Sven Cnattingius
email: sven.cnattingius@ki.se

Tobias Svensson

Fredrik Granath

Anastasia Iliadou

Maternal smoking during pregnancy and risks of

Keywords: pregnancy, prenatal, psychiatry, smoking, suicide

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

Each year, there are around 1 million completed suicides in the world. According to the WHO statistics, suicide is now one of the three leading causes of deaths among adolescents and young adults [START_REF]Suicide Prevention (SUPRE[END_REF]. Psychiatric disorders are commonly present in suicide victims or suicide attempters [START_REF] Nock | Cross-national prevalence and risk factors for suicidal ideation, plans and attempts[END_REF], including adolescent and young victims [START_REF] Bridge | Adolescent suicide and suicidal behavior[END_REF]. Both suicides and psychiatric disorders have strong hereditary components [START_REF] Baldessarini | Genetics of suicide: an overview[END_REF][START_REF] Kendler | Twin studies of psychiatric illness: an update[END_REF], but there are evidence for a familial clustering of suicidal acts even after psychiatric disorders have been taken into account [START_REF] Mann | Neurobiology of suicidal behaviour[END_REF].

Obstetric and neonatal complications may have a long-term influence on offspring mental health, including risks of schizophrenia [START_REF] Hultman | Prenatal and perinatal risk factors for schizophrenia, affective psychosis, and reactive psychosis of early onset: case-control study[END_REF], depression [START_REF] Gale | Birth weight and later risk of depression in a national birth cohort[END_REF], and suicidal acts [START_REF] Mittendorfer-Rutz | Restricted fetal growth and adverse maternal psychosocial and socioeconomic conditions as risk factors for suicidal behaviour of offspring: a cohort study[END_REF][START_REF] Riordan | Perinatal circumstances and risk of offspring suicide. Birth cohort study[END_REF][START_REF] Salk | Relationship of maternal and perinatal conditions to eventual adolescent suicide[END_REF][START_REF] Jacobson | Obstetric care and proneness of offspring to suicide as adults: casecontrol study[END_REF]. Maternal smoking has been recognized as one of the most important risk factors for adverse pregnancy outcomes, including placental complications, fetal growth restriction, preterm birth and associated conditions [START_REF] Cnattingius | The epidemiology of smoking during pregnancy: smoking prevalence, maternal characteristics, and pregnancy outcomes[END_REF]. Women with mental disorders are more often smokers than women without such disorders [START_REF] Diaz | Tobacco smoking behaviors in bipolar disorder: a comparison of the general population, schizophrenia, and major depression[END_REF]. Thus, compared with offspring of women without mental disorders, offspring of women with mental disorders are more likely to be prenatally exposed to smoking [START_REF] Nilsson | Women with schizophrenia: pregnancy outcome and infant death among their offspring[END_REF]. We are unaware of any study investigating if prenatal smoking influences offspring risks of suicidal acts. Prenatal smoking exposure may have a long-term influence on offspring serotonin metabolism. Chronic nicotine exposure reduces serotonin metabolism in animals [START_REF] Olausson | Involvement of serotonin in nicotine dependence: processes relevant to positive and negative regulation of drug intake[END_REF], smokers have reduced serotonin levels in the cerebrospinal fluid [START_REF] Malone | Cigarette smoking, suicidal behavior, and serotonin function in major psychiatric disorders[END_REF], and reduced serotonin levels is associated with increased risks of suicidal acts [START_REF] Hughes | Smoking and suicide: a brief overview[END_REF]. Using a large population-based data set, we studied the association between maternal smoking during pregnancy and risk of completed suicide and suicidal acts in young offspring after taking family history of psychiatric diseases into account. To account for residual family confounding, we also studied risks of suicidal acts in sibling pairs discordant for suicidal acts and prenatal smoking exposure.

Subjects and Method

Data sources

This cohort study is based on linkage of population-based registers from the Swedish National Board of Health and Welfare and Statistics Sweden. Individual record linkage across these registers was possible through the individually unique national registration number, assigned to each Swedish resident.

The Medical Birth Register includes information on 98 to 99% of all deliveries in Sweden. [START_REF]The Swedish Medical Birth Register: a summary of content and quality[END_REF] Starting with the first antenatal visit (generally at 8 to 12 gestational weeks), information is prospectively collected on standardized antenatal, obstetric and neonatal records. Copies of these records are forwarded to the Medical Birth Register after delivery.

Self-reported information on maternal smoking at the first visit to antenatal care has been collected since 1983, using checkboxes for non-daily smoking, 1 to 9 cigarettes per day and at least 10 cigarettes per day, respectively.

The Multigeneration Register contains information on children born in Sweden since 1932 and their parents [START_REF][END_REF]. This registry was used to identify fathers, full and half siblings. The Hospital Discharge Register provides a nation-wide coverage on hospital discharges and diagnoses for psychiatric care determined by the treating physician since 1973 [START_REF]Inpatient diseases in Sweden 1987-2008[END_REF]. The Cause of Death Register contains the date and cause of death on all Swedish residents. The completeness has been estimated to exceed 99% [START_REF]Causes of Death[END_REF]. Diseases and causes of death in the Medical Birth-, Hospital Discharge-and Cause of Death Registers are classified according to the International Classification of Diseases (ICD). The Education Register provides information on highest attained educational level for all Swedish residents [START_REF]Statistics Sweden, Educational attainment of the population[END_REF]. The National Population Register includes dates of death and migration. The study was approved by the research ethics committee at Karolinska Institutet (Stockholm, Sweden).

Outcomes

All individuals surviving until their tenth birth day were followed up with respect to completed determined or undetermined suicide or attempted suicide (ICD-9 codes E950-E959 and E980-E989; ICD-10 codes X60-X84 and Y10-Y34), as recorded in the Cause of Death Register and the Hospital Discharge Register, respectively.

Exposures

From the Birth Register, we retrieved information on maternal smoking and living or not living with the father-to-be at registration to antenatal care (yes/no), mother's age at delivery, parity, mother's country of birth, pregnancy complications (hypertensive diseases [ICD-8 code 637 and ICD-9 code 642] and placental abruption [ICD-8 codes 632.1 and 651.4 and ICD-9 code 641C]), mode of delivery, sex of infant, gestational age, birth weight, birth length and head circumference. Gestational age was based on the last menstrual period, since early ultrasound investigations to date pregnancies were not in regular use. If information about the last menstrual period was lacking, we used information about early second trimester ultrasound, when available. Birth weight-birth length-, and head circumference-for gestational age were standardized according to the Swedish birth weight-, birth length-, and head circumference standards [START_REF] Niklasson | An update of the Swedish reference standards for weight, length and head circumference at birth for given gestational age (1977-1981)[END_REF], and expressed in standard deviation scores (SDS).

Information about paternal age at delivery was retrieved from the Multigeneration Register and information about mother's education was retrieved from the Education Register in 1995.

Information about inpatient psychiatric care for the study population and their first degree relatives (parents, full and half siblings) was derived from the Hospital Discharge Register.

