period, 86 patients were recruited; 43 of these patients had bone graft harvested from the proximal tibia, while the other 43 patients had graft harvested from the iliac crest. There were 53 males, and 33 females. Mean age of patients was 41.2 ± 11 years and 40.8 ± 16 years, for proximal tibia and iliac crest group respectively. Average compressed volume of graft harvested from proximal tibia and iliac crest were 7cm3 and 5.5cm3 respectively. Non-unions were the commonest indications for bone grafting, femur was the commonest bone requiring bone grafting. Complications such as primary haemorrhage, pain and surgical site infection were commoner with iliac crest than proximal tibia donor sites, however bone graft harvested from both proximal tibia and iliac crest provided adequate bone union of the indicated procedure.Conclusion: Larger quantity of graft can be harvested from proximal tibia than the iliac crest. Though graft harvested from both the proximal tibia and the iliac crest have good healing properties, the proximal tibia donor site gave less complication than the iliac crest. Due to the problems associated with autograft, such as donor site complications and inadequate quantity available as often encountered, graft substitutes such as recombinant bone morphogenic protein (rhBMP-2) and ceramic-based bone graft substitutes have been developed and used3.Bone grafts can be vascularised or non-vascularised. The non-vascularised can further be subdivided into autografts when the graft is taken from the same individual where it is used, allograft when the graft is taken from another individual, usually referred to as cadaveric bone. The autogenous graft can be cortical graft or cancellous graft8. Autografts allow faster time to union, lesser needs for revision procedures, and lower infection rate compared to allograft or recombinant morphogenic protein. The disadvantages of autogenous graft include: increase anaesthesia/surgery time, additional surgical scar, and increased blood loss10.Autogenous bone graft is regarded as the gold standard because it is not prone to immunological rejection and it has high osteoconductive, osteoinductive and osteogenic properties compared to other types of grafts and graft substitutes12.Bone grafting is indicated in nonunion, a condition commonly encountered in orthopaedic practice especially in developing countries where traditional bone setters compete with orthopaedic surgeons in managing long bone fractures. Bone grafting is also indicated in joints arthrodesis and filling of bone defects15. The aims of this study were to compare the quantity of graft harvested from proximal tibia and iliac crest, and also to compare the complications at these bone graft donor sites.The reported complications following the use of bone graft are: pain at donor site, infection at the donor site, transmission of infection especially where allograft is used, and fracture at donor site has also been reportedThis is a prospective descriptive study, approval for which was obtained from the ethical committee of the study centre. The study spanned a period of one year from April, 2015 to March 2016. All consecutive patients that required bone grafting during the study period were recruited into the study after obtaining consent from them. Proximal tibia graft and iliac crest graft were written on equal number of pieces of papers and each consented patient who required bone graft for his/her procedure was instructed to blindly pick one from these pieces of paper kept in a big brown envelope. The bone graft was taken from either the iliac crest or the proximal tibia of the patients depending on the site on the paper picked by the patient. The quantity of the graft harvested was determined by collecting the harvested cancellous bone graft into the barrel of an empty 10 milliliters syringe.Intraoperative blood loss from the donor site was estimated using sterile gauze and abdominal packs; mildly soaked gauze = 5mls, moderately soaked = 10mls, and fully soaked gauze = 15mls. While mildly soaked abdominal pack = 50mls, moderately soaked = 100mls, and fully soaked abdominal pack = 150mls. Pain at the graft donor sites were assessed using the numerical rating scale within the 24 hours-postoperative period, and one month and three months post-surgery.0 = no pain, 1—3 = mild pain, 4—6 = moderate pain, 7—10 = severe pain.For the proximal tibia bone graft harvest, the patient was placed in supine position following regional or general anaesthesia, with prophylactic antibiotic was given at induction of anaesthesia. The leg was cleaned and draped separately to expose the proximal part of the leg, an incision of about 6 cm was made on the anteromedial surface of the proximal leg between the anterior and medial border of the tibia, about 6 cm below the knee joint line.The incision was deepened down to the periosteum which was incised and periosteal elevator was used to separate the incised periosteum from the bone. The skin and the periosteum were retracted medially and laterally to expose the proximal tibia. An osteotome was used to create a rectangular cortical window of about 2 cm in width and 3 cm in length between the medial and anterior border of the proximal tibia.Cancellous bone graft was scooped out from the marrow through the cortical window. The wound was irrigated, the removed cortex at the cortical window site was then reinserted to close the window and skin was closed with 2/o nylon suture and dressing was applied.For the iliac crest bone graft harvest, the patient was placed in supine position following regional or general anaesthesia, and prophylactic antibiotic was given at induction of anaesthesia. The iliac crest area was cleaned and draped separately. The anterior superior iliac crest was identified, the crest was palpated posterolaterally to get to the iliac tubercle (widest portion of iliac crest). About 6 cm skin incision was made and centered on the iliac tubercle, the skin and subcutaneous layers retracted, and the attached muscles on the lateral aspect of iliac tubercle were detached subperiosteally. Using an osteotome, an opening was created through the lateral aspect of the iliac crest with a medial cortical hinge. Cancellous bone graft was scooped out followed by wound irrigation with saline, the periosteum was apposed with O-vicryl suture, skin closed with 2/O nylon suture and dressing was applied.The data obtained were recorded in structured proforma which included the biodata, indications for grafting, types of procedure and complications at the graft donor sites. The data generated was analyzed by SPSS version 17. The test of significant association was done by using Chi Square or Fisher exact test as appropriate; the level of statistical significant was set at P < 0.05.Eighty-six patients who had procedures requiring cancellous bone grafting were recruited into the study. There were 53 males and 33 females, (F: M = 1: 1.6). The mean age of the patients who had bone graft harvested from the proximal tibia was 41.2 ± 11 years, while the mean age of those who had bone graft harvested from the iliac crest was 40.8 ± 16 years.3, while that from the proximal tibia was 7 cm3 . The commonest indication for cancellous bone grafting was nonunion found in 55 (64%) patients, followed by delayed union in 12 (14%) patients, Other indications included graft being used as space filler, in arthrodesis and malunion as shown in 2 = 8.52, P < 0.05).Proximal tibia and iliac crest bone graft were taken in 43 patients each, all grafts harvested were cancellous. The average amount of graft taken from the iliac crest was 5.5 cm2 = 0.03, P > 0.05). Superficial surgical site infections was seen in four patients (4.7%), one patient with proximal tibia graft and three patients with iliac crest graft . All patients had pain for 24 hours after surgery but with different severity; by one month post-surgery, three of the patients who had proximal tibia graft harvested had mild pain while moderate pain was noted in 12 patients with iliac crest graft, and by three months post-surgery none of the patient with proximal tibia graft had pain at their donor sites, while two patients who had iliac crest graft harvested reported mild pain at their donor sites, as shown in 2 = 0.06, Fisher exact P < 0.05). All the eighty-six patients had adequate bone union following the various procedures and none of them required re-operation.Complications observed were primary haemorrhage with an average of about 40 milliliters for