For both study subjects and first degree relatives, such information was dichotomized into having received inpatient care for a mental disorder or not (ICD-8 or ICD-9 codes 290-315 and ICD-10 codes F00-F99). For first degree relatives, we also collected information from the Hospital Discharge and Cause of Death Registers of attempted and completed suicide (ICD-8 and ICD-9 codes E950-959 and E980-989; ICD-10 codes X60-X84 and Y10-Y34). We only included information about psychiatric care and suicidal acts of relatives up to the time of suicidal act of the study subjects.

Statistical Analyses

The study base included 1 449,333 singletons born from 1983 through 1996, who survived until their tenth birth day. Risk time (person-years) was accrued from the time of the 10 th birth day until completed suicide or time for first suicidal act (also including attempted suicide), or censored at the date of first emigration from Sweden, death from another cause, or December 31, 2007.

We used Cox proportional hazard regression models to evaluate associations of maternal smoking with respect to suicidal acts by estimating hazard ratios with 95% confidence intervals (CI). All applied models were stratified by birth year cohort and sex, inplying adjustment for sex, attained age and calendar year of follow-up. For completed suicide, we calculated hazard ratios for the following variables: maternal smoking, maternal and paternal age at delivery, parity, living with the father-to-be or not, mother's country of birth, mother's education, complications during pregnancy (hypertensive diseases and placental abruption), mode of delivery, gestational age, birth weight for gestational age (Table 1), and for inpatient psychiatric care and suicidal acts among relatives as time-dependent co-variates (Table 2).

In multivariate models of the effects of maternal smoking and risks of completed suicide or suicidal acts, we further adjusted for parental characteristics, including maternal and paternal age at delivery, parity, living with infant's father or not at delivery, mother's education and mother's country of birth. In separate analyses, we also adjusted for first occurrence of either inpatient care for psychiatric disorders or suicidal acts in first degree relatives (i.e., mothers, father or siblings).

We also wanted to investigate two possible pathways for an association between maternal smoking and risks of completed suicide and suicidal acts. First, to investigate whether a possible association was mediated by pregnancy or birth characteristics, we adjusted for hypertensive diseases, placental abruption, mode of delivery, gestational age, and birth weight for gestational age. Second, to investigate whether a possible association was mediated by offspring psychiatric disease, we censored subjects at the time for their first admission to psychiatric care. In both these analyses, we also adjusted for parental characteristics and for previous in-patient care for psychiatric diseases or suicidal acts in first degree relatives. All adjustment variables were categorized as presented in Tables 1 and2.

Finally, in order to control for familial confounding, we performed a sib-pair analysis on maternal smoking and risk of suicidal acts. A Cox' regression model stratified on maternal identity numbers was used to estimate the effect of maternal smoking during pregnancy on the risk of suicidal act among the first and second sibling. In order to estimate the effect of smoking, this analysis only utilizes the sibling pairs that are discordant for maternal smoking during pregnancy and where at least one of the sibs has an event. The time scale used is attained age starting at the age of 10. This implies that only pairs where the unaffected sib is under observation at the age of the other sibling's suicidal act are informative. Potential confounders in this analysis were sibling order, calendar time at follow-up, and sex. When estimating the effects of these potential confounding factors, pairs concordant for maternal smoking are also informative. Thus, in the adjusted model, we included all sibling pairs discordant for suicidal acts, and adjusted for sibling order, sex and birth year cohort as categorical variables. Effect modification of sex and attained age, categorized by below 18 years and from 18 years of age, were tested by likelihood ratio tests. The estimated effects are presented as hazard ratios with 95% confidence intervals. All analyses were performed by SAS ® version 9.2.

Results

In the cohort of 1 449,333 singletons born from 1983 through 1996, who survived until their tenth birth day, 374 committed suicide during the time of follow-up, of whom 322 were assessed as determined and 52 as undetermined suicides.

Compared with offspring to non-smokers, both offspring to moderate and heavy smokers were at increased risk of completed suicide (Table 1). Other parental factors associated with an increased risk of suicide in offspring were teenage parenthood, if the mother was not living with the father-to-be in early pregnancy, and if the mother was born in another Nordic country (i.e., Denmark, Finland, Iceland or Norway). Offspring to mothers with medium-high (13 to 14 years) education and to mothers giving birth at 30 to 34 years were at reduced risk of suicide. Compared with females, males had an increased risk of suicide, but other birth or pregnancy characteristics did not influence risk. There were no associations between birth length-or head circumference for gestational age and risk of suicide (data not shown).

Table 2 shows risks of completed suicide with respect to in-patient psychiatric care and suicidal acts in first degree relatives. If a first degree relative previously had been hospitalized for a psychiatric disorder or performed a suicidal act (attempted or completed suicide), the risk of completed suicide among study subjects was generally substantially increased.

Compared with offspring to non-smoking mothers, the risk of completed suicide was increased by around 80% among offspring to smokers after adjusting for parental characteristics (Table 3a). When we also adjusted for previous psychiatric history and suicidal acts among first degree relatives, these risk estimates were reduced. We used identical analyses to analyze maternal smoking during pregnancy and offspring risk of suicidal acts (i.e., completed or attempted suicide), including 350 offspring with completed suicide and 8,060 offspring with attempted suicide and information on maternal smoking (Table 3b).

There was a dose-response relationship between amount smoked and risks of suicidal acts, but these risks were reduced when we also adjusted for previous psychiatric history and suicidal acts among first degree relatives.

Next, we wanted to investigate possible mediating factors for the association between maternal smoking and offspring risk of completed suicide or suicidal acts. First, we investigated whether the smoking-related risks were mediated by smoking-related pregnancy and birth complications. However, after adjusting for such complications, there was no attenuation in the smoking-related risks of completed suicide (adjusted hazard ratios for 1-9 cigarettes and >10 cigarettes per day were 1.67 [95% CI: 1.28, 2.17] and 1.53 [95% CI: 1.12, 2.10], respectively) or suicidal acts (adjusted hazard ratios for 1-9 cigarettes and >10 cigarettes per day were 1.29 [95% CI: 1.22, 1.36] and 1.50 [95% CI: 1.41, 1.59], respectively). Second, to investigate whether the association was mediated by psychiatric diseases in study subjects, we censored subjects admitted to psychiatric care at the date of their first admission. Of individuals with no previous admission for psychiatric care, there were in all 280 individuals with completed suicide and 7,291 individuals with suicidal acts, respectively. After censoring subjects admitted to psychiatric care, we found that the smoking-related risks remained increased: for completed suicide, the adjusted hazard ratios for 1-9 cigarettes and >10 cigarettes per day were 1.81 (95% CI: 1.34, 2.43) and 1.38 (95% CI: 1.09, 2.03), respectively, and for suicidal acts, corresponding adjusted hazard ratios were 1.28 (95% CI: 1.20, 1.36) and 1.50 (95% CI: 1.40, 1.59), respectively. Finally, in order to control for familial confounding, we analyzed the influence of maternal smoking on suicidal acts in sibling pairs. In 334,505 sibling pairs, there were 396 sibling pairs who were discordant for suicidal acts (12 completed suicides and 384 suicide attempts) and for maternal smoking (Table 4). Of cases, 37.1% were non-exposed to maternal smoking during pregnancy, while corresponding rate among sibling controls was 38.1%. Cases and sibling controls also had similar rates of prenatal exposure to moderate (30.1% in cases and 28.5% in controls) and heavy smoking (8.1% in cases and 8.6% in controls). In the sibling case-control analyses, prenatal smoking exposure was not associated with increased risks of suicidal acts (Table 4). There was no interaction between maternal smoking and offspring sex or attained age with respect to risks of suicidal acts (P=0.25 and P =0.30, respectively). Thus, in the sibling analysis, there was no association between maternal smoking and risk of suicidal acts in neither boys nor girls, and no association between maternal smoking and suicidal acts below 18 years or from 18 years and onwards (data not shown).

Discussion

In this nation-wide study, we initially found that maternal smoking during pregnancy was associated with increased risks of completed suicide and suicidal acts in young offspring.

These risks were reduced when we adjusted for effects of family history of psychiatric disorders and suicidal acts requiring inpatient care. However, using data on hospital admissions for such diagnoses does not allow us to capture the effect of family history of psychiatric illness or psychopathology not requiring inpatient care. In analysis within sibling pairs, we found no association between prenatal smoking exposure and risk of suicidal acts in offspring. This suggests that the association between prenatal smoking exposure and risk of suicidal acts obtained in the cohort analysis is explained by unmeasured familial factors.

Analyses restricted to sibling pairs discordant for suicidal acts, provide control for familial (genetic and environmental) factors shared by the siblings. Full siblings share on average 50% of their genes, but the segregation of alleles to different eggs is a random process. Thus, sibling analysis should account for genetic confounding. A hereditary component has been suggested for suicidal acts [START_REF] Bridge | Adolescent suicide and suicidal behavior[END_REF] and smoking [START_REF] Schnoll | Genetics and smoking behavior[END_REF], and the search for candidate genes has recently started for both suicidal [START_REF] Hawton | Suicide[END_REF][START_REF] Currier | Stress, genes and the biology of suicidal behavior[END_REF][START_REF] Rujescu | Molecular genetic findings in suicidal behavior: what is beyond the serotonergic system?[END_REF] and smoking [START_REF] Schnoll | Genetics and smoking behavior[END_REF] behaviors. Sibling analysis also control for important environmental factors shared by the siblings. Both smoking and suicidal acts are associated with family dysfunction, other life-style habits, psychological problems and psychiatric diseases not requiring in-patient care [START_REF] Bridge | Adolescent suicide and suicidal behavior[END_REF][START_REF] Diaz | Tobacco smoking behaviors in bipolar disorder: a comparison of the general population, schizophrenia, and major depression[END_REF][START_REF]Factors influencing tobacco use among women Women and smoking[END_REF][START_REF] Gilman | Social factors, psychopathology, and maternal smoking during pregnancy[END_REF]. Similar to the present findings, prenatal smoking exposure has recently been associated with increased risks of both attention deficit hyperactivity disorder (ADHD) and low intellectual performance in cohort analyses, but not in analysis within sibling pairs, suggesting underlying familial confounding [START_REF] Lindblad | ADHD after fetal exposure to maternal smoking[END_REF][START_REF] Lundberg | Maternal smoking during pregnancy and intellectual performance in young adult Swedish male offspring[END_REF].

One serious concern that cannot be addressed in the sibling analysis, is the validity of selfreported information about smoking exposure in early pregnancy. The validity of selfreported smoking is generally high, but is lower among women stating that they recently changed smoking habits [START_REF] George | Self-reported nicotine exposure and plasma levels of cotinine in early and late pregnancy[END_REF]. We only had information on smoking in early pregnancy, and during the 1980's in Sweden, around 10% of these smokers stopped smoking later during pregnancy [START_REF] Cnattingius | Who continues to smoke while pregnant?[END_REF]. Women who stated that they only smoked in one pregnancy may be more likely to temporarily stop smoking in the other pregnancy (and thus be recorded as nonsmokers), and later during pregnancy resume smoking [START_REF] Cnattingius | Who continues to smoke while pregnant?[END_REF]. Moreover, we cannot rule out differential misclassification of smoking exposure between mothers from dysfunctional and non-dysfunctional families. We also lack information on other specific exposures that may be related to maternal smoking during pregnancy. For example, we had no information on alcohol use or infections.

We also investigated two possible biological pathways by which prenatal smoking exposure may influence offspring risk of suicidal acts. First, smoking is causally associated with reduced fetal growth [START_REF] Kramer | Intrauterine growth and gestational duration determinants[END_REF], and low birth weight has previously been associated with suicidal acts [START_REF] Mittendorfer-Rutz | Restricted fetal growth and adverse maternal psychosocial and socioeconomic conditions as risk factors for suicidal behaviour of offspring: a cohort study[END_REF][START_REF] Riordan | Perinatal circumstances and risk of offspring suicide. Birth cohort study[END_REF]. However, we found no evidence that birth weight for gestational age (a measure of fetal growth) was associated with risk of suicide, or that the association between prenatal smoking exposure and risk of suicidal acts was attenuated after taking birth weight for gestational age into account. Second, maternal smoking has in some studies been associated with offspring risks of psychiatric disorders [START_REF] Huizink | Maternal smoking, drinking or cannabis use during pregnancy and neurobehavioral and cognitive functioning in human offspring[END_REF], which in turn is associated with high risks of suicidal acts among young men and women [START_REF] Bridge | Adolescent suicide and suicidal behavior[END_REF]. We found no support for the hypothesis that a possible association between prenatal smoking exposure and offspring risk of suicide is mediated by smoking-related effects of developing psychiatric diseases requiring inpatient care.

Strengths of the present investigation include the population-based design, prospectively collected information about smoking habits in early pregnancy, and that we were able to take family history of psychiatric diseases into account. Importantly, the analyses of prenatal smoking exposure within siblings provide control for confounding by unmeasured familial factors shared by the siblings.

Maternal smoking during pregnancy is in many countries recognized as the most important preventable risk factor for an unsuccessful pregnancy outcome. Much less is known about possible long-term effects of prenatal smoking exposure [START_REF] Cnattingius | The epidemiology of smoking during pregnancy: smoking prevalence, maternal characteristics, and pregnancy outcomes[END_REF][START_REF] Huizink | Maternal smoking, drinking or cannabis use during pregnancy and neurobehavioral and cognitive functioning in human offspring[END_REF]. Our results support the importance of familial factors for the association between prenatal smoking exposure and offspring risk of suicidal acts. Our discrepant results, when using cohort and sibling pair analyses, also emphasize the importance of study design when investigating long-term effects of prenatal smoking exposure, such as offspring mental health.