proximal tibia graft and about 100 milliliters for iliac crest graft . This may be due to the fact that males are more prone to injury than females due to the types of activities they are engaged in.The mean age of patients involved was 41.6 ± 16 years. This means that majority of the patients requiring bone grafting procedures to assist fracture healing were young adults who constitute the work force of the population, conforming the importance of this procedure.3) than those harvested from the proximal tibia (7cm3). This finding was statistically significant (P < 0.05). This means that more graft can be harvested from the proximal tibia than the iliac crest. This finding is contrary to Alt et al, who conducted an experimental study on human cadaver and found out that more graft can be harvested from the iliac crest than the proximal tibia16. Also Burk et al reported that graft harvested from proximal tibia was the least in quantity when compared to anterior and posterior iliac crest17. The study by Burk et al, harvested corticocancellous graft as against cancellous graft in this study, this may be the reason why the proximal tibia had the least quantity of graft in their study, because the cortical part of graft that can be taken in proximal tibia is less compare to those of the iliac crest.The average volume of graft taken from the iliac crest was less . This is because adult femoral fractures must be managed operatively because the surrounding large muscle bulks exert forces that distract the fracture ends and hence prevent healing. Delayed union was the second commonest indication in 12 patients (14%) with the tibia topping the list of bones with delayed union in five patients (5.8%) Other indications for bone grafting were in malunion, arthrodesis and as space filler.In our study, the commonest indication for bone grafting was nonunion, 64% n= 55 . This isThis study showed that it took longer time to harvest bone graft from iliac crest than the proximal tibia, which may be due to the excess subcutaneous fat, the surrounding muscle which need to be dissected and retracted away from the iliac crest before harvesting the graft, unlike the proximal tibia graft site which is subcutaneous. Achieving haemostasis took more time while harvesting graft from the iliac crest than the proximal tibia.Primary haemorrhage was more at the iliac crest graft site than the proximal tibia site, this is because the iliac crest has surrounding muscles which bleed easily when incised compared to the anteromedial part of the proximal tibia (where bone graft is harvested) that is subcutaneous in nature. However, this incidental finding was not statistically significant (p=0.778).Staphylococcus aureus was the organism isolated in three patients while pseudomonas eruginosa was isolated in one of the patient. The infections were treated with wound dressing and appropriate antibiotics based on sensitivity. All the infections resolved within two weeks of surgery.Superficial surgical site infections occurred in four patients (4.7%) who were known diabetics with controlled blood glucose prior to surgery. One of them had bone graft harvested from the proximal tibia while the remaining three patients had graft harvested from iliac crest. Folds of skin around the iliac crest containing excessive fat may be part of the reasons while infection was commoner at the iliac crest site. Despite the combination of parenteral analgesics , all patients had varying degrees of pain within the first 24 hours of surgery. By one month post surgery, three patient who had bone graft harvested from proximal tibia had mild pain, while twelve of those patients with iliac crest site had moderate pain. By the third month post surgery, none of the patient with proximal tibia graft site had pain, while two of those patients with iliac crest graft site still had mild pain. This showed that pain was more intense and lasted longer when bone graft was harvested from the iliac crest than the proximal tibia. This may be due to more soft tissue with nerve endings surrounding the iliac crest graft site than the proximal tibia site .Both groups of patients had adequate bone union with bone grafting for the procedures that they used for, none of the procedure requiring re-operation, confirming that the grafts from both sites have good bone healing properties.et al who reported that the tibia is biomechanically stable with full weight bearing ambulation following graft harvest from the proximal tibia19. Although the size of the cortical window used by Lim et al was smaller than the size used in this study, re-insertion of the osteotomized bony cortex of the cortical window after scooping out the cancellous graft might have contributed to the biomechanical stability of the tibia in this study.Interestingly, none of the patient who had bone graft harvested from the proximal tibia had complain of biomechanical instability of the tibia, this was similar to the findings of Lim Harvesting bone graft from the proximal tibia should be considered when carrying out procedures that may require larger amount of cancellous bone for grafting. Even though both proximal tibia and iliac crest grafts have good healing properties, the complications such as post-operative pain, surgical site infection and primary haemorrhage are less when graft is harvested from proximal tibia than the iliac crest.


Dear Colleagues,It is my extreme pleasure to compile an editorial for the journal entitled “Euroasian Journal of Hepato-Gastroenterology.” It is needless to emphasis the importance of scientific journal for dissemination of knowledge, providing guidelines, and interconnecting civil and scientific societies. The importance of journals is further manifested in developing and resource-constrained countries. I also view the importance of medical journal from a novel perspective of globalization. In this evolving world, new and novel information and advanced techniques have been piling up in developed and advanced countries. On the contrary, developing countries have been harboring most of the patients of different specialties and subspecialties. In fact, many of these patients have not been properly diagnosed and managed. A bridge between these realities of developed/ advanced countries versus developing/resource-constrained nations may be only be addressed by compiling and circulating medical journals.I am really excited to know that more than 10 countries of Europe and Asia are actively involved in designing and proper execution of regular publication of “Euroasian Journal of Hepato-Gastroenterology.” The journal has successfully completed its 6th birthday without any break or discontinuation. Medical scientists and clinicians of 25 countries enrolling continents of Asia, Africa, Europe, and America have sent their articles for this peer-reviewed journal.At the top of these facts, I am especially excited to know the fact that the journal is compiled by combined efforts of hepato-gastroenterologists of Asia and Central Europe: it is published from India. It is a well-known fact that the medical scientists of Asia and Central Europe are lagging behind those of Europe, USA, and Japan. However, the majority population of the world is from these parts of the globe. The entire approach of this journal is to provide what is needed but remain absent for decades. The journal is indexed in several citation bodies and the editors and publishers are doing their best to incorporate the journal in PubMed. I hope that their dream would come into reality and we would soon find the existence of this journal in PubMed and acquisition of an impact factor may not be a remote goal.I also feel proud that this journal has become an important publication pot for the researchers and clinicians of Bangabandhu Sheikh Mujib Medical Universities and Bangladeshi medical scientists of other institutions. Also, several medical scientists of Bangladesh have been working in the capacity of editors and reviewers for this journal.Taken all facts into a comprehensive outlook, I would congratulate the initiators of the journal, the editorial board, and publishers and hoping for eternal existence of this booklet of science for our development and searching for identity.Prof Kamrul Hasan KhanVice ChancellorBangabandhu Sheikh Mujib Medical UniversityDhaka, BangladeshE-mail: vc@bsmmu.edu.bd


TM Esthetic) specimens (2.0 mm × 20 mm × 10 mm) were exposed to different surface treatments prior to bonding to CoCr cylinders . All surfaces were sandblasted  and treated with alcohol , saliva (s), saliva + water (sw), or saliva + NaOCl + water (sn) before bonding was performed with the following adhesive luting systems: RelyXTM Unicem 2 (RX), ScotchbondTM Universal (SBU) + RelyXTM Ultimate (RU) or Monobond Plus (MP) + Multilink® Automix (ML). After 24 h, thermocycling  and 90 days of storage at 37 °C in distilled water, the shear bond strength (SBS) was evaluated according to ISO/TS 11,405:2015. Failure modes along bonding areas were characterized. Means and standard deviations (n = 10 per group) were determined and statistically analyzed with one-way ANOVA/Bonferroni (α = 0.05). (3) Results: The SBS after 24 h varied between 3.5 (sRX) and 69.4 MPa (snMP + ML). Values from 0 (sRX) to 70.3 MPa (swRX) were found after TC. Data after 90 days of storage showed the lowest values for sRX (0 MPa) and the highest values for alSBU + RU (75.5 MPa). Adhesive failure was noted at all aging conditions. (4) Conclusions: SBU + RU or RX and MP + ML including saliva decontamination of the ceramic surface with water or NaOCl + water allow efficient bonding to LavaTM Esthetic.(1) Background: This study evaluated the bonding performance of high translucency zirconia after diverse surficial decontamination and cleaning procedures. (2) Methods: High translucency zirconia (Lava Dental restorative materials for single crowns and fixed or partially fixed dentures are intended to mimic the visual nature and function of the original teeth. Silica-based ceramics, e.g., feldspathic  or lithium disilicate ceramic ≤400 MPa), are predominately utilized for single tooth restorations. Non-silica-based or oxide ceramics, such as high-strength yttria-stabilized tetragonal zirconia polycrystals 3Y-TZP; ≤1200 MPa), are increasingly being used as veneer frameworks or monolithic material to restore and rehabilitate partially edentulous patients. The inferior translucency of standard monolithic zirconia restorations adversely affects their clinical application. Aesthetic demand for high translucency zirconia produced by computer aided design and computer aided manufacturing (CAD/CAM) for monolithic restorations has increased. Improved translucency results from changes in zirconia structure due to an increase in yttria content from 3 to >5 mol %. The tetragonal zirconia phase decreases to more cubic phase particles, causing a reduction in flexural strength 600–800 MPa) 00 MPa, a,8. Ceram00 MPa 0000 MPa, ap < 0.001) differed from 3.5 ± 2.1 MPa (#4) to 75.7 ± 15.0 MPa (#14) after 24 h  + RelyXTM Ultimate (RU) was not statistically significant in contrast to non-saliva-contaminated systems. The shear bond strength (SBS) of high translucency zirconia, standard zirconia, and lithium disilicate ceramic significantly (24 h see . Only sap < 0.001) from 0 (#4) to 70.3 ± 10.6 MPa (#5). Again, saliva (s) contamination showed lowermost values between 0 and 7 ± 16.0 MPa for RX (#4) and MP + ML (#12), which were statistically significant in contrast to all other systems. With TC, 11 systems showed an increase in SBS, whereas the SBS of 5 systems decreased. Thus, the temperature difference of TC (5–55 °C) affects the chemical and mechanical bond between adhesive luting systems and ceramic. In comparison to the baseline, most systems showed statistically significant differences. After 90 days of storage, SBS values ranged from 0 (#4) and 10.8 ± 6.6 MPa (#12) due to saliva contamination to 75.7 ± 6.9 MPa (#7). Compared to the baseline, eight systems showed an increase in SBS. Compared to TC, four systems showed an increase in SBS. In general, statistically significant differences were identified between the different groups at each time point (p < 0.001). Moreover, adhesive failures were predominantly observed at all aging conditions (see After thermocycling (TC), the SBS varied significantly  different cleaning and activating procedures for high translucency zirconia surfaces allow for effective bond to adhesive luting agents for all systems. However, the results of the present study led us to partially reject the hypothesis in that (2) preliminary surficial treatment does not affect the bond strength and fracture mode after aging because there are significant differences in the SBS between different time points. The clinical long-term performance of ceramic restorations is related to an appropriate bonding procedure ,2,3 inflIn general, manufacturers recommend specific pre-treatments for the inner ceramic surface before bonding to achieve a sufficient micromechanical and chemical interlocking between ceramic restorations and adhesive luting systems. A recently introduced single-component ceramic primer Monobond Etch & Prime (ME), which includes an alcoholic aqueous solution of ammonium polyfluoride and silane methacrylate, allows for the etching and priming of silica-based ceramic in one step. Limited comparative studies (hydrofluoric acid (HF) + MP vs. ME) ,5 show cDuring the intraoral try-in procedure, restoration surfaces come in close contact with the gingiva, causing contamination with saliva, sulcus fluid, and blood. Different cleaning protocols for zirconia and their influence on bond strength have been reported in the literature. These include the application of water, alcohol, phosphoric acid, or universal cleaning paste ,11. SomePrevious studies conflict in their conclusions on saliva-contaminated conventional zirconia surfaces rinsed with water before a light-cured composite resin is applied, because saliva immediately coats surfaces with a proteinaceous layer . On the A decrease in SBS is usually expected after artificial aging (TC). SBS of MP + ML bonded high translucency zirconia was reduced by TC. When comparing the 24 h results with thermocycling RX or RBU + RX bonded Lava™ Esthetic, an increase in SBS was observed. There was no further increase after 90 days of water storage in any case, but the values were elevated compared to the baseline data. It can be assumed that, besides an initially and primarily micromechanical bond, the functional methacrylates of adhesive luting systems bond chemically to the ceramic. The comparison of SBS of primer (MP) + resin cement (ML) revealed a statistically significant reduction after 90 days, especially for the saliva-contaminated and subsequently cleaned zirconia, indicating the failure of the bond strength through hydrolysis. n = 60; Ivoclar Vivadent, Schaan, Liechtenstein) and a 3Y zirconia  were used as references. The shear surfaces of the ceramic materials were differentially treated prior to bonding. First, zirconia surfaces were sandblasted . To approximate the usual clinical situation surfaces were subsequently treated with 70% alcohol , saliva (s), saliva + water (sw), or saliva + 5% NaOCl + water (sn) for 3 min each. The lithium disilicate ceramic was either etched with a 5% hydrofluoric acid gel  or prepared with Monobond Etch & Prime . Following surface preparation, the specimens were bonded to CoCr cylinders  [TM Unicem 2 (RX), ScotchbondTM Universal (SBU) + RelyXTM Ultimate (RU), and Monobond Plus (MP) + Multilink® Automix (ML). Overall, 16 different combinations of activation and cleaning procedures were discriminated (n = 10 per group) were calculated and statistically analyzed with one-way analysis of variance (ANOVA) and a Bonferroni correction (α = 0.05).Whole unstimulated saliva was collected after informed consent from a single 52-year-old, healthy female volunteer by expectoration at different occasions and frozen immediately at −30 °C. Immediately prior to the experiments saliva was gently defrosted, pooled, and successively sterilized with single use filtration devices . All in all, 360 specimens were fabricated from the high translucency zirconia Lava™ Esthetic  with the following dimensions: thickness: 2 mm; length: 20 mm; width: 10 mm. A lithium disilicate ceramic   by meansiminated . At a crCleaning and decontamination procedures  are crucial for obtaining an effective bond between various adhesive luting systems and high translucency zirconia. Only the bond of the universal adhesive SBU, together with the resin cement RU, to zirconia withstood saliva contamination. Within the limitations of the present study, the use of a self-etching cement or adhesive bonding in combination with cleaning and decontamination appears to guarantee efficient bonding to high translucency zirconia. However, further studies are needed to predict the clinical long-term performance of the adhesive bond of formerly contaminated high translucency zirconia.