Table 1 .

 1 Numbers and Hazard Ratios (HR) of Completed Suicide as a function of Parental, Pregnancy and Birth

	Characteristics.
	Characteristic	Cohort		Completed suicide
		Number	(%)	Number	HR a	(95% CI)
	Parental characteristics
	Maternal smoking b
	None c	1 027,718	70.9	196	1.00
	1-9 cigarettes per day	207,419	14.3	92	1.85 (1.45-2.37)
	>10 cigarettes per day	126,717	8.7	62	1.97 (1.48-2.62)
	Missing information	87,479	6.0	24
	Maternal age (years)
	-19	40,628	2.8	29	2.47 (1.65-3.69)
	20-24	321,903	22.2	102	1.19 (0.92-1.54)
	25-29 c	540,483	37.3	133	1.00
	30-34	374,163	25.8	65	0.73 (0.54-0.98)
	>35	172,156	11.9	45	1.11 (0.79-1.56)
	Paternal age (years)
	-19	6,190	0.4	6	4.02 (1.77-9.17)

b Information was retrieved at time of registration to antenatal care.

c

Table 2 .

 2 Numbers and Hazard Ratios (HR) of Completed Suicide as a function of Previous Psychiatric InpatientCare and Suicidal Acts in First Degree Relatives.

	Characteristic	Cohort			Completed suicide
		N	(%)	N	HR a	(95% CI))
	Psychiatric care in first degree relatives
	No admission b	1 204,819 83.1	245 1.00
	Admission of one relative
	Mother	68,144	4.7		30 2.30	(1.59-3.35)
	Father	71,040	4.9		42 2.84	(2.05-3.94)
	Sibling	65,270	4.5		28 1.96	(1.32-2.89)
	Admission of at least two relatives	40,060	2.8		29 3.31	(2.25-4.87)
	Suicidal acts in first degree relatives
	No suicidal act b	1 353,843 93.4	305 1.00
	Suicidal act of one relative
	Mother	34,279	2.4		25 3.37	(2.24-5.07)
	Father	27,196	1.9		11 1.72	(0.94-3.14)
	Sibling	26,077	1.8		21 3.29	(2.11-5.12)
	Suicidal act of at least two relatives	7,938	0.5		12 6.91	(3.97-12.05)

Table 3a .

 3a Maternal Smoking during pregnancy and Offspring Risk of Completed Suicide.

			Hazard Ratio a (95 % CI)
		Model 1	Model 2 b
	Maternal smoking
	(cigarettes per day)
	0 c	1.00	1.00
	1-9	1.81 (1.40-2.35)	1.67 (1.29-2.16)
	>10	1.78 (1.31-2.42)	1.54 (1.12-2.10)

a Both models are adjusted for sex, age, calendar period, maternal and paternal age at delivery, parity, living with infant's father, mother's education and mother's country of birth. b Also adjusted for first event of either previous inpatient care for psychiatric diseases or suicidal acts in first degree relatives c Reference category 28

Table 3b .

 3b Maternal Smoking during pregnancy and Offspring Risk of Suicidal Acts. Sucidal acts include completed and attempted suicide. N denotes number of individuals with suicidal acts.Also adjusted for first event of either previous inpatient care for psychiatric diseases or suicidal acts in

				Hazard ratio a (95 % CI)
		N	Model 1	Model 2 b
	Maternal smoking
	(cigarettes per day)
	0 c	4,898	1.00	1.00
	1-9	1,925	1.42 (1.37-1.50)	1.28 (1.21-1.35)
	>10	1,587	1.76 (1.66-1.87)	1.48 (1.39-1.57)
	Missing	845
	first degree relatives
	c Reference category
				29

a Both models are adjusted for sex, age, calendar period, maternal and paternal age at delivery, parity, living with infant's father, mother's education and mother's country of birth. b

Table 4 .

 4 Associations between maternal smoking during pregnancy and suicidal acts in offspring discordant for suicidal a

	Sibling controls
	Maternal smoking	Hazard

a Analysis includes siblings discordant for suicidal acts and maternal smoking exposure. b Analysis includes siblings discordant for suicidal acts. Hazard ratios are adjusted for offspring sex, sibling order and birth c Reference category

Funding

The study was supported by grants from the Swedish Research Council (Project numbers K2007-70P-20518-01-4 and K2007-70K-20510-01-4).

a Hazard Ratios (HR) are adjusted for sex, age and calendar period.


Mehdi Jonoobi

Jalaluddin Harun

Aji P Mathew

Kristiina Oksman
email: kristiina.oksman@ltu.se

Mechanical properties of cellulose nanofiber (CNF) reinforced polylactic acid (PLA) prepared by twin screw extrusion

Keywords: A. Nanocomposites, Cellulose, B. Mechanical properties, C. Modeling, D. Dynamic mechanical thermal analysis, E. Extrusion

The aim of this study was to develop cellulose nanofiber (CNF) reinforced polylactic acid (PLA) by twin screw extrusion. Nanocomposites were prepared by premixing a master batch with high concentration of CNFs in PLA and diluting to final concentrations (1, 3, 5 wt%) during the extrusion. Morphology, mechanical and dynamic mechanical properties (DMA) were studied theoretically and experimentally to see how different CNF concentrations affected the composites' properties. The tensile modulus and strength increased from 2.9 GPa to 3.6 GPa and from 58 MPa to 71 MPa, respectively, for nanocomposites with 5 wt% CNF.

The DMA results were also positive; the storage modulus increased for all nanocomposites compared to PLA; being more significant in the high temperature region (70°C). The addition of nanofibers shifted the tan delta peak towards higher temperatures. The tan delta peak of the PLA shifted from 70°C to 76°C for composites with 5 wt% CNF.

Introduction

Polylactic acid (PLA) is a biodegradable, thermoplastic, aliphatic polyester which can be derived from renewable resources such as starch and is a sustainable alternative to petrochemical-derived products. PLA has been found to have good stiffness and strength and is being used in several applications, such as food packaging, water and milk bottles, degradable plastic bags as well as in automotive applications. The products made from PLA are bio-degradable and are found to fully disappear in less than 30 days in ideal conditions [START_REF] Meinander | Polylactide-degradable polymers for fibres and films[END_REF][START_REF] Oksman | Natural Fibres as Reinforcement in Polylactic Acid (PLA) Composites[END_REF][START_REF] Mathew | Mechanical properties of biodegradable composites from poly lactic acid (PLA) and microcrystalline cellulose (MCC)[END_REF].