Teleoperated robotic systems are widely spreading in multiple different fields, from hazardous environments exploration to surgery. In teleoperation, users directly manipulate a master device to achieve task execution at the slave robot side; this interaction is fundamental to guarantee both system stability and task execution performance. In this work, we propose a non-disruptive method to study the arm endpoint stiffness. We evaluate how users exploit the kinetic redundancy of the arm to achieve stability and precision during the execution of different tasks with different master devices. Four users were asked to perform two planar trajectories following virtual tasks using both a serial and a parallel link master device. Users' arm kinematics and muscular activation were acquired and combined with a user-specific musculoskeletal model to estimate the joint stiffness. Using the arm kinematic Jacobian, the arm end-point stiffness was derived. The proposed non-disruptive method is capable of estimating the arm endpoint stiffness during the execution of virtual teleoperated tasks. The obtained results are in accordance with the existing literature in human motor control and show, throughout the tested trajectory, a modulation of the arm endpoint stiffness that is affected by task characteristics and hand speed and acceleration. Teleoperated robotic systems are widely used in several long-short range application fields, from plant decommissioning  musculoskeletal model of the upper limb  and a serial link (SL) master device, to test how the differences between the two manipulators would elicit different levels of arm end-point stiffness during virtual teleoperation. During task performance, arm kinematics and EMG signals were acquired using an optical and a magnetic tracking systems).Our primary hypothesis is that end-effector stiffness would be modulated according to both the mechanical features of the master device and the task characteristics. In particular, we expect the users to generate higher overall end-point stiffness when teleoperating with the serial link master device, characterized by lower structural stiffness and apparent mass. We also expect the users to increase arm stiffness while performing the second task; due to the increased complexity of simultaneously controlling position and orientation of the virtual tool. Our second hypothesis is that end-point stiffness would be modulated accordingly to curvature variations throughout the trajectory. More specifically, we believe that high curvature can be associated with higher complexity in executing the task, leading to higher values of arm end point stiffness. As third and final hypothesis, we expect a correlation between the hand speed and acceleration and arm end-point stiffness so that the users would generate the maximal levels of stiffness during slow and non accelerated movements.In order to analyze the stiffness regulation during virtual tele-operated tasks, we created a simple teleoperation scenario in which the users interacted with master devices to control virtual tools used to perform specific tasks.Simple repeatable and cyclical planar virtual tasks were developed to challenge the users with different levels of complexity without requiring any surgical expertise.Half Cloverleaf (HC)mm < z < 1mm), red otherwise.Figure Shape Fitted Half Cloverleaf (SFHC)The second task was designed to include wrist rotation as the subjects were asked to navigate a virtual cylindrical shaped tool tip along the path, as shown in Figure The subjects were controlling the virtual tools position and orientation using two different master devices: a 7 DoFs parallel links haptic interface (PL) and a 6 DoFs serial links haptic interface (SL).N/mm.A Force Dimension Sigma7  was used as PL  was used as SL  using 10 bi-polar electrodes .Retroreflective and electromagnetic markers movements and EMG signals were acquired, recorded and synchronized using custom developed software based on the Robotic Operating System  who provided informed written consent, in accordance with the recommendations of Politecnico di Milano Ethical committee Board. All subjects gave written informed consent in accordance with the Declaration of Helsinki.The subjects were sitting in a comfortable chair without arm rests, in front of a 2D monitor screen where the virtual tasks were displayed  and the filtered data were used as input for the inverse kinematics. The inverse kinematic algorithm solves the minimization problem in Equation (1) for each set of marker coordinates.q is the vector of joint angles, kth marker, acquired using the optical or the magnetic tracking device, xk(q) is the position of the corresponding virtual marker on the model that depends on the vector of joint angles q, and wk is the kth marker weight thorough which is possible to account for the uncertainty related to each marker position detection. For each set of marker coordinates, the algorithm finds the best joint vector q that minimizes the error between the experimental marker position vector xexp and the virtual markers position vector x(q), which is a function of the fixed model geometry and joint vector, q.Where w =  with N = number of markers, we analyzed the markers positions during a static recording. The ratio between the standard deviations from optical markers and electromagnetic markers is comparable with the ratio between the RMSE characteristics of each device. We therefore weighted the thorax optical markers with a coefficient of 1, while the arm, forearm and hand electromagnetic markers with a coefficient of 0.52 .In order to define the correct weighting vector τ is the unknown (7 × 1) vector of joint torques, M is the system mass matrix, C is the vector of Coriolis and centrifugal forces, G is the vector of gravitational forces, while q, The obtained joint angles were filtered with an IIR second order Butterworth filter (cutoff frequency 4 Hz) twice, compensating the nonlinear phase shift. The filtered data were used as input for the inverse dynamic reconstruction, along with the model dynamic characteristics . Equation (2) shows the dynamic equation when no external forces are applied to the arm; A refined estimation of the joint torques was obtained using the Calibrated EMG-informed Neuro-Musculoskeletal (CEINMS) modeling toolbox in which the musculotendon unit (MTU) force arms obtained with the inverse kinematics are used with the joint torques estimated by the inverse dynamics and the recorded EMG signals.The tool firstly calibrates the experimental muscle excitations derived from the surface EMG acquisition with the muscle activation patters, obtained from the inverse dynamic algorithm  was computed using Equation (3), where τ(i) is the joint torque vector that is associated with the q(i) arm configuration at the ith time instant  matrix (Ke) has been obtained using the arm kinematic Jacobian J (7 × 6), as follows:In order to obtain the Cartesian representation of the arm stiffness, the arm end-point Cartesian stiffness ) in the ith time instant.Through the Singular Value Decomposition (SVD), the left singular vectors and non-zero singular values of The following performance-related parameters were evaluated:ith time frame, di: the distance is computed from the tool tip to the closest point on the trajectory center on the task plane target are the tool tip's 2D coordinates and tool are the 2D coordinates of the closest point on the trajectory. The z-component of the displacement from the trajectory was excluded from the distance metric computation due to the significant differences that the color visual feedback could introduce with respect to the other two dimensions.2D distance from the trajectory at the Kmax: the end-point stiffness ellipsoid main axis was computed in 29 (for HC) and 23 (for SFHC) equally spaced points along the task trajectory . For each task trial and for each point selected, the three closest virtual tool positions were searched and the corresponding maximal stiffness (Kmax(j), Kmax(j + 1) and Kmax(j − 1)) were averaged to obtain the mean maximal stiffness estimation as in Equation (6)j = 1…M − 1 number of points along the trajectory.Maximal stiffness C: In both tasks, the trajectory was created to fit the same Bernulli's Lemniscate function. The generic function of a Bernulli Lemniscate curve in a planar x-y plane with the main axis oriented along the x axis isa is the parameter that defines the position of the curve foci. The task trajectory curvature (C) can be computed in polar coordinates using:t that spans from 0 to 2π. For each 3D point of the trajectory selected for the stiffness analysis, the corresponding curvature was computed.Curvature x-y-z coordinates, smoothing the signal with a second order Butterworth filter with cutoff frequency of 4 Hz twice forward and backward. Acceleration was computed and filtered from hand speed following the same procedure previously described.Hand speed and acceleration: the position and orientation of the hand in the shoulder reference frame .Kmax were first of all studied as a function of the task type and controller. The distributions of the normalized stiffness data were compared between users and repetition, since no significant differences were found, the users data were grouped together for each experimentMaximal stiffness through the trajectory - The stiffness maximal values Hypothesis 1 & 2: To analyze whether the task and master device types, as well as level of trajectory curvature affected the maximal values of stiffness, the data from the 29 (HC) and 23 (SFHC) points on the trajectory for each experiment were grouped. The mean values of maximal stiffness for each region of curvature and repetition were extracted and a three-way ANOVA was performed on the log-normalized data . To further test the effects of the different factors on the end-point stiffness, the same ANOVA model was also applied to the stiffness variability in the various regions and repetitions. To test the presence of possible correlations between stiffness and curvature the Pearson rank test (α = 0.05) was adopted.Stiffness against hand speed and acceleration - Using two separate two-ways ANOVAs  the hand speed and acceleration distributions in the four experiments were analyzed. Hypothesis 3: we tested the