Cellulose nanofibers (CNFs) and nanowhiskers (CNWs) derived from renewable biomass have attracted much interest as an alternative to micro-sized reinforcements in composite materials . One challenge when using nanocelluloses is that, due to their polar surfaces, it is difficult to disperse them uniformly in a non-polar medium. This might be the reason why the processing of cellulose nanocomposites was first limited to solvent casting, where water soluble or dispersive polymers were the most common matrices [START_REF] Helbert | Thermoplastic Nanocomposites Filled with Wheat Straw Cellulose Whiskers[END_REF][START_REF] Heux | Nonflocculating and chiralnematic self-ordering of cellulose microcrystals suspensions in nonpolar solvents[END_REF][START_REF] Mathew | Morphological investigation of nanocomposites from sorbitol plasticized starch and tunicin whiskers[END_REF][START_REF] Morin | Nanocomposites of chitin nanofibrils from Riftia tubes and poly(caprolactone)[END_REF][START_REF] Gousse | Stable suspensions of partially silylated cellulose whiskers dispersed in organic solvents[END_REF][START_REF] Samir | Crosslinked nanocomposite polymer electrolytes reinforced with cellulose whiskers[END_REF][START_REF] Samir | Review of recent research into cellulosic whiskers, their properties and their application in nanocomposite field[END_REF][START_REF] Kvien | Orientation of Cellulose Nanowhiskers in Polyvinyl Alcohol (PVA)[END_REF][START_REF] Petersson | Structure and thermal properties of polylactic acid/cellulose whiskers nanocomposite materials[END_REF][START_REF] Chakraborty | Reinforcing potential of wood pulp-derived microfibres in a PVA matrix[END_REF]. However, the development of other, more flexible and industrially viable processing techniques is necessary to promote commercialization of these materials. Therefore, several interesting processing methods have recently been reported for these materials [START_REF] Oksman | Manufacturing process of cellulose whiskers/ polylactic acid nanocomposites[END_REF][START_REF] Mathew | The structure and mechanical properties of cellulose nanocomposites prepared by twin screw extrusion[END_REF][START_REF] Bondeson | Dispersion and characteristics of surfactant modified cellulose whiskers nanocomposites[END_REF][START_REF] Bondeson | Polylactic acid/cellulose whisker nanocomposites modified by polyvinyl alcohol[END_REF][START_REF] Bondeson | All Cellulose Nanocomposites Produced by Extrusion[END_REF][START_REF] Okubo | Multi-scale hybrid biocomposite: Processing and mechanical characterization of bamboo fiber reinforced PLA with microfibrillated cellulose[END_REF][START_REF] Iwatake | Cellulose nanofiber-reinforced polylactic acid[END_REF][START_REF] Suryanegara | The effect of crystallization of PLA on the thermal and mechanical properties of microfibrillated cellulose-reinforced PLA composites[END_REF][START_REF] Wang | Dispersion of soybean stock-based nanofiber in a plastic matrix[END_REF][START_REF] Seydibeyo Lu | Novel nanocomposites based on polyurethane and microfibrillated cellulose[END_REF][START_REF] Nakagaito | The effect of morphological changes from pulp fiber towards nano-scale fibrillated cellulose on the mechanical properties of high-strength plant fiber based composites[END_REF][START_REF] Nakagaito | Production of microfibrillated cellulose (MFC)-reinforced polylactic acid (PLA) nanocomposites from sheets obtained by a papermaking-like process[END_REF][START_REF] Petersson | Biopolymer based nanocomposites: Comparing layered silicates and microcrystalline cellulose as nanoreinforcements[END_REF].

One of the developed processing methods has been melt compounding [START_REF] Oksman | Manufacturing process of cellulose whiskers/ polylactic acid nanocomposites[END_REF][START_REF] Mathew | The structure and mechanical properties of cellulose nanocomposites prepared by twin screw extrusion[END_REF][START_REF] Bondeson | Dispersion and characteristics of surfactant modified cellulose whiskers nanocomposites[END_REF][START_REF] Bondeson | Polylactic acid/cellulose whisker nanocomposites modified by polyvinyl alcohol[END_REF][START_REF] Bondeson | All Cellulose Nanocomposites Produced by Extrusion[END_REF][START_REF] Okubo | Multi-scale hybrid biocomposite: Processing and mechanical characterization of bamboo fiber reinforced PLA with microfibrillated cellulose[END_REF][START_REF] Iwatake | Cellulose nanofiber-reinforced polylactic acid[END_REF][START_REF] Suryanegara | The effect of crystallization of PLA on the thermal and mechanical properties of microfibrillated cellulose-reinforced PLA composites[END_REF]. The melt compounding of cellulose nanocomposites presents several challenges [START_REF] Oksman | Manufacturing process of cellulose whiskers/ polylactic acid nanocomposites[END_REF]. The major difficulties are to feed the nanocelluloses into the extruder and achieve uniform dispersion in the polymer's matrix. The nanocelluloses have a very high surface area and have a tendency to aggregate when dried. That can be avoided by first mixing them in a suitable medium, either in a liquid, which is then fed into the extruder, or first dried and then extruded [START_REF] Oksman | Manufacturing process of cellulose whiskers/ polylactic acid nanocomposites[END_REF][START_REF] Mathew | The structure and mechanical properties of cellulose nanocomposites prepared by twin screw extrusion[END_REF][START_REF] Bondeson | Dispersion and characteristics of surfactant modified cellulose whiskers nanocomposites[END_REF][START_REF] Bondeson | Polylactic acid/cellulose whisker nanocomposites modified by polyvinyl alcohol[END_REF][START_REF] Bondeson | All Cellulose Nanocomposites Produced by Extrusion[END_REF][START_REF] Okubo | Multi-scale hybrid biocomposite: Processing and mechanical characterization of bamboo fiber reinforced PLA with microfibrillated cellulose[END_REF][START_REF] Iwatake | Cellulose nanofiber-reinforced polylactic acid[END_REF][START_REF] Suryanegara | The effect of crystallization of PLA on the thermal and mechanical properties of microfibrillated cellulose-reinforced PLA composites[END_REF][START_REF] Wang | Dispersion of soybean stock-based nanofiber in a plastic matrix[END_REF].

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Extensive studies of nanocelluloses as reinforcement in PLA have been carried out and the nanocomposites have been prepared by solvent casting as well as melt compounding [START_REF] Oksman | Manufacturing process of cellulose whiskers/ polylactic acid nanocomposites[END_REF][START_REF] Mathew | The structure and mechanical properties of cellulose nanocomposites prepared by twin screw extrusion[END_REF][START_REF] Bondeson | Dispersion and characteristics of surfactant modified cellulose whiskers nanocomposites[END_REF][START_REF] Bondeson | Polylactic acid/cellulose whisker nanocomposites modified by polyvinyl alcohol[END_REF]. In the first study, a slurry of plasticizer and CNWs was pumped into the melt PLA during extrusion to solve the feeding problem and improve the dispersion of CNWs in PLA [START_REF] Oksman | Manufacturing process of cellulose whiskers/ polylactic acid nanocomposites[END_REF]. The results showed that the CNWs improved the mechanical properties of the PLA, but the used additives had a negative impact. In the next study, cellulose microfibers (CMFs) were used and compared to CNWs [START_REF] Mathew | The structure and mechanical properties of cellulose nanocomposites prepared by twin screw extrusion[END_REF]. The results showed limited improvements in the mechanical properties and agglomerated CMFs in PLA. In the next study, PLA nanocomposites were successfully processed by extrusion using a surfactant, which facilitated the dispersion of nanowhiskers in PLA matrix [START_REF] Bondeson | Dispersion and characteristics of surfactant modified cellulose whiskers nanocomposites[END_REF]. In this case, both liquid feeding and dry feeding of CNWs into the polymer melt were attempted [START_REF] Bondeson | Dispersion and characteristics of surfactant modified cellulose whiskers nanocomposites[END_REF]. Later on, polyvinyl alcohol was tested as a carrier polymer for CNWs and compounded with PLA during extrusion [START_REF] Bondeson | Polylactic acid/cellulose whisker nanocomposites modified by polyvinyl alcohol[END_REF]. In this case, PVA formed CNW-rich domains in the continuous PLA matrix [START_REF] Bondeson | Polylactic acid/cellulose whisker nanocomposites modified by polyvinyl alcohol[END_REF], which led to relatively small improvements in mechanical properties.

Okubo and co-workers [START_REF] Okubo | Multi-scale hybrid biocomposite: Processing and mechanical characterization of bamboo fiber reinforced PLA with microfibrillated cellulose[END_REF] have also studied the process and properties of PLA nanocomposites, where the PLA water slurry was premixed with microfibrillated cellulose (MFC) and processed using a roller mixer followed by drying and injection molding. They reported that the addition of 1 and 2 wt% of MFC had a positive impact on the modulus as well as on composites' fracture energy, but the strength did not show any improvements. The well-dispersed fibrils with lower concentration improved the composites' modulus by 31% and the fracture energy by almost 200% [START_REF] Okubo | Multi-scale hybrid biocomposite: Processing and mechanical characterization of bamboo fiber reinforced PLA with microfibrillated cellulose[END_REF].

Yano and co-workers [START_REF] Iwatake | Cellulose nanofiber-reinforced polylactic acid[END_REF][START_REF] Suryanegara | The effect of crystallization of PLA on the thermal and mechanical properties of microfibrillated cellulose-reinforced PLA composites[END_REF] have also studied PLA nanocomposite reinforced with cellulose microfibrils and have reported promising results. The microfibrils were mixed with PLA in an organic solvent, and then roller-mixed to attain uniform dispersion of the cellulose fibrils. The Young's modulus and tensile strength of PLA increased using the microfibrils as reinforcements, being highest at 10 wt%, when the modulus and strength increased by 40% and 25%, respectively.

In the current study, nanofibers separated from kenaf pulp were used as reinforcing phase for PLA matrix. Nanocomposites were prepared using a two-step process: master batch preparation using a solvent mixture followed by extrusion process and injection molding. The effect of nanofiber content (1, 3, 5 wt%) on the composites' mechanical properties and thermal stability was evaluated theoretically and experimentally. Microscopic analysis was used to study of nanofibers' morphology and surface fracture of nanocomposites.

Experimental

Materials

Matrix: Poly (lactic acid) (PLA), ESUN™, extrusion grade was supplied by Shenzhen Bright China Industrial Co., China. The used PLA has a density of 1.24 g/cm 3 and the melt flow index (MFI) of 20 g/10 min at 190°C/ 2.16 kg, reported by the material supplier.

Reinforcement: Cellulose nanofibers were isolated from kenaf pulp. The details of the pulping process have been reported earlier by us [START_REF] Jonoobi | Chemical composition, crystallinity and thermal degradation of bleached and unbleached kenaf bast (Hibiscus cannabinus) pulp and nanofibers[END_REF]. The kenaf pulp was immersed in distilled water for 8 h. Diluted suspension: fiber concentration of 0.5% was distributed in the water using high shear mixing, Silverson L4RT (England), at 4000 rpm for 20 min. The suspension was grinded using a ultra-fine grinder, MKCA 6-3 from Masuko (Japan) at 3500 rpm in contact mode for approx. 15 min. to obtain cellulose nanofibers. Figure 1 shows the AFM images of the isolated nanofibers, indicating that the fibrillation process was successful and the pulp was isolated to an entangled network of nanofibers. The diameters of the fibers were measured using Nanoscope V software, showing that the diameters ranged from 40 to 70 nm, and the length was estimated to be several micrometers, from the AFM images .

Chemicals: Chloroform and acetone were purchased from Merck (Darmstadt, Germany).

Both of the chemicals were used in master batch preparation in order to dissolve PLA.

Processing of nanocomposites

Nanocomposites were prepared using a two-step process: master batch preparation using a solvent mixture followed by extrusion process.

Preparation of the master batch

Adequate amount of PLA (refer to Table 1) for each material was weighed and completely dissolved in a solvent mixture of acetone and chloroform in the ratio of 9:1 by stirring at 55°C. The nanofibers in aqueous medium were solvent-exchanged to acetone by a series of centrifuging and re-dispersing steps. After that, different mixtures of PLA and nanofibers were prepared by mixing the acetone-based nanofiber suspensions with dissolved PLA (see Table 1). Then, the mixture was casted in Petri-dishes and allowed to evaporate at room temperature overnight followed by oven vacuum-drying at 55°C for 8 h. The composite films obtained from the casting were crushed using a kitchen blender until a particulate material was obtained.

Extrusion process and sample preparation

Nanocomposites were manufactured using a twin screw extruder, Coperion W&P ZSK 18 MEGALab (Stuttgart, Germany), with a screw speed of 120 rpm and the temperature profile was varied from 165°C at the feeding zone to 200°C at the die. The crushed master batch was dry-mixed with PLA, diluting it to 1, 3 and 5 wt% nanofiber contents. The mixtures were vacuum-dried at 55°C for 8 h prior to the process whereby the PLA and its nanocomposites were compounded, followed by the pelletizing step. The formulations of prepared materials are presented in Table 1.

The extruded pellets were dried at 55°C for 6 h prior to injection molding (Haake MiniJet II) of the test specimens according to ASTM, D638 type 5, testing method. The barrel ACCEPTED MANUSCRIPT temperature of the injection molding machine was set at 200°C. The injection pressure was kept constant at 400 bar and the mold temperature was 70°C. The injection-molded specimens were used for evaluation of the mechanical and thermal properties.

Characterization

Microscopy

The microstructures of nanocomposites were investigated using scanning electron microscopy (SEM). Fractured surfaces from tensile testing were analyzed in a Jeol, JSM-6460 (Japan). The acceleration voltage was 5 kV and samples were sputter-coated with gold to avoid charging.

Tensile testing

The tensile tests were performed using a universal testing machine, Hounsfield H25KS, (United Kingdom), with a load cell of 10 kN and with a crosshead speed of 3 mm/min. The injection-molded specimens (ASTM, D638 type 5) were used for tensile testing and the gauge length was 30 mm. The elastic modulus was calculated from the initial part of the slope from stress-strain curves. At least five test samples were tested for each material and the average values are presented.