hypothesis that a relation between hand speed and acceleration with respect to maximal stiffness could exist. For each of the ten trajectory repetitions, 4 levels of hand speed and acceleration were obtained in the four experiments and for the 4 users. Two separated three-way ANOVAs were performed on the corresponding values of stiffness .Distance metric - The difference between different trials of the same experiment for each user was tested using a one-way ANOVA test (α = 0.05). Since no statistical difference emerged, the mean values of distance throughout each repetition and user were extracted. The four experiments' performance distributions were then modeled with a two-way ANOVA statistical model, where task and master device type were considered as fixed factors, while user number was considered as random effect factor.N/m were removed from the analysis: the threshold was obtained from previous arm end-point stiffness estimations  = 10.06, p < 0.005] while no significant difference was found between the master devices . Overall, users elicited significantly higher arm stiffness when performing the HC task with respect to SFHC. Figure F = 7.75, p < 0.01] and master devices . Interestingly while the stiffness variance increases in the SFHC task when teleoperating with PL, when teleoperating with the SL master device, instead, users decreased the arm stiffness variance when performing SFHC with respect to HC. Overall, users explored the highest range of end-point stiffness in HC when teleoperating with SL and showed the lowest variance with the same master device in the SFHC task.Figure Kmax value is represented as a colored column  and 23 (for SFHC) points along the trajectory while teleoperating with the PL and SL master devices, Figure F = 28.19, p < 0.0001] between the four different levels of curvature. The box-plots in Figure Kmax mean values are registered where the normalized curvature is <0.25 while the maximal values appear in the range between 0.75 and 1. Interestingly, while for the hybrid parallel-serial link master device (PL) the end-point stiffness behavior in HC and SFCH shows the same trend, with an apparent plateau in the middle curvature zones, different behaviors can be seen with SL. In this case, HC shows a clear increase in the mean value of maximal stiffness with increasing levels of curvature, while in SFHC users were eliciting very small variations of arm end-point stiffness. Due to the high variance in the data, Pearson correlation analysis showed non-significant (p > 0.05) correlations for all the experiments. Furthermore, regarding the stiffness variability in the four regions of curvature, the three-way ANOVA test showed no significant differences .Regarding the end-point stiffness mean values relation with curvature, the three-way ANOVA showed significant difference  and acceleration . The two separate three-way ANOVAs showed no significant interaction among task, master device type and level of speed or acceleration; a main significant effect was found for the task type  while no significant effects were found for the master device type and the levels of speed or acceleration. Figure p| ranging from 0.1 to 0.3) non significant (ρ > 0.1) negative correlations.Figure F = 548 p < 0.0001] and no difference between PL and SL [F = 2.43 p = 0.1214]. The users were capable of achieving the best performances, testified by lower distances, with the parallel link master device (PL) and in the first task (HC). Similarly as seen with the maximal stiffness values  to a cylinder (in SFHC). These differences could have decreased the users perception of the distance from the target trajectory thus reducing the feedback on their actual performance. Consequently the users performed the task with higher speed. This explanation seems to be endorsed by the statistically higher mean distances recorded in SFHC with both master devices. The differences in mean speed and acceleration between the master devices are affected by the task request: during HC users where slower using PL while significantly faster using SL. On the contrary, the maximal speed for SFHC was obtained with PL, showing how different tasks may require different types of master devices in order to be efficiently executed. No statistical difference was found between the arm stiffness at different levels of speed and acceleration, but the higher end-point stiffness values and variability that were seen for some experiments, suggest that, in those experiments, high stiffness is more likely to occur at lower speed and accelerations. On the other hand, it appears that, even though joint speed and acceleration are fundamental components in the joint stiffness computation, their relation with arm end-point stiffness can't be modeled with simple correlations.A limitation of this work can be found, first of all, in the small number of subjects; the number of trials for each task may not have been enough to account for the high variability that characterizes human motor control. Another limitation lies in the described differences between the two tasks; the SFHC task, in fact, differs from HC not only for the necessity or re-orienting the tool end-effector, but also in terms of trajectory thickness and tool-tip dimension.In conclusion, the results obtained suggest that the users tend to modulate the arm endpoint stiffness with respect to different tasks and interfaces and that this modulation is influenced by both the trajectory characteristics and the users' hand kinematics. The users were coping with the difference in task design and master device by adapting their arm stiffness modulation both in terms of central tendencies and variability. Although affected by some limitations, these findings prove that the arm dynamic proprieties are highly variable and that there could be significant benefits from the estimation of the kinetic proprieties of the users' arm during teleoperation. For instance, knowing how the users modulate their stiffness would allow to develop master devices able to match and to enhance this modulation, potentially reducing the energetic cost of the teleoperators while maintaining high precision. The results obtained could also be used to improve human-robot interactions during cooperative tasks, as in Beretta et al. ,b.An interesting possible future development would be the inclusion in the research of a higher number of subjects as well as expert teleoperators, in order to study and compare the stiffness modulation strategies that they may have developed with expertise. To further understand which are the characteristics and parameters that play a fundamental role in the human-robot interaction, it would also be advisable to study how stiffness relates to other dynamic parameters such as damping and inertia whose relation to human-robot system stability have been recently investigated (Dyck et al., JB developed the acquisition framework, system synchronization software, performed the experiments, analyzed data and wrote the paper; with ED, he conceived the methods and analysis. JJ helped in designing and performing the experiments, also writing the corresponding portion of the manuscript. GF and ED supervised, reviewed and approved the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Miscanthus is a perennial bioenergy crop that offers a sustainable alternative to fossil fuels. We sought to identify candidate metabolic biomarkers of productivity that may be used as a method of screening for superior individuals in breeding programmes. Our experiments were carried out over two years and two sites in four genotypes. The concentration of fructose positively correlated whereas starch and the ratio of soluble sugars to starch negatively correlated with three biomass traits: yield, stem height and growth rate. Our results show the potential of the carbohydrate metabolic profile as a biomarker of productivity in a perennial energy crop. Miscanthus is a perennial energy grass predominantly used for combustion but with the current advancement of ligno-cellulosic fermentation technologies there is an interest in using Miscanthus for bioethanol production. Currently, the only commercially grown genotype of Miscanthus is M.× giganteus; a high yielding, interspecific hybrid of M. sacchariflorus and M. sinensis. As M.× giganteus is a sterile triploid, it cannot be used as a parent so Miscanthus breeding effort is focused on producing new interspecific varieties that out-perform M.× giganteus. The carbohydrate profiles of four genotypes of Miscanthus, including M. sacchariflorus (Sac-5), M.× giganteus (Gig-311), M. sinensis (Sin-11) and M. sinensis (Goliath), were characterized at replicated field sites in Aberystwyth, West Wales and Harpenden, south-east England. Our hypothesis was that a distinctive carbohydrate profile underlies enhanced biomass accumulation. Biomass accumulation is greatest when day-lengths and solar intensity are highest; so, observations were made in the middle of UK summer (July) for 2 years. Gig-311 had a greater abundance of fructose in its stems at both sites, and both Gig-311 and Sac-5 had low abundance of starch. At both sites, the highest yielding genotype was Gig-311 and Sac-5 was also high yielding at Harpenden, but performed comparatively poorly at Aberystwyth. At both sites Gig-311 had a distinctly high concentration of fructose, low starch and a high ratio of soluble sugars: starch, and at Harpenden, Sac-5 was similar. We conclude that the abundance of starch and fructose and a greater partitioning of soluble sugars, relative to starch, are candidate biomarkers of productivity in Miscanthus.There is a pressing need to find a sustainable alternative to fossil fuels that will not compromise food security or require extensive use of agrochemicals.  