Dynamic mechanical properties (DMA)

The dynamic mechanical properties of the nanocomposites were carried out using the dynamic mechanical analyzer, DMA Q800 TA Instruments (New Jersey, USA), in tensile mode. The measurements were performed at a constant frequency of 1 Hz with a temperature range from 25°C to 110°C at a heating rate of 3°C/ min. The test specimen dimensions were approx. 2.0 x 5.0 x 30.0 mm (thickness x width x length). The presented data is based on three measurements.

Results and discussion

Nanocomposite morphology

SEM micrographs of fractured surfaces of the PLA and its nanocomposites are presented in Figure 2. The aim was to investigate if any CNF aggregates were visible in the fractured surfaces. Compared to pure PLA, the surface of the PLA-CNF1 and PLA-CNF3 nanocomposites showed no significant differences, but the nanocomposite with 5 wt% CNF (PLA-CNF5), had a rougher surface compared to those with lower nanofiber contents (Fig 2d). A more detailed micrograph of this composite is shown is Figure 3. The image shows small areas which are attributed to small aggregates of CNFs. Generally, a relatively good dispersion was achieved in composites with low fiber contents (1 and 3 wt%).

Mechanical properties

Figure 4 shows typical stress-strain curves of neat PLA and PLA-CNF composites with different fiber contents and Table 2 shows the average values and standard deviations of the mechanical properties. The tensile properties indicated than the strength and modulus were improved with increased nanofiber contents. The modulus of the PLA was increased from 2.9 GPa to 3.6 GPa with the addition of 5 wt% nanofibers, which corresponds to a 24% increase.

Similarly, a 21% increase in tensile strength was observed for PLA-CNF5 composites compared to pure PLA. Table 2 shows that the average value of maximum strain was reduced by increased nanofiber contents. The positive impact of the nanofibers especially in the composite strength indicated that the used nanofibers have good mechanical properties and that there is interaction between the PLA and the CNFs. However the large standard deviations especially on the modulus and strain values indicated that the nanofibers were not homogeneously distributed.

Yano and co-workers reported similar improvements for PLA-nanocomposites with 5 wt% fiber content [START_REF] Iwatake | Cellulose nanofiber-reinforced polylactic acid[END_REF][START_REF] Suryanegara | The effect of crystallization of PLA on the thermal and mechanical properties of microfibrillated cellulose-reinforced PLA composites[END_REF].

In order to see the reinforcing potential of CNFs, common theoretical models developed by Halpin-Tsai and Krenchel were used and compared to experimental data.

The theoretical modulus for the composites (E comp ) based on the Halpin-Tsai equation given below, see Eq (1) -Eq (3) [START_REF] Agarwal | Analysis and performance of fibre composites[END_REF].

E comp = E m (1+ Ø) / (1-Ø) (1) = (E r / E m -1)/(E r / E m + ) (2)
E m is the modulus of the matrix, E r is the modulus of the reinforcements and Ø is the volume fraction. The Halpin-Tsai equation is usually used for aligned fiber composites, but it has also been used before to predict the modulus of nanocomposites [START_REF] Helbert | Thermoplastic Nanocomposites Filled with Wheat Straw Cellulose Whiskers[END_REF][START_REF] Petersson | Biopolymer based nanocomposites: Comparing layered silicates and microcrystalline cellulose as nanoreinforcements[END_REF].

= ( 2 x l) / d (3)
is a shape parameter, depending on the geometry and aspect ratio of the reinforcement.

The the length of the nanofibers was predicted to be 10 µm and the diameter to be 100 nm.

The diameter value is based on our microscopy study (AFM) where the measured diameters were below 100 nm while the length is estimated from the AFM study considering that nanofibers are several micrometers in length though the exact measurement of the length is not possible because of the entangled structure.

The modulus prediction by Krenchel [START_REF] Krenchel | Fibre reinforcement[END_REF] might be a more reasonable model for the CNF composites prepared by compounding and injection molding because we do not expect aligned nanofibers after the injection molding, but rather that they are randomly distributed in plane. In this case the modulus is predicted by following Eq (4) [START_REF] Krenchel | Fibre reinforcement[END_REF], which have also been used for carbon nanotube composites [START_REF] Río | Influence of single-walled carbon nanotubes on the effective elastic constants of poly(ethylene terephthalate)[END_REF].

E comp = E m ( 1-Ø )+ l o E r Ø (4)
l is the length correction factor and o orientation factor, being 3/8 when the fibers are oriented randomly in plane. The l is calculated from the following expression Eq [START_REF] Heux | Nonflocculating and chiralnematic self-ordering of cellulose microcrystals suspensions in nonpolar solvents[END_REF][START_REF] Mathew | Morphological investigation of nanocomposites from sorbitol plasticized starch and tunicin whiskers[END_REF],

l = 1-((tanh ( l / 2) / ( l / 2 ) (5) = 1 / r ( E m / 2 E r ln (R / r )) 1/2 (6)
packing.

The volume fractions of nanoreinforcements were calculated using the Eq [START_REF] Morin | Nanocomposites of chitin nanofibrils from Riftia tubes and poly(caprolactone)[END_REF],

Ø = (w r / r ) / ((w r / r )+ (1-w r )/ m ) (7)
where, w r is weight % of reinforcements, r represent the density of the fibers and m is the density of the matrix polymer. The following values for the matrix and the cellulose nanofibers were used for theoretical calculations: E m = 2.9 GPa, E r = 138 GPa [START_REF] Nishino | Elastic modulus of the crystalline regions of cellulose polymorphs[END_REF], m = 1.25 g/cm 3 , and r = 1.58 g/cm 3 [START_REF] Ganster | Physical constants of cellulose[END_REF], the length of the nanofibers was predicted to be 10 µm and the diameter was chosen to be 100 nm. The volume fractions of nanofibers were determined to be 0.8%, 2.4%, and 4%, respectively.

The comparison between the experimental data and predictions by the Halpin-Tsai and Krenchel are presented in Figure 5. When comparing the results one has to keep in mind that the theoretical calculations by Halpin-Tsai are based on systems where the reinforcements are fully dispersed and aligned in the longitudinal direction and have perfect interfacial adhesion to the matrix, or where the reinforcing phase is randomly distributed in the matrix (Krenchel).

Dynamic mechanical properties

Figure 6 shows the storage modulus and tan delta as a function of temperature of the prepared materials. Generally, it is possible to see that the PLA has a transition around 60°C.

The storage modulus of PLA is increased with increased nanofiber content, in glassy as well as in rubbery state. The storage modulus of PLA has a significant drop on the transition region, whereas the drop is relatively less for the nanocomposites. Above the transition region, around 60°C, the modulus of the PLA nanocomposite was remarkably higher than PLA and the improvement is most significant at 5 wt% of nanofibers, indicating entangled nanofibers. Table 3 shows the storage modulus of the composites at two different temperatures. The storage modulus of the CNF composites was higher in the glassy and rubbery regions compared to pure PLA. The storage modulus data of PLA and the nanocomposites at 25°C support the data obtained from tensile testing and the trend as well as the values are found to be comparable. The improvement is most obvious at 70°C, where the modulus was 2.5 GPa for PLA-CNF5 compared to 100 MPa for pure PLA. The large modulus improvement after the relaxation, forming a "plateau" indicates entangled nanofibers and this is most obvious for the composites with highest nanofiber content; similar tangling behavior for nanofibers have been reported by Dufresne et al [START_REF] Samir | Tangling effect in fibrillated cellulose reinforced nanocomposites[END_REF].

Figure 6b shows how the addition of different content of nanofibers influenced the tan delta peak position of PLA. As can be seen from Figure 6b, the tan delta peak is shifted to higher temperature with increased nanofiber contents. The tan delta peak temperatures are given in Table 3. The peak position for PLA was at 70°C and increased to 76°C for the composites with 5% CNF. Additionally, Figure 6b shows that the intensity of the tan delta peak decreased with the concentration of nanofiber content compared to neat PLA. This indicates that fewer polymer chains are participating in this transition. The increase in modulus, together with positive shift in tan delta peak position, can be attributed to physical interaction between the polymer and reinforcements that restrict the segmental mobility of the

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polymer chains in the vicinity of the nanoreinforcements [START_REF] Bondeson | All Cellulose Nanocomposites Produced by Extrusion[END_REF]. It may also be noted that results from the viscoelastic behavior of the composites agree with the mechanical properties, suggesting that the reinforcing effect of nanofibers in PLA led to restriction of molecular chain mobility.

Film transparency

The visual study of the transparency of PLA and the PLA-CNF nanocomposites with different nanofiber contents is presented in Figure 7. Generally, the transparency of the PLA-CNF nanocomposites was good but as seen the the Figure 7 some white spots are visible in the PLA-CNF5 composite. These spots are expected be CNF aggregates in the PLA matrix.

Conclusions

Cellulose nanocomposite processing via a master batch with high content of nanofibers in PLA was attempted. The master batch and the bulk PLA were premixed and compounded during extrusion to obtain nanocomposites with different fiber compositions.

The morphology studies of PLA and its nanocomposites showed that a relatively good dispersion was achieved as no CNF aggregates were visible in the fracture surfaces of the PLA-CNF1 and PLA-CNF3 nanocomposites. The PLA-CNF5 however shows small aggregates, indicating that the nanofibers were not as well dispersed in this composite.

The visual characterization of the prepared materials showed small white spots, especially in composites with 5 wt% CNF, which further indicated that nanofibers were aggregated in that composite.

The evaluation of the mechanical properties of the PLA and its nanocomposite showed a trend that the tensile strength and modulus were improved with increased nanofiber content.

The modulus of the PLA was increased from 2.9 GPa to 3.6 GPa with the addition of 5 wt% nanofibers, and a 21% increase of tensile strength was observed. theoretical and experimental results showed that CNF has good potential as a reinforcement, especially if the nanofibers can be aligned but also that it might be possible to improve the properties even for randomly oriented nanofibers if the dispersion can be improved.

The DMA study indicated that the increased modulus, together with positive shift in tan delta peak position, is attributed to physical interaction between the polymer and nanofibers that restrict the segmental mobility of the polymer chains in the vicinity of the CNFs. Further more, storage modulus showed a plateau after the relaxation, especially when the nanofibers content was 5 wt% indicating entangled nanofibers.

This study shows that the melt compounding process using the master batch is a very promising method to reach good processability of cellulose nanocomposites and achieve improved mechanical and thermal properties for PLA. However, further work is needed to improve the dispersion of the nanofibers in the PLA.

Figure 5

 5 Figure5show that CNFs have a potential as reinforcement in polymers, especially if the

Figure captions Figure 1 .

 captions1 Figure captions Figure 1. AFM images of isolated nanofibers after grinding process, a) height image with measured fiber dimensions, b) fiber diameter measurements and c) amplitude image.

Figure 2 . 5 Figure 3 .

 253 Figure 2. Fractured surfaces of neat PLA and its nanocomposites with different CNF contents: a) PLA, b) PLA-CNF 1, c) PLA-CNF 3 and d) PLA-CNF 5 Figure 3. Detailed view of the fractured surface of PLA-CNF5 nanocomposite showing CNF aggregates.

Figure 4 .Figure 5 .

 45 Figure 4. Tensile stress-strain curves of PLA and PLA nanocomposite based on cellulose nanofibers Figure 5. Experimentally measured tensile modulus for nanocomposites compared to theoretical estimations by Halpin-Tsai and Krenchel.

Figure 6 .Figure 7 .

 67 Figure 6. DMA analysis of PLA and PLA nanocomposites; a) storage modulus; b) tan curves Figure 7. Visual comparison of the transparency of PLA and its nanocomposites

Table captions Table 1 .

 captions1 Material formulations in the processing and in the final compositions

Table 2 .

 2 Mechanical properties of the PLA and its nanocomposites with different compositions

Table 3 .

 3 Storage modulus and tan delta peak temperatures of the PLA and its nanocomposites

Table 1 .

 1 Material formulations in the processing and in the final compositions : Solvents were evaporated during drying of the master batch

	Materials		Master batch	Bulk PLA (g)	Final composition (%)
		PLA	CNF	Solvent mixture
		(g)	(g)	(g)*
	PLA	15	-	300	85	PLA (100)
	PLA-CNF1	14	1	300	85	PLA-CNF (99\1)
	PLA-CNF3	12	3	300	85	PLA-CNF (97\3)
	PLA-CNF5	10	5	300	85	PLA-CNF (95\5)

*

Table 2 .

 2 Mechanical properties of the PLA and its nanocomposites with different compositions

	Materials	Tensile modulus (GPa)	Tensile strength (MPa)	Max strain (%)
	PLA	2.9 ± 0.6	58.9 ± 0.5	3.4 ± 0.4
	PLA-CNF1	3.3 ± 0.4	63.1 ± 0.9	2.8 ± 0.3
	PLA-CNF3	3.4 ± 0.1	65.1 ± 0.6	2.7 ± 0.2
	PLA-CNF5	3.6 ± 0.7	71.2 ± 0.6	2.7 ± 0.1

Table 3 .

 3 Storage modulus and tan delta peak temperatures of the PLA and its nanocomposites

	Materials	E' at 25°C (MPa)	E' at 70°C (MPa)	Tan δ peak (°C)
	PLA	2600 ± 100	100 ± 00	70 ± 0
	PLA-CNF1	2800 ± 100	170 ± 30	71 ± 1
	PLA-CNF3	3100 ± 200	340 ± 10	73 ± 1
	PLA-CNF5	4400 ± 400	2500 ± 300	76 ± 0

Acknowledgements

The authors are grateful to the Ministry of Plantation Industry and Commodity (MPIC), Government of Malaysia as well as Kempe Stiftelserna, Sweden, for funding of this research.