There is a global urgency to replace fossil fuels with a sustainable source of bioenergy without compromising soil health, food security or requiring the heavy use of agrochemicals. Dedicated energy crops can produce high yields with low inputs and therefore provide a means by which sustainable energy can be generated. The use of perennial crops can also improve water retention, increase climate change adaptation and promote biodiversity compared with traditional annual systems .Miscanthus is a giant perennial grass native to Eastern Asia but cultivated for bioenergy production in Europe and North America. Currently, Miscanthus is mainly used for combustion as a substitute for coal, but with the advancement of ligno-cellulosic fermentation technologies there is an increasing interest in using Miscanthus as a sustainable source of bioethanol  is being developed for Miscanthus . In the first study, combinations of metabolites were found to correlate with biomass; metabolites of particular significance were intermediates of the hexose pool such as fructose-6-phosphate, members of the tricarboxylic acid cycle and sucrose .Our experiment utilized four clones of The field trials described in this paper were established on private land belonging to the Institute of Biological Environmental and Rural Sciences (IBERS), Aberystwyth, West Wales  and Rothamsted Research in Harpenden, south-east England . Both sites are on land dedicated for agricultural research so no specific permission was required to establish the trials. No endangered or protected species were affected either by the establishment of the trials or subsequent experimental work.Miscanthus sinensis (Sin-11) is a diploid clone selected in 1988 from temperate Japan (Honshu Island) by Danish plant collector Dr Poul Erik Brander. It was part of the European Miscanthus Improvement (EMI) programme (1997–2000) and is the female parent of the Mx2 mapping family (Miscanthus sinensis (Goliath) is a triploid intraspecific hybrid of M. sinensis and was originally selected as a vigourous seedling from a cross (parents unknown) by Ernst Pagels and marketed as a ‘large-type’ horticultural variety since the 1970s. Miscanthus sacchariflorus (Sac-5) is a tetraploid that was part of a seed population collected from central Japan, by TINPLANT, in 1992. It was also part of the EMI programme. Miscanthus× giganteus (Gig-311) is a naturally occurring triploid hybrid of diploid M. sinensis and a tetraploid M. sacchariflorus. It was supplied to Aberystwyth from Bical Farms Ltd (Taunton) in 2005. The physical appearance of the four genotypes in July can be seen in Fig. All genotypes used in this study are of Japanese origin. nsis Sin-1 is a dihttp://www.bsbec-biomass.org.uk/) at the Institute of Biological Environmental and Rural Sciences (IBERS) West Wales  and Rothamsted Research in Harpenden, south-east England . At both sites plants were arranged in randomized block designs consisting of four blocks, each block containing four plots, one for each Miscanthus genotype described above. Each plot contained 121 plants (7.8× 7.8 m2) with areas designated for: non-destructive measurements, annual yield harvest and destructive harvests. Plants were grown from rhizome pieces cut from mature stands in modules before planting at a density of 2 plants m−2. Surrounding each plot was a row of guard plants of the same genotype. The soil type at Aberystwyth is classified as a silty clay loam. The soil type at Harpenden is classified as a silty clay loam (18–27 % clay) with high flint content. Prior to the establishment of the BSBEC field trial, the Aberystwyth site was under grassland and the Harpenden site in cereal cultivation. Both sites were ploughed, power harrowed and then planted. The surrounding paths were then re-sown with grass.Two dedicated trials were established in May 2009 as part of the BSBEC-BioMASS project  fitted with a CR1000 data-logger and a multiplexer. Rainfall was recorded on-site at Aberystwyth whereas at Harpenden these data were obtained from the central weather station located 1.7 km from the site across flat ground.n = 4). It is recognized in a number of species, including Miscanthus, that strong diurnal flux in carbohydrate occurs . Measurements were made on the same four plants per plot over the 8-week period. These values were then averaged on a per plot basis to provide a single value per plot to avoid pseudoreplication.Throughout the months of June and July, measurements of canopy height were made fortnightly. The average growth rate for the 2-weeks preceding the destructive harvest was then calculated as the change in height divided by the number of days  . Briefly, the dried pellet was resuspended in 0.4 mL of 0.2 M KOH, vortexed vigorously and heated to 90 °C in a water bath for 15 min to facilitate gelatinization of the starch. A total of 1.28 mL of 0.15 M NaOAc (pH 3.8) was added to each tube  before the addition of 20 µL α-amylase and 20 µL amyloglucosidase . After incubation at 50 °C for 30 min and centrifugation for 5 min, a 0.02 mL aliquot was combined with 0.6 mL of GOPOD reagent (Megazyme). A total of 0.2 mL of this reaction was assayed photometrically  on a 96-well microplate at 510 nm against a water-only blank. Starch was quantified from known standard curves on the same plate. Each sample and standard was tested in duplicate. Each plate contained a n = 4). To identify differences in leaf and stem carbohydrate concentrations and ratios, data for Aberystwyth from both years was used in a single analysis  and the value of the eight individual plants harvested per genotype was used to generate the mean and SE. At Harpenden, only a single year was analysed for leaf and stem carbohydrate concentration (2012) and therefore n = 3–4. For all statistical tests, significant differences equal P≤0.05. Regression analyses were performed in Excel, significant correlations were determined by ANOVA, P≤0.05.Statistical analyses were carried out using GenStat Version 13 . To identify genotypic and annual differences in biomass traits and total aboveground carbohydrates, two-way analyses of variance (ANOVA) were used for each site in which year (2011 and 2012) and the four genotypes were grouped as factors. Associated Tukey honestly significant difference (HSD) tests were performed to identify specific differences between genotypes for each year's data  that were consistent between sites included a negative correlation of −0.4–0.5 between stem starch and both growth rate and final yield, and a positive correlation between leaf fructose and growth rate. The ratios of hexose : starch and sucrose : starch showed significantly positive correlations of 0.3–0.7 for all biomass traits in both tissue types at both sites   exhibited a distinct carbohydrate profile showing a high ratio of hexose : starch and sucrose : starch and a low ratio of glucose : fructose owing to a greater abundance of fructose. The same was observed at Harpenden but many of these features were also observed in Sac-5 at this site. This is particularly interesting when the final yields of the different genotypes were compared; whilst Gig-311, Sin-11 and Goliath all yielded similarly (<50 g DW difference) between the two sites, Sac-5 yielded an average of 200 g per plant (30 %) more at Harpenden. Therefore, when Sac-5 was high yielding it possessed a similar carbohydrate fingerprint to Gig-311. This provides strong evidence that a high ratio of sucrose : starch and hexose : starch is an indicator, or biomarker, of high productivity. These finding support our first and second starting questions; that individual carbohydrates and the partitioning between different pools can be correlated with biomass traits.At Aberystwyth, M.× giganteus are unknown, it is known that this hybrid was derived from a cross between a Japanese, diploid M. sinensis and a Japanese, tetraploid M. sacchariflorus  (In a study to investigate the effects of elevated COstem (i) . Owing tThe stem of all genotypes at both sites contained glucose : fructose ratios of >2 : 1, with the exception of the stem of Gig-311, which was closer to 1 : 1. Fructose is exclusively produced by the metabolism of sucrose through the action of both sucrose synthase (SUSY) and INVs . GlucoseArabidopsis and maize that have also identified a negative relationship between starch and biomass  carbohydrates are in greater abundance it is due to reduced utilization, i.e. slower growth  are confirmed, it will be important to address at what stage in the plants' development this relationship can be observed. Our study was carried out on plants in their third and fourth complete growing season, but if the relationship could be identified in seedlings, or 1-year-old plants, then such markers could be used as a method of selection to accelerate breeding programmes in a similar way to the potential of molecular markers. However, if relationships between carbohydrates and biomass do not become apparent until maturity, it would be of limited benefit to use them directly as markers but they could prove important quantitative traits for genome mapping studies. This method has been successfully used in Arabidopsis where a co-localization of quantitative trait loci for metabolites and dry weight has been discovered in two different mapping families  grant (BB/G016216/1) working within the BSBEC-BioMASS (http://www.bsbec-biomass.org.uk/) programme of the centre, the BBSRC Energy Grasses & Biorefining Institute Strategic Programme (BBS/E/W/00003134) at IBERS and the Cropping Carbon Institute Strategic Programme at Rothamsted Research and Ceres Inc.This work was supported by the S.J.P. conceived the starting hypotheses for this manuscript. S.J.P., A.L.M., J.C., I.D. and J.C.-B. designed the experiments. A.L.M. carried out the carbohydrate analyses. S.J.P., A.L.M. and J.C. carried out the field work and sample collection. S.J.P. wrote the paper with significant input and editing from all other authors.The BSBEC-BioMASS project was primarily funded by the Biotechnology and Biological Sciences Research Council (BBSRC) but also received funding from an industrial partner, Ceres Inc. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.The following additional information is available in the online version of this article –Figure S1. The total monthly rainfall from May–July 2011 and 2012 and the 19-year averages for both sites. The 1981–2010 rainfall data are sourced from the UK Met Office which has weather stations located at both research institutes.Table S1. The concentration of carbohydrates in the total aboveground in July 2011 and July 2012 at Aberystwyth and Harpenden.Table S2. Linear regression analysis of carbohydrate concentrations and ratios against the biomass traits in the total above ground biomass in 2012 at Aberystwyth and Harpenden.


Rhodococcus erythropolis to produce bioflocculant. Kinetics of cell growth and bioflocculant production were firstly constructed. After fermentation for 60 h, 0.97 g of bioflocculant with polysaccharides nature was extracted from 1 L of fermentation liquor. Kinetics characteristics showed that cell growth and bioflocculant production could be simulated well with Logistic and Luedeking-Piret equations, respectively. When R. erythropolis was in logarithm growth phase, COD, ammonium, and TP of the potato starch wastewater medium were rapidly down to 1736, 188, and 146 mg/L, respectively, from 7836, 975, and 712 mg/L, while the medium’s exactly pH value was almost not changed. Furthermore, bioflocculant flocculation can be used as an effective pretreatment way for potato starch wastewater, and it was feasible in actual treatment projects in Ronghua Starch Co., Ltd., Sichuan Province.Potato starch wastewater was used as fermentation medium for  Although traditional flocculants were effective and economical in wastewater treatment, their degraded monomers always posed serious health and environmental concerns5. Compared with traditional flocculants, bioflocculant, secreted by microorganisms during their active secretion and cell lysis, has been attracted scientific attention due to its biodegradable and less harmful to human and ecosystems. Literatures reported that the bioflocculant was effective in the treatment of low temperature drinking water6, swine wastewater7, and so on, on laboratory scales.Flocculation technique, an effective method in aggregating colloids, has been widely used in industrial settingsBacillus licheniformis, Lipomyces starkeyi, and Ochrobactium ciceri9. However, there were no comprehensive studies on variation of the fermentation medium’s composition, which directly related to the characteristics of cell growth, metabolites generation, and fermentation kinetics10. It is precisely that the investigation of these elements would be beneficial for improving bioflocculant yield and flocculating activities in practical application10. Besides, high production cost was still the major limitation for bioflocculant in large scale production and commercial applications, and so far, with the aim of commercialization, a considerable effort has gone into reducing the production cost through using some wastes rich in organic matter, nitrogen, and phosphorus. For instance, Guo et al. produced bioflocculant by using swine wastes and rice stover11, Wang et al. produced bioflocculant by using dairy wastewater12, More et al. produced bioflocculant by using excess sludge13.Over time, different kinds of microorganisms and their bioflocculant have been reported, such as 14. Utilization of potato starch wastewater to produce bioflocculant, can reduce the production cost, and further reduce the pollution caused by uncontrolled emissions of this type of wastewater.Potato starch wastewater, usually produced in the manufacturing process of potato starch and some related products, was one of the most serious and unwieldy pollution sources in food industry, in which the large amount of organic pollutants could be used to cultivate microorganisms to produce bioflocculantR. erythropolis to produce bioflocculant by using potato starch wastewater. Based on the optimization of the fermentation process, kinetics of cell growth and bioflocculant production were simulated. Meanwhile, concentrations of COD, ammonium, total phosphorus (TP), and pH value of the potato starch wastewater medium were examined. Furthermore, actual applications of the bioflocculant were discussed.This study effectively applied Paenibacillus polymyxa15, effects of extra phosphate salts, nitrogen source, carbon source, and initial pH value of the potato starch wastewater medium on bioflocculant production were investigated in turn. From Figs 4)2SO4, urea, and yeast extract) were beneficial for bioflocculant yield and flocculating activity, while extra carbon sources  almost have no promotion, compared with the fermentation liquor from the potato starch wastewater medium (0.12 g/L of bioflocculant and 66.8% of flocculating activity can be obtained). Especially, the bioflocculant yield and flocculating activity improved to 0.75 g/L and 78.4% respectively when the total added phosphate salts were 6 g/L (4 g/L of K2HPO4 and 2 g/L KH2PO4), which were further improved to 0.96 g/L and 92.8% respectively when 2 g/L urea was extra added as the nitrogen source. The role of carbon during the bioflocculant production was inconsistent with conclusions reported by Xia et al. and Li et al., in which glucose was the most preferred carbon source for Proteus mirabilis and Bacillus licheniformis16. This may be due to the high-levels of organics in potato starch wastewater. When adjusted the initial pH value in range of 6.5–8.5, R. erythropolis can effectively produce bioflocculant, especially at pH point of 7.5, bioflocculant yield and flocculating activity were appeared as 1.18 g/L and 95.6%, respectively. It is noteworthy that pH range of 6.5–8.5 just covered the pH value of the potato starch wastewater (6.8), so, pH adjustment was not needed from a practical standpoint.Reference to our previous study about the cultivation of 600), and colonies number were increased rapidly to 1.58 g/L, 0.86 and 5.3 × 107 cfu/mL, respectively. Cells entered stationary phase since 60 h, and on 78 h and onward, the cells were in death phase, cell dry weight and density were still increased, while colonies number was reduced. As known to all, cell dry weight contained both the weight of live and death bacteria, so it was increased. The increasing in cell density was due to the increased turbidity after bacteria died in the medium. Colonies number was expressed in the number of viable cells, which was reduced when the cells were entered into death phase, due to the rapid reduction of carbon source, ammonium, and phosphorus, and the cytotoxicity chemicals derived from the accumulation of metabolites in medium17. From Fig. As seen from the cell growth curve in Fig. 9. A similar bioflocculant production style was observed by Aspergillus parasiticus18.Above results reflected that cell growth as well as bioflocculant yield increased sharply in the period of 0–60 h. Both bioflocculant yield and colonies number were reached their peaks at 60 h and then stabilized after 60 h, indicated that bioflocculant production was almost simultaneously accompanied with cell growth. Afterwards, the bioflocculant yield was decreased monotonically to 0.77 g/L at 90 h, which may be due to cell autolysis and the decrease of enzymatic activity11. This was ascribed to the different nutrition components and biophysical environments, which may account for the discrepancy in bioflocculant components19. Gel permeation chromatography indicated that approximate molecular weight of the bioflocculant was 3.85 × 105 Da.Proportions of total sugar and protein contents were detected as 92.3% and 7.6% (w/w), respectively. Further chemical analysis revealed that the total sugar mainly including 49.7% of neutral sugar, 13.2% of uronic acid, 12.4% of amino sugar, and so on, different from our previous research in which bioflocculant was prepared by using swine wastesBacillus subtilis remained its 89% at 100 °C20.Results of thermal stability test showed that the bioflocculant was thermostable and retained more than 89.7% of flocculating activity in a temperature range of 10–70 °C. Further, residual flocculating activities of 80.3% or more could still be achieved after heating for 30 min at 80, 100, and 120 °C, respectively. These thermo-stable characteristics suggested that active ingredients in the bioflocculant were mainly polysaccharide. Literatures reported that in temperature range of 30–120 °C, polysaccharide chains can remain extended and exposed its flocculating sites, and ensure the flocculating activity, for example, flocculating activity of the bioflocculant produced by −1, which was assigned to –OH and –NH221, in accordance with that of NOC-1 (mainly glycoprotein)18. The peak around 1632 cm−1 may be assigned to the –CO stretching in –CONH2 group, and the peaks around 1080 cm−1 were characteristic of C–O groups. The peak at 1405 cm−1 is probably an indication of –COO− symmetric stretching vibration. Typical functional groups of polysaccharide, namely, hydroxyl (–OH), amino (–NH2), carboxyl (C–O) groups, and acylamino (–CONH2) groups were all observed in the FTIR spectrum, revealed that the bioflocculant mainly contains polysaccharide. The FTIR spectrum of this bioflocculant was similar to that of Sun et al.’s research, in which functional groups of a polysaccharide flocculant made from sludge cells directly were investigated21. But the bioflocculant was somehow different from the R. erythropolis sludge bioflocculant (RSF), which was prepared by using sludge and livestock wastewater19, even though the FTIR spectrum of the RSF showed the typical functional groups of polysaccharide, the former exhibited absorption at 582 cm−1, corresponding to the existence of protein.Infrared spectrum in Fig. R. erythropolis was in logarithm growth phase, while in stationary phase (60–78 h), the down-ward trends of COD and ammonium were became slowly and maintained at a low level at last , due to the buffer effect of phosphorus15.During fermentation process, COD and ammonium contents were rapidly down to 1736 and 188 mg/L from 7836 and 975 mg/L, respectively when X0, Xm) were detected as 0.02 and 1.72 g/L, respectively. Cell growth kinetics which described the cell dry weights was obtained from the non-linear fit  were detected as 0.05 and 0.92, respectively. Cell growth kinetics which described the biomass concentrations was obtained from the non-linear fit  was constructed to treat potato starch wastewater, due to the better anti-shock loading capability and system stability. Under the condition of influent average COD of 7836 mg/L, the temperature of 25 °C, continual aerations of 2.5 h and sludge density of 2000–3000 mg/L, the reactor had a good degeneration ability of COD and ammonium in potato starch wastewater, and the corresponding removal rates could reach 65% or more (without addition of bioflocculant) , indicated that the bioflocculant flocculation can be used as an effective pretreatment way for the potato starch wastewater. The improvement of removal rate of COD from 68.2% to 97.3% and ammonium from 66.1% to 94.6% when the 30 mg/L of bioflocculant was applied to combine with the sequencing batch reactor (SBR), indicated that the bioflocculant was feasible in actual projects of potato starch wastewater treatment in Ronghua Starch Co., Ltd., Sichuan Province. Some other bacteria were also effective in pollutants removal from wastewaters, for example, Coelastrella sp. QY01 can remove 90% of ammonium and 90% of TP from aerobically treated swine wastewater after cultivated for 10 days23; Rhodobacter blasticus and Rhodobacter capsulatus can remove 83.3% of COD from aerobically treated swine wastewater after cultivated for 72 h24.Further, from a practical standpoint, at natural pH value of the potato starch wastewater of 6.8, after treated by 30 mg/L bioflocculant, the residual COD, BODPotato starch wastewater used in this study was taken from Ronghua Starch Co., Ltd., Sichuan Province, and its water quality was shown in Table R. erythropolis, deposited in China Center for Type Culture Collection (No. 10543), was specified to produce bioflocculant. Before bioflocculant production, R. erythropolis was first inoculated in 150 mL seed medium consisted of peptone 10 g, beef extracts 3 g, and NaCl 5 g dissolved in 1 L distilled water, and incubated on a reciprocal shaker  at 150 r/min and 30 °C for 24 h. After cultivation, 2.0% v/v of the above inoculums was used to inoculate the potato starch wastewater medium of composition: 1 L potato starch wastewater, 4 g K2HPO4, 2 g KH2PO4, 0.2 g MgSO4, 0.1 g NaCl, and 2 g urea, and incubated in the same procedure to produce bioflocculant. Main characteristics of the potato starch wastewater medium was also shown in Table n No. 1053, was spet al.11, with the following modifications: the fermentation liquor with flocculating components was centrifuged at 3000 r/min for 30 min to obtain supernatant containing slime bioflocculant and pellet containing capsular bioflocculant. To determine the slime bioflocculant, the supernatant was precipitated with 2 volumes of chilled absolute acetone (containing 0.07% β-mercaptoethanol) by incubating the mixture at −20 °C for 4 h, then, the resulting precipitate was collected by centrifugation at 3000 r/min for 30 min and the slime bioflocculant was obtained. To determine the capsular bioflocculant, the pellet was re-suspended in phosphate solution, and heated at 40 °C for 15 min followed by centrifugation at 3000 r/min for 30 min, then, the supernatant was treated in the same the same procedure as the determination of slime bioflocculant to obtain the capsular bioflocculant. Sum of the slime and the capsular was denoted as the bioflocculant produced by Rhodococcus erythropolis in this study.After fermentation for 60 h, the fermentation liquor was obtained, from the whole of which the bioflocculant was extracted by using the methods proposed by Guo 25. Protein content was measured by the Bradford method26. Molar mass was determined by gel permeation chromatography (GPC) using a Hitachi L-6200 system controller. Functional groups were determined by using a Fourier transform infrared spectrophotometer . Thermal stability of the bioflocculant was determined as follows: the bioflocculant was dissolved in a suitable volume of deionized water to achieve an initial flocculating activity of over 90% and divided into 12 aliquots, which were placed at 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, and 120 °C for 30 min, respectively. Subsequently, their flocculating activities were measured. The thermal stability was judged according to the changeable of the flocculating activities.Total sugar content of the purified bioflocculant was determined by the phenol-sulfuric acid method15, in which 4 g/L of kaolin clay suspension was chosen as the suspended solid, whose optical density (OD) were measured with a spectrophotometer  at 550 nm before and after treated by the bioflocculant. The control experiment was conducted in the same manner without adding bioflocculant.Flocculating activity was measured in jar tests referenced to the method reported in our previous publication17. Cell dry weight was determined as follows: the fermentation liquor at different fermentation time was centrifuged at 3000 r/min for 10 min, and the resulting precipitate (strain cell) was collected and then dried in an oven at 105 °C until a constant weight, which was denoted as cell dry weight.Biomass concentration was measured by turbidimetry method using a spectrophotometer  at 600 nm, numbers of colonies were counted using the plate count method27.COD, ammonium, and TP of the potato starch wastewater medium were determined according to the EPA Standard MethodsX0 and Xm were the initial and the maximum cell dry weight (or biomass concentration), respectively; X(t) was the cell dry weight (or biomass concentration) at fermentation time t (g/L); μm was a constant that characterized the maximum specific growth rate (h−1); t was the fermentation time (h).Generally, Logistic equation could nicely describe the dynamic process of cell growth, and was normally given as follows:P was the bioflocculant yield (g/L); m1 and m2 were the equation parameters, they were constants when the fermentation conditions were determined.Luedeking-Piret equation was always applied to simulate the process of bioflocculant production, and was normally given as follows:15, the residual COD, BOD5, ammonium, TP, turbidity, and chroma of the potato wastewater were determined after the pretreatment.Potato starch wastewater from Ronghua Starch Co., Ltd., Sichuan Province was selected to identify the potential of the bioflocculant in actual application. Bioflocculant dose and pH values of the potato starch wastewater were used to optimize the flocculating conditions. A sample of 1.0 L wastewater was poured in a beaker and the pH value was adjusted using 1.0 mol/L NaOH or HCl if necessary. The bioflocculant was then added, and the mixture was stirred at the design agitation speed for 10 min, and then allowed to stand 30 min. Referenced to the treatment process reported in our previous publicationAll the measurements through this study were carried out in triplicates and the average values were presented (with standard error less than 5% of the mean).Supplementary data